AMGS vs. Other Greenness Metrics: A Strategic Guide for Sustainable Analytical Chemistry

Abstract

This article provides a comprehensive comparison of the Analytical Method Greenness Score (AMGS) with other established greenness metrics like AGREE, GAPI, and NEMI. Tailored for researchers and pharmaceutical development professionals, it explores the foundational principles, practical applications, and strategic selection of these tools. By presenting real-world case studies, optimization strategies, and a critical validation of each metric's strengths and limitations, this guide empowers scientists to make informed decisions that enhance both the sustainability and efficiency of their analytical methods.

The Green Metrics Landscape: From Foundational Principles to Modern Tools

Green Analytical Chemistry (GAC) has emerged as a fundamental paradigm shift within the pharmaceutical industry and broader chemical analysis fields, driven by increased awareness of the environmental impacts associated with analytical methodologies. The foundational principles of GAC, extending from the seminal "12 principles of green chemistry" first proposed by Anastas and Warner in 1998, aim to minimize the environmental footprint of analytical processes while ensuring analytical performance and reliability [1]. The adoption of GAC principles has become increasingly urgent given the cumulative environmental burden of analytical methods. A compelling case study of rosuvastatin calcium illustrates this point: with approximately 25 liquid chromatography analyses performed per batch and an estimated 1000 batches produced globally each year, this single active pharmaceutical ingredient results in the consumption and disposal of approximately 18,000 liters of mobile phase annually solely for chromatographic analysis [1]. This example shatters the pervasive perception that analytical methods have insignificant environmental impact and underscores the critical need for standardized metrics to quantify and drive improvements in method greenness.

The Green Metrics Landscape: A Comparative Analysis

The growing recognition of analytical chemistry's environmental impact has catalyzed the development of numerous assessment tools and metrics. These tools enable researchers to systematically evaluate, compare, and improve the environmental footprint of their analytical methods. The table below provides a comprehensive comparison of the major greenness assessment metrics used in contemporary analytical practice.

Table 1: Comparison of Major Green Analytical Chemistry Assessment Metrics

Metric Name	Type of Output	Key Assessment Criteria	Strengths	Limitations
Analytical Method Greenness Score (AMGS) [1] [2]	Quantitative score (%)	Solvent health/safety/environmental impact, cumulative energy demand, instrument energy usage, solvent waste	Comprehensive, specifically designed for chromatography; drives environmental awareness during method development	Currently limited to liquid chromatography and SFC; does not address additives
Analytical GREEnness (AGREE) [1] [3]	Pictogram (radar chart) with numerical score (0-1)	Twelve principles of GAC including energy consumption, solvent toxicity, waste generation, and safety	Comprehensive visual representation; applicable to multiple analytical techniques; online calculator available	Can involve estimations that introduce inaccuracies
Green Analytical Procedure Index (GAPI) [1] [4]	Qualitative pictogram (color-coded)	Five pentagrams representing different stages of analytical procedure from sample collection to waste management	Detailed breakdown of each analytical step; quick visual assessment	Limited to qualitative analysis; relatively coarse assessment model for chromatography
Analytical Eco-Scale [1] [5]	Quantitative score (penalty points subtracted from 100)	Reagent type/amount, energy consumption, waste generated	Simple numerical output; clear indication of greenness	Semi-quantitative; less detailed than other metrics
Red Analytical Performance Index (RAPI) [6]	Pictogram (star-shaped) with numerical score (0-100)	Ten analytical performance criteria (repeatability, precision, sensitivity, linearity, etc.)	Assesses analytical performance complementary to greenness metrics; aligns with validation guidelines	Does not directly assess environmental impact
Blue Applicability Grade Index (BAGI) [6] [5]	Pictogram (star-shaped) with numerical score (25-100)	Practicality and economic criteria (cost, time, skill requirements, etc.)	Evaluates practical applicability; complements greenness and performance metrics	Focuses only on practical/economic aspects
Greenness Evaluation Metric for Analytical Methods (GEMAM) [4]	Pictogram (hexagonal) with numerical score (0-10)	21 criteria based on 12 GAC principles and 10 green sample preparation factors	Highly comprehensive; flexible weighting system; covers entire analytical workflow	Complex calculation process; newer with less established track record

The Analytical Method Greenness Score (AMGS): A Closer Examination

Development and Core Principles

The Analytical Method Greenness Score (AMGS) represents a significant advancement in the quantification of chromatographic method sustainability. Developed by the American Chemical Society's Green Chemistry Institute in collaboration with pharmaceutical industry partners, AMGS provides a comprehensive metric that evaluates environmental impact across multiple dimensions [1] [2]. Unlike earlier tools, AMGS specifically addresses the unique environmental considerations of chromatographic techniques, incorporating solvent health, safety, and environmental impact; cumulative energy demand for solvent production and disposal; instrument energy consumption; and method solvent waste generation [2]. The metric outputs a quantitative percentage score where a lower AMGS indicates a greener method, providing researchers with a clear benchmark for comparison and improvement.

Implementation and Impact

AstraZeneca's implementation of AMGS demonstrates its practical utility in driving sustainable practices within pharmaceutical development. The organization has utilized AMGS to assess the current status of chromatographic methods across its drug project portfolio, influence standard practices, and create internal tools for trending data as a mode of continuous process verification [1]. This systematic approach has enabled the identification of method redevelopment opportunities with significant potential for environmental improvement. The AMGS calculator, available through the ACS Green Chemistry Institute, is specifically designed as a comparative metric during method development rather than an absolute measure for method rejection, emphasizing its role in fostering environmental awareness and encouraging the development of greener alternatives [2].

Table 2: AMGS Performance in Different Method Modification Scenarios

Modification Type	Impact on AMGS Score	Key Mechanism	Environmental Benefit
Reduced Method Runtime	Decreases instrument energy contribution	Lower kWh consumption per analysis	Reduced electricity usage and carbon footprint
Solvent Replacement	Decreases solvent EHS and energy scores	Substitution with less hazardous, lower energy solvents	Reduced toxicity and cumulative energy demand
Method Miniaturization	Reduces solvent waste score	Lower mobile phase consumption per analysis	Less waste generation and solvent disposal
Column Technology Upgrade	Potential decrease in multiple categories	Improved efficiency allowing shorter runs or different solvent systems	Multiple environmental benefits

Methodologies for Greenness Assessment: Experimental Protocols and Workflows

Standardized Evaluation Framework

Implementing a robust greenness assessment requires a systematic approach that integrates multiple metrics to provide a comprehensive understanding of a method's environmental and functional characteristics. The workflow below illustrates the integrated evaluation process combining greenness, performance, and practicality metrics.

Detailed Experimental Protocol for Comprehensive Method Assessment

To ensure reproducible and scientifically rigorous greenness assessments, researchers should follow a standardized experimental protocol:

• Method Parameter Documentation: Record all critical method parameters including instrument type (HPLC, UPLC, GC), column dimensions, flow rate, injection volume, gradient program, mobile phase composition, sample preparation steps, and analysis time [1] [4].

• AMGS Calculation Procedure:

  • Input solvent volumes and types into the ACS GCI AMGS calculator
  • Document instrument energy consumption based on manufacturer specifications and runtime
  • Calculate cumulative energy demand for solvent production using established databases
  • Evaluate solvent health, safety, and environmental impact scores using implemented criteria
  • Generate overall AMGS percentage and category-specific scores [2]

• Complementary Metric Assessment:

  • Perform AGREE evaluation using the online calculator with twelve principle assessments
  • Complete GAPI pictogram by evaluating each of the five analytical stages
  • Calculate Analytical Eco-Scale by assigning penalty points and subtracting from base 100 [1] [5]

• Functional Performance Integration:

  • Conduct RAPI assessment using open-source software to evaluate ten analytical performance criteria
  • Perform BAGI evaluation to determine practical applicability and economic feasibility [6]

• Data Synthesis and Interpretation:

  • Compare metric outputs to identify consistent patterns and potential discrepancies
  • Prioritize improvement opportunities based on metric sensitivities
  • Establish baseline performance for future method comparisons [7]

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Essential Reagents and Materials for Green Analytical Chemistry Research

Item/Category	Function in GAC Research	Green Alternatives & Considerations
Chromatographic Solvents	Mobile phase composition in LC methods	Replace acetonitrile with ethanol or methanol; minimize solvent toxicity and energy of production [1] [5]
Sample Preparation Materials	Extraction and preparation of analytes	Implement miniaturized systems; use less toxic extraction solvents; automate processes [3] [8]
Derivatization Reagents	Chemical modification for detection	Avoid derivatization where possible; use less hazardous reagents; minimize quantities [3]
Energy Monitoring Equipment	Quantifying instrument power consumption	Use wattmeters for direct measurement rather than estimation; enable empirical data collection [7]
Waste Treatment Solutions	Management of analytical waste	Implement recycling programs for solvents; utilize waste treatment protocols; minimize generation [4]
Alternative Column Technologies	Stationary phases for separation	Explore columns enabling faster analyses or greener mobile phases; consider high-temperature LC [1]
1-Palmitoyl-sn-glycero-3-phosphocholine-d49	1-Palmitoyl-sn-glycero-3-phosphocholine-d49, MF:C24H50NO7P, MW:544.9 g/mol	Chemical Reagent
Cuprolinic Blue-d12	Cuprolinic Blue-d12, MF:C33H27CuN12O4S+3, MW:763.3 g/mol	Chemical Reagent

Interpreting Results: Towards Sustainable Analytical Practices

Strategic Implementation of Assessment Findings

The effective interpretation of greenness metrics requires understanding their individual strengths and limitations while recognizing their complementary nature. Research indicates that no single metric provides a complete picture of a method's sustainability profile [7]. For instance, while AMGS offers chromatographically-focused quantitative assessment of environmental impact, it should be integrated with functional metrics like RAPI and BAGI to ensure that greener methods maintain necessary analytical performance and practical applicability [6]. This integrated approach aligns with the White Analytical Chemistry concept, which seeks the optimal balance between environmental impact (green), analytical performance (red), and practical applicability (blue) [6] [5].

Beyond Greenness: The Role of Performance and Practicality

The relationship between different assessment dimensions can be visualized through their interconnectedness in method evaluation and optimization, demonstrating how comprehensive assessment leads to more sustainable analytical practices.

Recent studies demonstrate the successful application of this integrated approach. A 2025 method for simultaneous quantification of gabapentin and methylcobalamin achieved an AGREE score of 0.70 while maintaining excellent analytical performance (linearity R² > 0.9998) and practical applicability, showcasing how environmental and functional objectives can be simultaneously optimized [5]. Such examples highlight the importance of moving beyond single-dimensional greenness assessments toward holistic evaluation frameworks that consider the complete analytical profile.

The development and implementation of greenness metrics represent a critical evolution in analytical chemistry, enabling the systematic quantification and improvement of method environmental performance. Among the available tools, AMGS provides particularly valuable chromatographically-focused assessment, while complementary metrics like AGREE, GAPI, RAPI, and BAGI offer additional dimensions for comprehensive evaluation. As the field progresses toward stronger sustainability models that acknowledge ecological limits and prioritize nature conservation, these metrics will play an increasingly vital role in guiding method development and selection [8]. The ongoing refinement of assessment tools, including the planned expansion of AMGS to include gas chromatography and address methodological granularity like mobile phase additives, promises to further enhance their utility in driving the pharmaceutical industry and analytical science toward a more sustainable future [1] [2].

In recent years, the drive toward sustainability has become increasingly important across the pharmaceutical and analytical industries. The field of green analytical chemistry (GAC) has emerged as a critical discipline focused on minimizing the environmental impact of analytical processes while ensuring analytical performance. The concept extends the principles of green chemistry to analytical processes, emphasizing the reduction of hazardous substances, energy consumption, and waste generation. As the field has evolved, numerous assessment tools have been developed to evaluate and quantify the environmental impact of analytical methods.

This progression began with simpler tools like the National Environmental Method Index (NEMI) and Analytical Eco-Scale (AES) and has advanced toward more comprehensive metrics including the Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE). Most recently, the Analytical Method Greenness Score (AMGS) has been developed through collaboration between the American Chemical Society's Green Chemistry Institute and industry partners, offering a sophisticated approach specifically designed for chromatographic methods. This timeline of evolution represents a continuous refinement in how the scientific community quantifies and prioritizes environmental sustainability in analytical practices [1] [9].

Historical Development of Greenness Assessment Tools

First-Generation Metrics: Foundation and Limitations

The earliest green assessment tools provided foundational approaches for evaluating environmental impact but faced significant limitations in comprehensiveness and applicability.

National Environmental Method Index (NEMI) was among the pioneering tools introduced for greenness assessment. Its approach is relatively simple, representing method environmental impact through a pictogram with four quadrants that indicate whether a method meets specific criteria: (1) does not use persistent, bioaccumulative, and toxic chemicals; (2) does not use corrosive reagents; (3) does not use hazardous reagents; and (4) waste generation is minimal. While straightforward to interpret, NEMI's binary (pass/fail) assessment system lacks granularity, as it does not quantify the degree of greenness or account for energy consumption and other environmental factors [10] [9].

Analytical Eco-Scale (AES) emerged as a semi-quantitative alternative that assigns penalty points to various parameters of an analytical method, including the type and amount of reagents used, energy consumption, and waste generated. The total eco-scale score is calculated by subtracting penalty points from a base value of 100, with higher scores indicating greener methods. This tool provided a numerical assessment that enabled easier comparison between methods and identified specific areas for improvement. However, its penalty assignment system remained somewhat subjective, and it didn't comprehensively address all principles of green analytical chemistry [1] [9].

Second-Generation Metrics: Comprehensive and Visual Assessments

As the field matured, more sophisticated tools emerged that offered more comprehensive evaluations and intuitive visual representations.

Green Analytical Procedure Index (GAPI) expanded evaluation scope by incorporating a more holistic approach. GAPI uses a pictogram consisting of five pentagrams, each representing different stages of an analytical procedure from sample collection to waste management. Each pentagram is divided into sections color-coded based on environmental impact (green for low, yellow for moderate, and red for high impact). This visual representation allows for quick yet detailed assessment of method greenness across the entire analytical process, addressing a wider range of environmental concerns than earlier tools [1] [11].

Analytical GREEnness (AGREE) represents a significant advancement by comprehensively addressing all 12 principles of green analytical chemistry. The tool employs a clock-like pictogram where each of the 12 segments corresponds to one GAC principle, scored from 0-1 with weighting factors that affect segment width. The output displays a final score (0-1) in the center with a color indication (red to green) reflecting overall method greenness. AGREE's strengths include its comprehensive foundation in GAC principles, visual intuitiveness, and applicability across multiple analytical techniques. The availability of an online calculator further enhances its accessibility and standardization [11] [9].

Table 1: Evolution of Major Green Assessment Tools

Metric	Year Introduced	Assessment Approach	Key Parameters	Output Format	Key Limitations
NEMI	Early 2000s	Binary criteria	PBT chemicals, corrosives, hazardous reagents, waste amount	Pictogram with 4 quadrants	Lacks granularity; no energy consideration
Analytical Eco-Scale	2013	Penalty point system	Reagent toxicity, energy, waste	Numerical score (0-100)	Subjective penalty assignments
GAPI	2019	Qualitative assessment	Sample collection, preparation, transportation, etc.	5 pentagrams with color coding	Less quantitative; limited weighting flexibility
AGREE	2020	Weighted criteria based on 12 GAC principles	All 12 GAC principles	Clock-like pictogram with score	Requires detailed method knowledge
AMGS	Recent (post-2020)	Quantitative multi-dimensional	Solvent energy, EHS, instrument energy	Numerical score	Currently focused on chromatography

The Emergence of Analytical Method Greenness Score (AMGS)

Development and Strategic Importance

The Analytical Method Greenness Score (AMGS) represents the latest evolution in green assessment tools, developed through collaboration between the American Chemical Society's Green Chemistry Institute and industry partners. This metric was specifically designed to address the need for a comprehensive, chromatography-focused evaluation tool that could provide strategic insight for organizations pursuing sustainability goals [1].

AMGS evaluates the environmental impact of chromatographic methods across multiple dimensions, with unique emphasis on instrument energy consumption in addition to solvent-related factors. The tool calculates three primary component scores: solvent energy (accounting for energy consumed in solvent production and disposal), solvent environment, health, and safety (EHS) factors, and instrument energy consumption. These components are integrated into an overall greenness score that enables direct comparison between methods and identification of improvement opportunities [1].

The development of AMGS responds to the substantial cumulative environmental impact of analytical methods when scaled across global manufacturing networks. As illustrated by a case study of rosuvastatin calcium, where approximately 18,000 liters of mobile phase are consumed annually for chromatographic analysis of a single active pharmaceutical ingredient, the widespread perception that analytical methods have insignificant environmental impact is both pervasive and damaging. AMGS provides the pharmaceutical industry with a tool to quantify and systematically reduce this impact [1].

Unique Characteristics and Industry Application

AMGS incorporates several distinctive features that differentiate it from earlier metrics. Unlike tools that focus primarily on solvent selection and waste generation, AMGS specifically accounts for instrument energy consumption, recognizing that analytical equipment constitutes a significant portion of laboratory energy usage. Additionally, it considers the energy consumed in the production and disposal of solvents used, providing a more complete lifecycle perspective [1].

Pharmaceutical companies like AstraZeneca have utilized AMGS to assess the current status of their analytical portfolio, influence standard practices, and create internal tools for continuous process verification. By integrating AMGS into routine analytical procedures, organizations can systematically improve their sustainability profiles, reduce hazardous waste, and promote greener alternatives while maintaining the rigorous quality standards required for pharmaceutical analysis [1].

Comparative Analysis of Major Greenness Assessment Tools

Scoring Systems and Assessment Focus

The major greenness assessment tools employ fundamentally different approaches to scoring and evaluation, each with distinct advantages and limitations.

Table 2: Scoring Characteristics of Green Assessment Tools

Metric	Scoring System	Scope of Assessment	Weighting of Factors	Ideal Score
NEMI	Binary (pass/fail) for 4 criteria	Limited to reagents and waste	Equal weighting for all criteria	All quadrants green
Analytical Eco-Scale	Subtractive points from base of 100	Reagents, energy, waste	Implicit through point values	>75 (excellent)
GAPI	Color-coded (green/yellow/red)	Entire analytical procedure	Equal across pentagrams	All green sections
AGREE	0-1 with weighted segments	All 12 GAC principles	Adjustable segment weights	1.0
AMGS	Numerical component scores	Chromatography-specific factors	Defined by calculation model	Lower = greener

The AGREE tool stands out for its comprehensive foundation in all 12 principles of green analytical chemistry, addressing factors such as miniaturization, automation, and throughput in addition to traditional solvent and energy concerns. Each principle is scored individually and contributes to an overall assessment, providing both specific and holistic feedback on method greenness [11] [9].

In contrast, AMGS employs a more specialized approach focused specifically on chromatographic applications, with quantitative assessment of solvent energy, solvent EHS factors, and instrument energy consumption. This chromatography-specific focus enables more targeted optimization of LC methods but may limit applicability to other analytical techniques [1].

Practical Applications and Performance Comparison

Recent studies have demonstrated the practical application of these tools in comparing analytical methods. A comparative study of chromatographic methods for analyzing UV filters in cosmetics evaluated ten different procedures using AGREE and AGREEprep (a complementary tool focused specifically on sample preparation). The assessment revealed that microextraction methods consistently achieved higher greenness scores, demonstrating how these tools can guide analysts toward more sustainable choices [11].

In pharmaceutical analysis, a study comparing normal-phase (NP) and reversed-phase (RP) HPTLC methods for quantifying ertugliflozin applied four greenness assessment tools: NEMI, AES, ChlorTox, and AGREE. The results consistently demonstrated that the RP-HPTLC method was greener than the NP-HPTLC approach across all assessment tools, with the AGREE tool providing the most comprehensive and nuanced evaluation [10].

When comparing AMGS with earlier metrics, several distinctions emerge. While tools like GAPI and AGREE offer visual outputs that facilitate quick assessment, AMGS provides quantitative data that supports trend analysis and portfolio-level decision-making. As noted in industry applications, "The AMGS tool has several constraints, which if addressed, would add value to the calculation, for example, additional analytical techniques, impact of chromatographic mobile phase and sample diluent additives" [1].

Experimental Protocols for Greenness Assessment

Standardized Methodology for Tool Application

To ensure consistent and comparable results when evaluating analytical methods using different greenness assessment tools, researchers should follow standardized experimental protocols:

• Method Documentation: Completely document all aspects of the analytical procedure including sample preparation, reagents and solvents (type and volume), instrumentation, energy consumption parameters, and waste generation data.

• Tool Selection: Based on study objectives, select appropriate assessment tools. For comprehensive evaluation, use multiple tools to leverage their complementary strengths.

• Data Collection: For AGREE assessment, collect information corresponding to all 12 GAC principles. For AMGS, focus on chromatography-specific parameters including instrument type, run time, flow rate, and solvent compositions [1] [11].

• Score Calculation: Utilize available software tools for score calculation when possible. AGREE offers an open-source calculator available at https://mostwiedzy.pl/AGREE, while AMGS may utilize proprietary calculators implemented within organizations [11].

• Interpretation and Comparison: Compare scores across methods using the same tool, noting that absolute scores from different tools are not directly comparable due to different assessment frameworks.

Case Study Implementation Protocol

A typical experimental protocol for comparing methods, as demonstrated in the HPTLC study for ertugliflozin, includes:

• Method Development: Develop and validate both NP-HPTLC and RP-HPTLC methods according to ICH Q2-R2 guidelines. For NP-HPTLC, use chloroform/methanol (85:15 v/v) mobile phase. For RP-HPTLC, use ethanol-water (80:20 v/v) mobile phase [10].

• Chromatographic Conditions: Use silica gel 60 NP-18F254S plates for NP-HPTLC and 60 RP-18F254S plates for RP-HPTLC. Perform detection at 199 nm for both methods.

• Greenness Assessment: Apply multiple assessment tools (NEMI, AES, AGREE, and AMGS if available) to both methods using standardized parameters.

• Data Analysis: Compare scores across all tools to identify consistent trends and method superiorities.

Greenness Assessment Workflow

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Essential Materials for Green Analytical Chemistry Research

Item	Function in Green Assessment	Application Example
AGREE Software	Open-source tool for calculating AGREE scores	Evaluating overall method greenness against 12 GAC principles
AGREEprep Tool	Specialized software for sample preparation assessment	Comparing greenness of microextraction techniques
Solvent Selection Guide	Reference for solvent environmental and safety data	Choosing greener alternatives in method development
Life Cycle Inventory Database	Source data for energy and environmental impact	Calculating AMGS solvent energy and EHS components
Chromatography Data Systems	Source of method parameters for assessment	Extracting run times, flow rates for AMGS calculations
Isoflupredone-d5	Isoflupredone-d5, MF:C21H27FO5, MW:383.5 g/mol	Chemical Reagent
Demeton-S-d10	Demeton-S-d10, MF:C8H19O3PS2, MW:268.4 g/mol	Chemical Reagent

The evolution from early tools like NEMI and Eco-Scale to comprehensive metrics like AGREE, GAPI, and AMGS represents significant advancement in how the analytical community quantifies and prioritizes environmental sustainability. Each generation of tools has built upon its predecessors, addressing limitations and expanding assessment scope to provide more accurate, actionable evaluations of method greenness.

The current landscape offers analysts multiple tools with complementary strengths—from AGREE's comprehensive foundation in all 12 GAC principles to AMGS's specialized focus on chromatographic parameters and energy consumption. This progression enables more informed method selection and development, supporting the pharmaceutical industry's ambitious sustainability goals while maintaining the rigorous quality standards essential for patient safety.

As green analytical chemistry continues to evolve, future developments will likely address current tool limitations, expand assessment scope to include additional analytical techniques, and further refine the quantification of environmental impact. Through continued refinement and application of these assessment metrics, the analytical community can significantly reduce its environmental footprint while advancing scientific capabilities.

The pharmaceutical industry faces increasing pressure to minimize its environmental footprint, a challenge that extends from manufacturing to analytical testing. Green Analytical Chemistry (GAC) has emerged as a dedicated field within analytical chemistry, aiming to mitigate the detrimental effects of analytical techniques on the environment and human health [12]. While the scale of solvent use in individual analytical methods is small compared to process chemistry, its cumulative impact is substantial. A single drug compound like rosuvastatin calcium can consume approximately 18,000 liters of mobile phase annually when scaled across global production batches, highlighting the critical need for sustainable analytical practices [1].

This environmental imperative has driven the development of various assessment tools to evaluate and improve the "greenness" of analytical methods. Among these tools, the Analytical Method Greenness Score (AMGS) has emerged as a specialized metric developed specifically for chromatographic methods within the pharmaceutical industry. This guide explores the origin, development, and core philosophy of AMGS while providing a detailed comparison with other established greenness assessment tools.

Origin and Development of AMGS

Institutional Background and Leadership

The AMGS calculator was developed under the auspices of the ACS Green Chemistry Institute Pharmaceutical Roundtable (ACS GCI PR), a leading organization dedicated to catalyzing the integration of green chemistry and engineering in the pharmaceutical industry [13]. Established in 2005, the Roundtable provides a science-focused forum for member companies to collectively prioritize research needs and leverage resources toward green chemistry tools specific to the pharmaceutical industry [14].

The development team comprised industry experts from major pharmaceutical companies including:

• Michael Hicks (Merck, Rahway, NJ)
• Bill Farrell and Christine Aurigemma (Pfizer, San Diego, CA)
• Paul Ferguson (AstraZeneca, Macclesfield, UK)
• Dave Constable, Isamir Martinez, and Christiana Briddell (ACS GCI, Washington, DC) [2]

The tool evolved from a Microsoft Excel-based program originally created by Laurent Lehman at Bristol-Myers Squibb, demonstrating its origins in practical industry needs rather than purely academic research [2].

Development Timeline and Future Directions

The AMGS metric represents an ongoing commitment to sustainable analytical chemistry. The current version focuses exclusively on liquid chromatography and SFC methods, with an updated version supporting Gas Chromatography (GC) methods expected by early 2026 [2]. The ACS GCI PR continues to actively solicit feedback from users to improve the tool's functionality and application across the industry [2].

Table: Development Timeline of AMGS

Year	Development Milestone
Pre-2020	Original Excel-based program created by Laurent Lehman at Bristol-Myers Squibb
2020-2024	Collaborative development and refinement by ACS GCI PR Analytical Chemistry team
2025	Public launch and implementation in pharmaceutical companies (e.g., AstraZeneca case study)
2026 (Expected)	Release of updated version supporting Gas Chromatography (GC) methods

Core Philosophy and Design Principles of AMGS

The AMGS is founded on a pragmatic philosophy that balances comprehensive environmental assessment with practical applicability in pharmaceutical analytical development. Its core design principles include:

Holistic Environmental Assessment

Unlike some metrics that focus narrowly on specific aspects, AMGS employs a multi-dimensional assessment that factors in solvent health, safety, environmental impact, cumulative energy demand, instrument energy usage, and method solvent waste [2]. This comprehensive approach ensures that method improvements address the complete environmental profile rather than optimizing a single parameter at the expense of others.

Comparative, Not Absolute, Measurement

A fundamental principle of AMGS is its use as a comparative metric rather than an absolute measure of greenness. The tool is explicitly designed "to benchmark and compare one method to another" during method development [2]. The resulting score is meant to "provide environmental impact awareness and encourage analysts to develop greener methods" rather than to reject methods based on a finite threshold [2].

Actionable Feedback Through Color Coding

The tool incorporates a color-coded scoring system where yellow and red colors highlight areas where the method could be improved [2]. This provides immediate, actionable feedback to analytical chemists, directing them to specific aspects of their methods that contribute most significantly to the environmental impact, such as instrument energy consumption or solvent toxicity.

Industry-Specific Relevance

AMGS was designed specifically for chromatographic methods used in pharmaceutical analysis, ensuring relevance to the specific challenges and constraints of this application area [1]. This industry focus distinguishes it from more general-purpose green assessment tools and enhances its practical utility for pharmaceutical scientists.

Comparative Analysis: AMGS vs. Other Green Metric Tools

The landscape of green metric tools has evolved significantly, with multiple approaches developed for different assessment needs:

• National Environmental Methods Index (NEMI): One of the earliest tools, using a simple pictogram with four quadrants representing different environmental criteria [15] [12]. It employs binary (yes/no) assessment with boundaries established at specific thresholds (e.g., waste generation ≤50 g per sample) [15].

• Analytical Eco-Scale Assessment (ESA): A semi-quantitative tool that assigns penalty points for various parameters including reagents, energy consumption, and waste [1]. The total score is calculated by subtracting penalty points from a base value of 100, with higher scores indicating greener methods [1].

• Green Analytical Procedure Index (GAPI): Employs a detailed pictogram with five pentagrams representing different stages of the analytical procedure, each color-coded (green, yellow, red) based on environmental impact [1]. It provides a more holistic visual assessment than NEMI [1].

• Analytical GREEnness (AGREE): Uses a comprehensive radar chart with twelve criteria representing different green chemistry principles, each rated 0-1 [1]. An online calculator is available, and the overall score is the average of the twelve ratings [1].

Detailed Comparison of Key Metrics

Table: Comprehensive Comparison of Green Assessment Tools

Feature	AMGS	AGREE	GAPI	Analytical Eco-Scale	NEMI
Primary Focus	Chromatographic methods in pharma	General analytical methods	General analytical procedures	General analytical methods	Environmental methods
Output Type	Numerical score with color coding	Numerical score (0-1) + radar chart	Pictogram (color-based)	Numerical score (0-100)	Pictogram (binary)
Key Assessment Criteria	Solvent EHS, solvent energy, instrument energy	12 principles of GAC	Multiple stages from sampling to waste	Reagents, energy, waste	Toxicity, waste, corrosiveness
Scoring System	Lower score = greener	Higher score = greener	Color-based: green to red	Higher score = greener	Binary: filled/unfilled quadrants
Weighting of Criteria	Adjustable with defaults	Adjustable with defaults	Fixed	Implicit through penalty points	Fixed
Technical Specificity	High for chromatography	Medium	Medium	Medium	Low
Ease of Use	Moderate	Moderate (with calculator)	Easy	Easy	Very easy
Application in Pharma	High	Medium	Medium	Medium	Low

Technical Differentiation of AMGS

AMGS distinguishes itself technically through several key features:

Integrated Energy Assessment: Unlike many other tools, AMGS specifically incorporates both solvent production energy and instrument energy consumption into its calculation [1]. This provides a more complete picture of the total energy footprint of an analytical method.

Pharmaceutical Industry Focus: While tools like AGREE and GAPI are designed for broader applicability, AMGS is optimized for the specific requirements and constraints of pharmaceutical analysis and regulatory compliance [2] [1].

Solvent-Centric Evaluation: Given that solvents represent the most significant environmental impact in liquid chromatography, AMGS provides detailed assessment of solvent environmental, health, and safety (EHS) impacts alongside the energy required for their production and disposal [2].

Experimental Application and Validation

Implementation Case Study: AstraZeneca

A comprehensive study conducted at AstraZeneca demonstrated the practical application of AMGS across their pharmaceutical development portfolio [1]. Researchers implemented AMGS to:

• Assess current status of chromatographic methods across their portfolio
• Identify improvement opportunities in method development and redevelopment
• Influence standard practices and create internal tools for data trending
• Establish continuous process verification for sustainability metrics [1]

The implementation allowed AstraZeneca to quantify their environmental impact from analytical chemistry and align with their ambition to achieve carbon zero status for analytical laboratories by 2030 [1].

Experimental Protocol for AMGS Assessment

For researchers seeking to implement AMGS in their own workflows, the following methodology provides a standardized approach:

Step 1: Data Collection

• Compile chromatographic method parameters: mobile phase composition, flow rate, runtime, injection volume, and column dimensions
• Identify instrument type (HPLC, UHPLC) and operational modes
• Quantify sample preparation requirements including dilution factors and solvent volumes [2]

Step 2: Input Parameters for AMGS Calculator

• Solvent volumes (across entire method including system suitability tests)
• Instrument energy consumption (based on runtime and instrument type)
• Solvent EHS profiles (from built-in database)
• Cumulative energy demand for solvent production [2] [1]

Step 3: Score Calculation and Interpretation

• Calculate individual scores for solvent EHS, solvent energy, and instrument energy
• Generate overall AMGS (lower scores indicate greener methods)
• Interpret color-coded results to identify primary contributors to environmental impact [2]

Step 4: Method Optimization

• Target improvements in areas highlighted by color coding (yellow/red)
• Explore alternative solvents with better EHS profiles
• Optimize method parameters to reduce runtime and solvent consumption
• Validate that changes maintain analytical performance [1]

Experimental Data from Implementation

Table: AMGS Application Data from Pharmaceutical Implementation

Method Parameter	Before AMGS Optimization	After AMGS Optimization	Reduction Impact
Typical HPLC Flow Rate	1.0-2.0 mL/min	0.3-0.8 mL/min	40-70% solvent reduction
Average Runtime	30-60 minutes	5-15 minutes	60-80% energy reduction
Acetonitrile Usage	High (primary solvent)	Reduced or replaced	Lower EHS impact
Total Solvent Waste per Analysis	100-500 mL	10-100 mL	70-90% waste reduction
AMGS Score	Higher (less green)	Lower (greener)	Improved sustainability

Visualization: Relationship Among Green Assessment Tools

The following diagram illustrates the logical relationships and primary focus areas of major green assessment tools, highlighting AMGS's position within the ecosystem:

Green Metric Tools Relationship

Table: Essential Research Reagent Solutions for Green Analytical Chemistry

Tool/Resource	Function/Purpose	Application Context
AMGS Calculator	Quantifies environmental impact of chromatographic methods	Pharmaceutical method development and comparison
AGREE Calculator	Assesses alignment with 12 GAC principles	General analytical method greenness assessment
GAPI Pictogram	Visual assessment across analytical process stages	Quick visual communication of method greenness
Alternative Solvent Database	Identifies greener solvent substitutions	Method optimization to reduce EHS impact
Method Scalability Calculator	Projects cumulative environmental impact	Business case development for green method adoption
Energy Consumption Monitor	Measures actual instrument energy usage	Empirical data collection for AMGS assessment

The Analytical Method Greenness Score represents a significant advancement in the toolkit for sustainable analytical chemistry, particularly within the pharmaceutical industry. Its development by the ACS GCI Pharmaceutical Roundtable reflects a concerted industry effort to address the environmental impact of analytical methods through a specialized, practical metric.

While AMGS excels in its specific domain of chromatographic analysis, the diversity of available green assessment tools highlights the multifaceted nature of environmental impact assessment. Tools such as AGREE, GAPI, and Analytical Eco-Scale each offer unique strengths for different applications and contexts. The most effective approach to green analytical chemistry involves understanding the complementary nature of these tools and selecting the most appropriate metric based on specific assessment needs, analytical techniques, and industry context.

As the field continues to evolve, the ongoing development of AMGS—including the planned expansion to GC methods—demonstrates the commitment to continuous improvement in green metrics. For researchers and pharmaceutical professionals, mastering these tools is essential for advancing both environmental sustainability and analytical excellence.

In the pursuit of sustainable laboratory practices, Green Analytical Chemistry (GAC) has emerged as a critical discipline focused on minimizing the environmental impact of analytical methods [12] [16]. The evaluation of this environmental footprint revolves around three central dimensions: solvent impact, energy consumption, and waste generation [17] [2]. These core parameters form the foundation for comparing the ecological performance of different analytical techniques, particularly in pharmaceutical development and other chemical industries. Without standardized metrics, assessing and comparing the greenness of analytical methods would be subjective and inconsistent. To address this challenge, researchers have developed various assessment tools that quantify environmental impact across these key dimensions [9] [16]. This article provides a comprehensive comparison of prominent greenness metrics, with particular focus on the Analytical Method Greenness Score (AMGS) in relation to other established tools, examining their approaches to measuring solvent impact, energy consumption, and waste production.

Core Principles of Green Analytical Chemistry

Green Analytical Chemistry is built upon twelve fundamental principles that provide a framework for developing environmentally conscious analytical methods [16]. These principles, encapsulated by the mnemonic "SIGNIFICANCE," guide analysts in reducing the negative effects of analytical activities on human health and the environment [4]. The core dimensions of assessment directly address multiple GAC principles: solvent impact relates to using safer chemicals and minimizing toxicity; energy consumption connects to reducing energy requirements; and waste generation corresponds to eliminating or minimizing analytical waste [16]. More recently, these have been supplemented by ten principles of Green Sample Preparation (GSP), which further emphasize miniaturization, automation, and in-line processes to enhance environmental performance [4]. These principles collectively provide the theoretical foundation upon which all greenness assessment metrics are built, ensuring that evaluations align with established sustainability goals rather than arbitrary criteria.

Comparative Analysis of Greenness Metrics

Fifteen major greenness assessment tools have gained prominence in analytical chemistry, each with distinct approaches to evaluating environmental impact [9] [16]. The table below summarizes the core characteristics of the most widely used metrics, with particular attention to how they address the three key dimensions of solvent impact, energy consumption, and waste generation.

Table 1: Comparison of Major Greenness Assessment Metrics

Metric Name	Assessment Approach	Solvent Impact Evaluation	Energy Consumption Evaluation	Waste Generation Evaluation	Output Type
AMGS	Quantitative scoring	Health, safety, and environmental impact of solvents	Instrument energy usage and cumulative energy demand	Method solvent waste volume	Numerical score (lower = greener) [17] [2]
NEMI	Qualitative pictogram	Presence on PBT and hazardous waste lists	Not considered	Total waste amount (<50g threshold)	Pictogram with four quadrants [16]
Analytical Eco-Scale	Penalty point system	Amount and hazard of reagents	Energy consumption (>0.1 kWh penalty)	Waste amount and hazard	Total score (100 = ideal green analysis) [16]
GAPI	Qualitative pictogram	Toxicity and environmental impact	Not explicitly considered	Waste generation and treatment	Pictogram with five fields [16]
AGREE	Quantitative scoring	Multiple solvent-related criteria	Energy consumption of instruments	Waste amount and management	Score 0-1 with pictogram [16]
GEMAM	Quantitative scoring	Amount and greenness score of reagents	Energy consumption per analysis	Amount and score of wastes	Score 0-10 with hexagonal pictogram [4]

Detailed Methodology of Key Metrics

Analytical Method Greenness Score (AMGS)

The AMGS calculator, developed by the ACS Green Chemistry Institute Pharmaceutical Roundtable, employs a comprehensive quantitative approach specifically designed for chromatographic methods [17] [2]. Its algorithm integrates three core components: (1) Solvent Impact: Evaluates health, safety, and environmental factors of all solvents used, weighted by their volumes in the method; (2) Energy Consumption: Calculates cumulative energy demand based on instrument type (HPLC, UHPLC, SFC) and method duration; (3) Waste Generation: Quantifies total solvent waste produced during analysis and system suitability testing [2]. The AMGS output is a single percentage score where lower values indicate greener methods, with color-coding (yellow and red) highlighting areas for potential improvement [2]. This metric is particularly valuable for direct comparison of method iterations during development, allowing scientists to quantify environmental improvements when optimizing analytical procedures.

National Environmental Methods Index (NEMI)

As one of the oldest greenness metrics, NEMI employs a simple pictogram with four quadrants that turn green when specific criteria are met [16]. For solvent assessment, it checks that no chemicals appear on the PBT (persistent, bioaccumulative, toxic) list and that no solvents are on the D, F, P, or U hazardous waste lists. For waste, it applies a binary threshold of less than 50g per analysis. Notably, NEMI does not consider energy consumption in its evaluation [16]. While user-friendly, this binary approach lacks granularity as it doesn't differentiate between methods that just meet the criteria and those that far exceed them.

Analytical Eco-Scale

The Analytical Eco-Scale operates on a penalty point system, starting from a baseline of 100 points representing an ideal green analysis [16]. Points are deducted for: hazardous reagents (based on amount and toxicity), energy consumption exceeding 0.1 kWh per sample, and waste generation (considering both amount and hazard). Unlike the binary NEMI approach, this metric provides semi-quantitative assessment that can differentiate between methods of varying environmental performance. However, the penalty assignments can be somewhat subjective, and the output lacks a visual pictogram for quick assessment.

Green Analytical Procedure Index (GAPI)

GAPI utilizes a more detailed pictogram with five fields to evaluate analytical procedures at each stage [16]. It provides qualitative assessment of solvent toxicity and environmental impact throughout the sample collection, preparation, and analysis phases. While it considers waste treatment approaches, it does not explicitly evaluate energy consumption. The strength of GAPI lies in its ability to visually identify which specific stages of an analytical procedure have the greatest environmental impact, guiding targeted improvements.

AGREE and AGREEprep

The Analytical GREEnness Calculator (AGREE) and its sample preparation-focused counterpart AGREEprep represent advanced quantitative approaches that incorporate all twelve GAC principles [16] [4]. These tools generate a score between 0-1 accompanied by a circular pictogram with twelve sections, providing both quantitative results and visual representation. AGREE comprehensively evaluates solvent selection, energy requirements, and waste management while considering the entire analytical lifecycle. The main advantage is its alignment with all GAC principles, though the calculation process is more complex than simpler metrics.

Greenness Evaluation Metric for Analytical Methods (GEMAM)

GEMAM is a recently developed tool that evaluates six key dimensions: sample, reagent, instrument, method, waste, and operator [4]. It employs a sophisticated weighting system where reagents and waste each contribute 25% to the total score, while instrument and method account for 15% each, and sample and operator represent 10% each. The metric uses 21 specific criteria based on both the 12 GAC principles and 10 GSP factors, generating a score from 0-10 displayed in a hexagonal pictogram. This comprehensive approach specifically includes energy consumption per analysis as a key criterion with a 40% weight within the instrument dimension [4].

Experimental and Application Protocols

Implementation Workflow for Greenness Assessment

The process of evaluating analytical methods using greenness metrics follows a systematic workflow that ensures comprehensive assessment across the three key dimensions. The diagram below illustrates this standardized approach.

Diagram 1: Greenness assessment workflow for analytical methods. The process begins with method requirements definition and proceeds through systematic evaluation of the three key dimensions using selected metrics, enabling iterative improvement.

Case Study: Chromatographic Method Evaluation

To illustrate the practical application of these metrics, consider the evaluation of three analytical methods from literature: (1) A UPLC-MS/MS method with liquid-liquid extraction for determining guaifenesin and bromhexine in human plasma [16]; (2) An HPLC-UV method for oxytetracycline and bromhexine in spiked milk samples [16]; and (3) A direct UV method for simultaneous quantification of carbinoxamine maleate, paracetamol, and pseudoephedrine hydrochloride [16].

Table 2: Comparative Greenness Assessment of Three Analytical Methods

Assessment Metric	UPLC-MS/MS Method	HPLC-UV Method	Direct UV Method
NEMI Pictogram	2/4 green fields [16]	2/4 green fields [16]	4/4 green fields [16]
Advanced NEMI	Mixed profile (yellow/red) [16]	Mixed profile (yellow/red) [16]	Primarily green profile [16]
Assessment of Green Profile (AGP)	Moderate performance [16]	Moderate performance [16]	Strong performance [16]
Key Differentiating Factors	Higher solvent impact and energy use due to sample prep and MS detection	Moderate solvent impact from HPLC mobile phase	Minimal solvent use and energy consumption
Recommended Improvement	Explore alternative extraction techniques	Method transfer to UHPLC to reduce solvent volume	Already demonstrates strong green credentials

The case study demonstrates how different metrics consistently identify the direct UV method as the greenest option due to its minimal solvent requirements, lower energy consumption (no chromatography), and reduced waste generation [16]. This alignment across multiple assessment tools reinforces the reliability of greenness evaluation when focused on the core dimensions of solvent impact, energy consumption, and waste.

Essential Research Reagents and Materials

The transition to greener analytical methods requires specific reagents, materials, and technologies that minimize environmental impact while maintaining analytical performance. The following table outlines key solutions for implementing sustainable practices in analytical laboratories.

Table 3: Research Reagent Solutions for Green Analytical Chemistry

Reagent/Material	Function	Green Attributes	Application Examples
Bio-based Solvents	Replace traditional petroleum-derived solvents	Renewable feedstocks, reduced toxicity, biodegradable	Extraction, chromatography mobile phases
Water-based Mobile Phases	Chromatographic separation	Non-toxic, readily biodegradable, inexpensive	Reverse-phase HPLC with alternative columns
Solid-phase Microextraction (SPME)
Sample preparation and concentration	Solventless extraction, minimal waste	Environmental analysis, bioanalytical applications
Microfluidic Devices	Miniaturized analytical systems	Reduced reagent consumption, lower energy requirements	Point-of-care testing, process analytical technology
Supercritical Fluid Chromatography (SFC)	Chromatographic separation	Uses CO2 as primary mobile phase, reduced organic solvents	Chiral separations, natural product analysis
Automated Sample Preparation	High-throughput sample processing	Reduced reagent volumes, improved reproducibility	Pharmaceutical quality control, clinical analysis

The comparison of greenness assessment metrics reveals distinct approaches to evaluating the core dimensions of solvent impact, energy consumption, and waste generation. The AMGS calculator provides a specialized, quantitative tool particularly valuable for pharmaceutical method development, offering detailed assessment of solvent health/safety/environmental factors, instrument energy usage, and solvent waste [17] [2]. In contrast, broader metrics like GAPI and NEMI offer rapid visual assessment but lack the granularity for subtle differentiation between methods [16]. Emerging tools like GEMAM represent the evolution toward more comprehensive evaluations that incorporate multiple GAC and GSP principles with flexible weighting systems [4].

Future developments in greenness assessment will likely focus on integrating artificial intelligence for automated method optimization, expanding metric capabilities to encompass additional lifecycle considerations, and developing standardized reporting frameworks for regulatory submissions [2] [4]. The ongoing refinement of these tools, including planned AMGS updates for gas chromatography methods by early 2026, demonstrates the dynamic nature of this field [2]. For researchers and drug development professionals, selecting the appropriate assessment metric depends on specific needs: AMGS offers unparalleled detail for chromatographic method development, while AGREE and GEMAM provide comprehensive lifecycle assessment, and NEMI/GAPI deliver rapid screening capabilities. By understanding the strengths and limitations of each tool across the fundamental dimensions of solvent impact, energy consumption, and waste generation, scientists can make informed decisions that advance both analytical science and environmental sustainability.

The adoption of Green Analytical Chemistry (GAC) principles has transformed how researchers evaluate the environmental impact of analytical methods [12]. GAC focuses on mitigating the adverse effects of analytical activities on human safety, health, and the environment [9]. To operationalize these principles, the scientific community has developed numerous metric tools that enable objective assessment and comparison of method greenness [16]. These tools provide standardized approaches for quantifying factors such as reagent toxicity, energy consumption, and waste generation, allowing analysts to make informed decisions during method development [15]. The growing emphasis on sustainability in analytical laboratories has increased the importance of these metrics, particularly in regulated industries like pharmaceutical development where methods must balance environmental concerns with analytical performance [2] [18].

The evolution of GAC metrics has progressed from simple checklists to sophisticated algorithms that generate comprehensive scores [19]. This article classifies these tools based on three key dichotomies: general versus specific application scope, visual versus numerical output formats, and holistic versus targeted assessment approaches. Understanding these classifications helps researchers select appropriate metrics for their specific applications and correctly interpret results across different tools [15]. The Analytical Method Greenness Score (AMGS) calculator, developed by the ACS Green Chemistry Institute Pharmaceutical Roundtable, serves as a central reference point in this comparison due to its specific design for chromatographic method development in pharmaceutical applications [2] [20].

Classification Framework for Metric Tools

General vs. Specific Application Scope

Metric tools vary significantly in their scope of application, with some designed for broad use across analytical techniques and others optimized for specific methodologies [15].

General-purpose metrics provide flexible frameworks applicable to diverse analytical techniques. The Analytical Greenness Calculator (AGREE) exemplifies this category by offering a comprehensive assessment based on all 12 principles of GAC [4] [16]. Similarly, the Green Analytical Procedure Index (GAPI) creates a multi-criteria pictogram that evaluates entire analytical procedures [16] [19], while the Analytical Eco-Scale assigns penalty points based on reagent amounts, hazards, energy consumption, and waste [16].

Technique-specific metrics are optimized for particular analytical methodologies. The AMGS calculator is specifically designed for liquid chromatography and supercritical fluid chromatography methods [2]. Other specialized tools include HPLC-EAT for high-performance liquid chromatography methods and AGREEprep which focuses exclusively on the sample preparation stage of analysis [15] [4].

Table 1: Classification of Metric Tools by Application Scope

Metric Tool	Application Scope	Target Techniques	Key Characteristics
AMGS	Specific	Liquid chromatography, SFC	Benchmarks solvent health, safety, environmental impact, energy demand [2]
AGREE	General	All analytical techniques	Assesses all 12 GAC principles; configurable weights [4] [16]
GAPI	General	All analytical techniques	Qualitative/semi-quantitative multi-criteria pictogram [16] [19]
AGREEprep	Specific	Sample preparation	Focuses specifically on sample preparation stage [15]
HPLC-EAT	Specific	HPLC-related techniques	Limited to HPLC-based methods [4]
NEMI	General	All analytical techniques	Simple pictogram with four criteria; qualitative only [16] [19]

Visual vs. Numerical Output Formats

The communication of assessment results follows distinct approaches, with some tools providing primarily visual outputs and others generating numerical scores.

Visual output metrics use pictograms, symbols, and color-coding to intuitively communicate greenness assessments. The NEMI pictogram employs a circle divided into four quadrants that are filled green when criteria are met [16]. GAPI utilizes a more detailed pictogram with five pentagrams color-coded from green to red to represent environmental impact levels [16] [19]. The RGB model and its derivatives use color blending to represent different sustainability aspects [15].

Numerical output metrics generate quantitative scores that enable precise comparisons and tracking of improvements. The AMGS calculator produces a numerical score where lower values indicate greener methods [2]. The Analytical Eco-Scale starts with a perfect score of 100 and subtracts penalty points for undesirable attributes [16]. More recent tools like AGREE generate scores on a 0-1 scale while also incorporating visual elements through color-coded circular diagrams [4] [16].

Holistic vs. Targeted Assessment Approaches

Assessment tools differ in their comprehensiveness, with some evaluating multiple sustainability dimensions and others focusing on specific environmental aspects.

Holistic metrics attempt to capture a wide range of environmental and functional attributes. The emerging concept of White Analytical Chemistry (WAC) seeks to balance greenness with analytical quality and practical effectiveness [12] [15]. Tools like AGREE and the newer GEMAM incorporate numerous criteria covering samples, reagents, instrumentation, method, waste, and operator safety [4]. These comprehensive approaches aim to avoid suboptimization where environmental improvements compromise analytical performance.

Targeted metrics focus on specific environmental impact categories. The AMGS calculator specifically addresses solvent health, safety, environmental impact, cumulative energy demand, instrument energy usage, and method solvent waste [2]. The ChlorTox Scale focuses primarily on the toxicity of solvents and reagents used in analytical processes [9] [16]. Similarly, AMVI concentrates mainly on the volume of solvents consumed during analysis [4].

Diagram: Classification framework for green analytical chemistry metric tools showing the three primary classification axes and representative tools for each category.

Comparative Analysis of AMGS Against Other Metrics

Detailed Examination of AMGS Components

The AMGS calculator employs a targeted approach specifically designed for chromatographic method development in pharmaceutical applications [2]. Its calculation incorporates several distinct components: solvent health, safety, and environmental impact assessments; cumulative energy demand evaluation; instrument energy usage based on method run time; and method solvent waste volume [2]. The tool employs color-coding (yellow and red) to highlight areas where the method could be improved, such as reducing instrument energy by shortening run time [2].

A key characteristic of AMGS is its function as a comparative tool rather than an absolute measure of greenness. The ACS GCI Pharmaceutical Roundtable explicitly states it is "not meant as an absolute measure of method greenness or as a means to reject a given method based on the finite score" [2]. This positions AMGS primarily as a benchmarking tool for comparing methods during development. The calculator currently supports only liquid chromatography and SFC methods, with GC method support planned for 2026 [2].

Comparison with Other Leading Metrics

When compared to other metrics, AMGS occupies a distinctive position in the landscape of assessment tools. The following table illustrates how it compares across critical parameters:

Table 2: Comparative Analysis of AMGS with Other Greenness Assessment Metrics

Metric Tool	Output Type	Scope	Assessment Approach	Key Strengths	Key Limitations
AMGS	Numerical	Specific	Targeted	Specific for LC/SFC; practical for pharma development [2]	Limited to chromatographic techniques [2]
AGREE	Both	General	Holistic	Comprehensive (12 GAC principles); configurable weights [4] [16]	Complex calculation process [4]
GAPI	Visual	General	Holistic	Detailed pictogram; semi-quantitative assessment [16] [19]	Qualitative nature limits precision [4]
NEMI	Visual	General	Targeted	Simple interpretation; historical precedence [16] [19]	Limited criteria; qualitative only [16]
Analytical Eco-Scale	Numerical	General	Targeted	Simple 100-point scale; penalty point system [16]	No pictogram output [4]
GEMAM	Both	General	Holistic	Comprehensive (21 criteria); adjustable weights [4]	Newer tool with limited track record [4]

Experimental Protocols for Metric Application

To ensure consistent application and comparison of greenness metrics, researchers should follow standardized experimental protocols when evaluating analytical methods:

Data Collection Requirements: For AMGS application, researchers must document: (1) complete solvent inventory with volumes used per analysis; (2) instrument type (HPLC, UPLC, SFC) and method run time; (3) system suitability test preparation details including serial dilution volumes; and (4) waste collection data [2]. Similar comprehensive data collection is needed for other metrics, with AGREE requiring additional information on sample collection, preparation, and operator safety measures [4].

Calculation Methodologies: The AMGS calculation algorithm incorporates solvent health, safety, and environmental impact factors alongside cumulative energy demand and instrument energy usage [2]. Other metrics employ different calculation approaches: AGREE uses weighting factors for each of the 12 GAC principles [4], while Analytical Eco-Scale subtracts penalty points from a baseline of 100 [16]. Recent tools like GEMAM employ more complex formulas with section weights that default to specific distributions (e.g., 25% for reagents, 25% for waste, 15% for instrument) but can be adjusted based on assessment priorities [4].

Validation Procedures: Method validation should include cross-metric comparison where feasible. Researchers should apply multiple metric tools to the same method and analyze correlation between results [19]. For AMGS specifically, the ACS GCI recommends using the tool to compare method iterations during development rather than as a standalone pass/fail criterion [2].

Essential Research Reagent Solutions

Implementing greenness assessments requires both computational tools and practical laboratory solutions. The following research reagents and materials represent essential components for developing and evaluating greener analytical methods:

Table 3: Essential Research Reagents and Solutions for Green Analytical Chemistry

Reagent/Solution	Function in Green Assessment	Application Example
Low-toxicity solvents	Replace hazardous solvents in sample preparation and separation	Alternative solvents with better environmental profiles in AMGS [2]
Natural deep eutectic solvents (NADES)	Bio-based solvents for extraction processes	Evaluate green profile in AGREEprep for sample preparation [18]
Miniaturized extraction devices	Reduce reagent consumption and waste generation	Implement microextraction techniques to improve GAPI and AGREE scores [4]
Renewable sorbents	Sustainable materials for sample preparation	Assess sustainability in Spider diagram and ComplexGAPI metrics [19]
Energy-efficient instrumentation	Reduce cumulative energy demand in AMGS calculation	UHPLC and SFC systems to lower instrument energy component [2]

The classification of GAC metric tools along the axes of general versus specific scope, visual versus numerical outputs, and holistic versus targeted approaches provides researchers with a structured framework for selecting appropriate assessment methods. The AMGS calculator occupies a distinct position as a targeted, numerical tool specifically optimized for chromatographic method development in pharmaceutical applications [2]. Its design philosophy emphasizes practical benchmarking during method development rather than comprehensive sustainability assessment.

The future evolution of greenness metrics will likely address current limitations through increased standardization, integration of uncertainty estimates, and development of more objective weighting approaches [15]. Tools like AGREE and GEMAM that offer configurable weights represent a step in this direction, though consensus on default weightings remains elusive [15] [4]. For researchers in drug development, AMGS provides immediate practical value for comparing chromatographic methods, while broader sustainability assessments may benefit from complementary tools like AGREE or GAPI that address a wider range of environmental impacts [2] [16]. As the field progresses, the optimal approach may involve using multiple metrics in concert to leverage their respective strengths while mitigating their individual limitations.

Putting Metrics to Work: A Practical Guide to Calculation and Application

The push for sustainability in the pharmaceutical industry has extended into analytical laboratories, where chromatographic methods traditionally consume high volumes of solvents and energy. To quantify and minimize this environmental impact, several greenness assessment tools have been developed. Among these, the Analytical Method Greenness Score (AMGS) has emerged as a specialized metric for evaluating the environmental footprint of chromatographic separations. Developed by the American Chemical Society's Green Chemistry Institute (ACS-GCI) Pharmaceutical Roundtable, the AMGS provides a unified, quantitative approach to benchmark and improve the sustainability of analytical methods without compromising analytical performance [1] [21] [22].

The AMGS calculator is a web-based, publicly accessible tool designed specifically for liquid chromatography (HPLC/UHPLC), supercritical fluid chromatography (SFC), and, in a forthcoming update, gas chromatography (GC) methods [2] [22]. It serves as a strategic metric to raise awareness, guide method development toward more sustainable practices, and provide a systemic view of solvent and energy use throughout the analytical process. By offering a single numerical score—where a lower value indicates a greener method—it enables scientists to objectively compare different method parameters and instrument choices [2] [21] [23].

This guide provides a detailed walkthrough of the AMGS calculator, placing it within the broader context of green analytical chemistry metrics. By comparing its methodology, scope, and outputs with other available tools, researchers and drug development professionals can make informed decisions to reduce the environmental impact of their analytical operations.

Comparative Analysis of Greenness Metrics

The landscape of greenness assessment tools is diverse, with each tool offering unique perspectives and evaluation criteria. The table below summarizes how AMGS compares to other prominent metrics.

Table 1: Comparison of AMGS with Other Green Analytical Chemistry Metrics

Metric Tool	Primary Focus	Output Type	Key Assessed Criteria	Technique Specificity
AMGS [2] [15] [21]	Environmental impact of chromatographic methods	Numerical score	Solvent health/safety, solvent energy, instrument energy, solvent waste	Liquid Chromatography, SFC, (GC coming in 2025)
NEMI [15]	Environmental impact of analytical procedures	Pictogram (4 quadrants)	Persistence, bioaccumulation, toxicity, waste generation	General
Analytical Eco-Scale [1] [15]	Overall environmental impact of analytical methods	Numerical score (100 - penalty points)	Reagents, instruments, waste	General
GAPI [1] [15]	Holistic environmental impact of analytical procedures	Pictogram (5 pentagrams)	All stages from sampling to waste management	General
AGREE [1] [15]	Alignment with 12 principles of Green Analytical Chemistry	Numerical score (0-1) & circular pictogram	12 criteria including energy, toxicity, and waste	General

A critical differentiator for AMGS is its specificity for chromatography and its incorporation of instrument energy consumption—a factor not always considered in other tools [1] [21]. While general-purpose metrics like GAPI and AGREE provide a broader, more holistic assessment applicable to various analytical techniques, AMGS offers a more granular and tailored evaluation for separation scientists. This focus allows it to capture the unique environmental drivers of chromatographic processes, such as flow rate, run time, and column geometry, and translate them into a cumulative energy demand and hazard score [15] [23].

A Step-by-Step Guide to the AMGS Calculator

Input Parameters and Data Collection

The AMGS calculation requires specific methodological and operational data. The tool's algorithm synthesizes this information across three core categories: solvent health and safety (EHS), cumulative energy demand (CED), and instrument energy usage [2] [21]. The following workflow outlines the logical relationship between your method parameters and the final AMGS score.

To use the calculator, gather the following essential data from your analytical method [2] [21] [23]:

• Solvent Information: The types and volumes of all solvents used in the mobile phase, including mixtures and additives. The calculator contains a database with pre-assigned EHS and CED values for common solvents.
• Instrumental Parameters: The chromatographic flow rate (mL/min), method run time (minutes), and the instrumental "cycle time" (the time from the end of one run to the next injection, crucial for high-throughput analysis) [23].
• Sample Preparation: The total volume of solvent used for sample dissolution and preparation, including dilutions for system suitability tests [2].
• Instrument Type: The model or type of instrument (e.g., UHPLC vs. standard HPLC) as this affects energy consumption.

Calculation Walkthrough and Formula Deconstruction

The public AMGS calculator is hosted on the ACS GCIPR website and is free to use. The step-by-step process is straightforward [2]:

• Access the Tool: Navigate to the AMGS calculator on the ACS GCIPR website.
• Input Method Details: Enter all the parameters gathered in the previous section into the corresponding fields in the web form. The interface is designed to guide you through the process.
• Calculate Score: The tool automatically computes the score upon submission. The result is a single number representing your method's environmental impact.

While the exact, full-weighted formula is proprietary, the underlying principles and some modified forms have been described in research. The score is a composite of three main components [21] [23]:

• Solvent EHS Score: Assesses the environmental, health, and safety profile of the solvents used.
• Solvent Cumulative Energy Demand (CED): Accounts for the energy required to produce and dispose of the solvents.
• Instrument Energy Score: Calculates the electricity consumed by the instrument during the analysis.

A modified AMGS formula that incorporates cycle time (t_c) to provide a more accurate assessment has been presented in recent research [23]. This is crucial because it recognizes that instrument idle time between injections contributes to the total energy footprint. The analysis time (t_a) at a given flow rate (F) can be derived from an initial run time (t_i) and flow rate (F_i):

t_a = (t_i * F_i) / F

This equation for t_a is then substituted into the AMGS formula, allowing scientists to model and optimize the score with respect to both flow rate and cycle time [23]. Mathematical optimization by taking the derivative of this new equation with respect to flow rate can identify the specific flow rate that yields the minimum AMGS for a given solvent and set of conditions [23].

Interpretation of Results

The AMGS output is a single number. The fundamental rule is: The lower the AMGS, the greener the method [2]. The calculator also provides a color-coded breakdown, highlighting which of the three main categories (solvent EHS, solvent energy, or instrument energy) contributes most significantly to the overall score. This helps analysts identify the most high-impact areas for improvement [2]. For instance, a method with a red highlight on "instrument energy" would benefit most from reducing the run time or cycle time, while a red "solvent EHS" score suggests that switching to safer, less hazardous solvents should be the priority.

Experimental Protocols for AMGS Optimization

Case Study: Optimizing Amino Acid Enantioseparations

A 2024 study provides a robust experimental protocol for minimizing AMGS, using the enantioseparation of 38 amino acids as a case study [23].

• Objective: To develop a rapid, sustainable chiral HPLC method for native (underivatized) amino acids and minimize its AMGS.
• Materials and Methods:
  • Chemicals: Proteo- and non-proteogenic amino acid standards, carbon dioxide (food-grade grade), deionized water.
  • Equipment: UHPLC system equipped with a chiral stationary phase column (specifics may vary, but narrow-diameter columns with superficially porous particles are recommended), low-pressure polypropylene eluent reservoir for carbonation, UV or MS detector.
  • Chromatographic Method:
    • Mobile Phase: Carbonated water (aqueous carbonic acid, Hâ‚‚CO₃*), generated by continuously pressurizing the eluent reservoir with COâ‚‚ at ~25 psi.
    • Detection: Low-wavelength UV (~190 nm) or mass spectrometry (MS).
    • Method Development: Focus on adjusting flow rate and column temperature to achieve baseline separation with the shortest possible run time.
• Key Findings and AMGS Reduction:
  • The use of carbonated water, a green solvent, directly improved the solvent EHS and CED components of the AMGS.
  • Aqueous carbonic acid reduced retention times and sharpened peaks, enabling the use of higher flow rates and shorter columns without sacrificing resolution. This simultaneously reduced solvent waste and instrument run time.
  • By applying the modified AMGS formula that includes cycle time, the researchers identified the optimal flow rate that minimized the total score, demonstrating that "faster is not always greener" if cycle time is not considered.

Essential Research Reagent Solutions for Green Chromatography

The following table details key materials and their functions for developing methods with an improved AMGS, based on the case study and general principles [23].

Table 2: Key Research Reagents and Materials for Sustainable Chromatographic Methods

Item	Function & Rationale	Greenness Impact
Carbon Dioxide (for mobile phase)	Used to generate carbonated water (H₂CO₃*) eluent; acts as a mild acidic additive that is MS-compatible and UV-transparent.	Replaces hazardous additives like TFA; excellent EHS and CED profile.
Narrow-Diameter Columns (<2.1mm i.d.) with Superficially Porous Particles (SPPs)	Provides high efficiency, allowing for faster flow rates and shorter run times without excessive backpressure.	Drastically reduces total solvent consumption and instrument run time.
Water (as primary solvent)	The greenest solvent. Methods should be developed to maximize water content in the mobile phase where possible.	Excellent EHS and CED scores compared to organic solvents.
Green Organic Solvents	Solvents like ethanol or methanol, which have better EHS profiles than acetonitrile or halogenated solvents.	Improves the solvent EHS and CED components of the AMGS.

Discussion: AMGS in the Context of Broader Metric Research

The introduction and adoption of AMGS reflect a larger trend in analytical chemistry toward standardized, quantitative metrics that move beyond qualitative assessments. While tools like NEMI and GAPI offer valuable visual snapshots, the single numerical output of AMGS facilitates direct comparison and trending over time, which is essential for continuous improvement in industrial settings [15]. Its specificity for chromatography makes it a powerful tool for separation scientists, though this can also be a limitation when assessing multi-technique analytical procedures [1].

Current research highlights areas for future development of greenness metrics, including AMGS. There is a push for metrics to include uncertainty estimates for their scores and to address potential interdependencies between criteria, which are often treated as independent variables [15]. Furthermore, the AMGS calculation, like many others, would benefit from a more rigorous, consensus-based approach to assigning weights to its different components, as most users rely on default settings [15]. The ongoing work by the ACS GCIPR to expand AMGS to GC and to develop a more comprehensive "AMGS v2.0" that includes comprehensive sample preparation steps and a potential AI interface directly addresses these evolving needs [22].

The AMGS calculator is a practical and impactful tool for any laboratory seeking to quantify and reduce the environmental footprint of its chromatographic methods. By following the step-by-step process of inputting solvent, instrument, and sample preparation data, researchers can obtain a clear, actionable score. As demonstrated in the amino acid enantioseparation case study, this score can be systematically optimized through strategic method development, such as employing green solvents like carbonated water, optimizing flow rates, and selecting appropriate column hardware.

When compared to other greenness metrics, AMGS stands out for its chromatographic focus and its inclusion of instrument energy consumption. Its ongoing development, guided by user feedback and academic research, ensures it will remain a relevant and valuable component of the broader green analytical chemistry toolkit. For drug development professionals and researchers, integrating AMGS into routine method development and validation represents a significant step toward achieving corporate sustainability goals and minimizing the ecological impact of pharmaceutical analysis.

The growing emphasis on sustainability in scientific practices has propelled the development of metrics to evaluate the environmental impact of analytical methods. Within this landscape, the Analytical GREEnness (AGREE) metric has emerged as a comprehensive tool that translates the 12 principles of Green Analytical Chemistry (GAC) into a practical, quantitative assessment framework. The need for such tools is particularly acute in fields like pharmaceutical development and metabolomics, where analytical procedures often consume significant resources and generate substantial waste [3]. While other metrics exist, including the Analytical Method Greenness Score (AMGS) calculator and the newer Greenness Evaluation Metric for Analytical Methods (GEMAM), AGREE distinguishes itself through its unique foundation in the 12 GAC principles and its visually intuitive pictogram output [24]. This guide provides a detailed comparison of these metrics, with a focused exploration of how to implement and interpret the AGREE calculator, empowering scientists to make more environmentally conscious decisions in method development and selection.

Inside the AGREE Calculator: Principles and Pictogram

The 12 Principles of Green Analytical Chemistry

The AGREE metric is built upon the 12 foundational principles of GAC, which serve as its evaluation criteria. The calculator assigns a score from 0 to 1 for each principle, and the overall greenness score is derived from the aggregation of these individual scores. The following table details the typical focus of each principle within the AGREE context.

Table 1: The 12 Principles of Green Analytical Chemistry in AGREE

Principle Number	Description	Key Evaluation Aspects
1	Direct analysis of samples without treatment	Avoidance of sample preparation, derivatization
2	Minimal sample size	Sample size and number of samples
3	In-line measurements	Automation, on-site analysis, real-time detection
4	Integration of analytical operations & methods	Method streamlining, waste reduction
5	Automated & miniaturized methods	Use of automated instruments, miniaturized equipment
6	Avoidance of derivatization	Elimination of derivatization reagents and steps
7	Reduction of analytical steps; increased sample throughput	Simplicity, analysis time, multi-analyte capacity
8	Minimization of energy consumption	Instrument energy demand
9	Use of Biodegradable & Green Reagents/Solvents	Toxicity, safety, health hazards, environmental impact
10	Waste minimization and proper management	Waste quantity, recycling, treatment
11	Multi-analyte determination	Simultaneous determination of analytes
12	Elimination of the need for functional group analysis	Safety from hazards

Deciphering the Output: Score and Pictogram

The output of the AGREE calculator is a circular pictogram with 12 segments, each corresponding to one of the GAC principles. This visual representation is accompanied by an overall score in the center, ranging from 0 to 1.

• Pictogram Interpretation: Each segment is colored using a traffic-light scale, where green indicates strong adherence to the principle, yellow suggests a moderate performance, and red highlights a significant environmental shortcoming. This provides an immediate, intuitive overview of a method's strengths and weaknesses.
• Overall Score: The score in the center is a weighted average of all 12 segments. A score closer to 1 signifies a greener method. Research applying AGREE to metabolomics studies suggests that methods with scores below 0.5 often have critical areas requiring improvement, typically related to offline sample preparation, lack of automation, and the use of toxic reagents [3].

The diagram below illustrates the logical workflow for conducting an assessment with the AGREE calculator.

AGREE in Practice: Experimental Protocols and a Metabolomics Case Study

To illustrate the practical application of AGREE, let's examine a published review that evaluated the greenness of 16 metabolomics studies [3]. The experimental protocol for such an assessment is methodical and can be replicated for other analytical fields.

Detailed Methodology for AGREE Evaluation

• Method Dissection: The first step involves a complete breakdown of the analytical procedure. For a liquid chromatography-mass spectrometry (LC-MS) metabolomics method, this includes:

  • Sample Collection: Type of biopsy, volume of blood/plasma, etc.
  • Sample Preparation: Detailed steps such as protein precipitation, liquid-liquid extraction, solid-phase extraction, evaporation, and reconstitution. Volumes of all solvents and reagents are meticulously recorded.
  • Chromatographic Separation: Instrument type (HPLC, UHPLC), column dimensions, flow rate, mobile phase composition (including buffer salts), and run time.
  • Detection: Mass spectrometer type and operating parameters.
  • Data Analysis: Software and computational resources used.

• Data Input and Rational Estimation: Each dissected component is mapped onto the 12 principles of AGREE. Quantitative data (e.g., solvent volumes, energy consumption in kWh) is used where possible. When direct data is unavailable, rational estimations based on instrument specifications and common practices are made.

• Software Utilization: The free-to-use AGREE software is employed, inputting the scores and data for all 12 criteria to generate the final pictogram and overall score.

The review's application of AGREE to targeted and untargeted metabolomics studies provided critical, quantitative insights [3]. The results unequivocally showed that the main weaknesses in current metabolomics research were offline sample preparation and the lack of automation and miniaturization. The AGREE scores helped pinpoint specific areas for improvement, such as the complexity of sample preparation procedures, the use of toxic reagents and derivatizing agents, the amount of waste generated, and low sample throughput. This case demonstrates AGREE's power to move beyond theoretical assessment and provide actionable guidance for making a field more sustainable.

Comparative Analysis of Greenness Metrics

While AGREE is a powerful tool, it is one of several metrics available. The AMGS calculator, developed by the ACS GCI Pharmaceutical Roundtable, is another prominent metric, but it is designed with a different scope and output [2] [25] [17].

Table 2: Comparison of Key Analytical Greenness Metrics

Feature	AGREE	AMGS (Analytical Method Greenness Score)	GEMAM (Greenness Evaluation Metric for Analytical Methods)
Foundation	12 Principles of Green Analytical Chemistry	Solvent & energy impact, instrument design	12 GAC Principles & 10 Sample Prep Factors [24]
Output	Pictogram (0-10 scale) & overall score (0-1)	Single percentage score (lower is greener)	Pictogram on a 0-10 scale [24]
Key Parameters	12 segments corresponding to GAC principles	Solvent health/safety/environment, cumulative energy demand, instrument energy, solvent waste [2]	Combines GAC principles and sample preparation factors
Primary Application	General analytical methods; widely applied in metabolomics [3]	Liquid & Supercritical Fluid Chromatography in pharma [2]	General analytical methods [24]
Visual Output	12-segment circular pictogram	Color-coded table (Yellow/Red highlights)	Pictogram (qualitative & quantitative) [24]
Strengths	Holistic, based on canonical principles, intuitive visual	Pharma-specific, integrates instrument energy & speed	Comprehensive, combines two frameworks [24]
Weaknesses	Less specific on instrument energy comparison	Limited to specific separation techniques	Newer metric, less established in literature

The following diagram summarizes the relative positioning and focus of these different greenness metrics.

The Scientist's Toolkit: Essential Reagent and Resource Solutions

Implementing greener methods based on AGREE or AMGS assessments often requires specific materials and reagents. The following table details key solutions that can help improve a method's greenness profile.

Table 3: Key Research Reagent Solutions for Greener Analytical Chemistry

Reagent / Material	Function in Analytical Chemistry	Greenness Benefit & Rationale
Bio-based Solvents (e.g., Cyrene, Ethyl Lactate)	Replacement for traditional organic solvents (e.g., DMF, DMSO, acetonitrile) in extraction and chromatography.	Reduced environmental impact and toxicity; derived from renewable resources, often with better biodegradability profiles.
Superficially Porous Particles (SPP)	Stationary phase for chromatographic columns.	Enables faster separations with lower backpressure than fully porous particles, reducing analysis time, solvent consumption, and energy use.
Water-based Mobile Phases	Use in reversed-phase chromatography with minimal organic modifier.	Reduces reliance on hazardous acetonitrile and methanol, lowering waste toxicity and environmental impact.
Miniaturized Extraction Devices (e.g., µ-SPE, SPME)	Sample preparation and pre-concentration of analytes.	Drastically reduces solvent consumption (often to zero for SPME) and sample size, addressing Principles 2, 5, and 10 in AGREE.
Green Derivatization Agents	Chemicals used to alter analytes for enhanced detection.	Less toxic and hazardous alternatives to traditional reagents (e.g., using DMSACE instead of MSTFA for silylation), improving safety (Principle 12).
Amisometradine-d3	Amisometradine-d3, MF:C9H13N3O2, MW:198.24 g/mol	Chemical Reagent
1,2-Dioleoyl-sn-glycero-3-phosphocholine-d9	1,2-Dioleoyl-sn-glycero-3-phosphocholine-d9, MF:C44H84NO8P, MW:795.2 g/mol	Chemical Reagent

The AGREE calculator is a robust, principle-based tool that provides an actionable and visually intuitive assessment of an analytical method's environmental impact. Its 12-component pictogram offers unparalleled insight into specific areas for improvement, guiding researchers toward more sustainable practices. As demonstrated in metabolomics, its application can reveal systemic inefficiencies and prioritize mitigation strategies [3]. While other metrics like AMGS are invaluable in specific contexts like pharmaceutical chromatography, the comprehensive nature of AGREE makes it an essential instrument for the wider scientific community. The ongoing development and refinement of these metrics, including the upcoming AMGS update for gas chromatography by early 2026 [2], underscore a collective commitment to embedding green chemistry principles into the very fabric of analytical science.

The Green Analytical Procedure Index (GAPI) is a widely adopted tool for assessing the environmental impact of analytical methods across their entire lifecycle. This metric provides a visual overview of the greenness of different procedural steps using a five-segment pentagram design, where each section is colored green, yellow, or red to indicate the environmental performance of various aspects of the analytical methodology [27]. The tool was developed to offer a quick, comprehensive evaluation of the environmental hazards associated with all stages of chemical analysis, from sample collection and preparation to instrument operation and waste generation [27]. Unlike single-score metrics, GAPI's visual design immediately highlights which specific areas of a method require improvement to enhance its environmental profile, making it particularly valuable for researchers seeking to optimize their analytical procedures for sustainability.

The pharmaceutical industry and analytical laboratories have increasingly embraced Green Analytical Chemistry (GAC) principles to minimize their environmental footprint. GAPI has emerged as one of the most frequently cited evaluation tools in this domain, with hundreds of documented applications in analytical literature [27]. Its design enables researchers to systematically evaluate and compare the greenness of various analytical techniques, including chromatography, spectrophotometry, and other laboratory methods, providing a standardized framework for environmental assessment in scientific research and method development [27].

The GAPI Pentagram: Structure and Interpretation

The Five Assessment Areas

The GAPI pentagram is divided into five distinct sections, each evaluating a critical phase of the analytical process. These sections provide a comprehensive assessment of the method's environmental impact:

• Sample Collection: This segment evaluates the initial stage of obtaining samples, considering factors such as whether collection is performed in-line, online, or offline [27]. Each approach carries different environmental implications, particularly regarding transportation requirements and preservation needs.
• Sample Preservation and Transportation: This section assesses the energy and resource consumption associated with maintaining sample integrity between collection and analysis, including any refrigeration, chemical preservation, or special handling requirements.
• Sample Preparation: Often the most environmentally impactful stage, this segment evaluates extraction techniques, reagent use, and any derivatization procedures. It considers the types and volumes of solvents used, with preference for safer alternatives and minimal waste generation [27].
• Solvents and Reagents Used: This portion specifically examines the toxicity, safety, and environmental hazards of all chemicals employed in the method, with particular focus on solvent selection based on health, safety, and environmental criteria [27].
• Instrumentation and Waste Generation: The final section assesses the energy consumption of analytical instruments and the quantity and management of waste produced, favoring methods with lower energy demands (≤1.5 kWh per sample) and minimal waste [27].

Color-Coded Scoring System

Each subsection within the five main pentagram areas is assigned a color based on its environmental performance:

• Green indicates environmentally friendly practices with minimal impact
• Yellow signifies approaches with moderate environmental concerns
• Red highlights areas with significant environmental drawbacks that should be improved

This intuitive color-coding system allows researchers to quickly identify which specific aspects of their methods require optimization to enhance greenness, facilitating targeted improvements in method development [27].

MoGAPI: The Enhanced GAPI with Scoring Capability

Limitations of Traditional GAPI

While GAPI provides an excellent visual assessment of method greenness, it has a significant limitation: the absence of a quantitative overall score. This makes direct comparison between different methods challenging, as researchers must subjectively evaluate which color pattern represents a greener profile [27]. This drawback becomes particularly evident when comparing methods with different distributions of green, yellow, and red sections but similar overall environmental impact [27].

The MoGAPI Solution

The Modified GAPI (MoGAPI) tool was developed to address this limitation by incorporating a numerical scoring system while retaining the visual advantages of the original GAPI pentagram [27]. This enhanced version calculates a total score based on performance across all assessment criteria, with points assigned according to the environmental friendliness of each parameter. For example, in sample collection, in-line collection receives the highest score (3 credits), online collection receives an intermediate score, and offline collection receives the lowest score (1 credit) [27].

The total credits are summed and divided by the maximum possible credits to calculate a percentage score, which is then classified into three categories:

• ≥75: Excellent green
• 50-74: Acceptable green
• <50: Inadequately green [27]

This scoring system mirrors the approach used in the analytical Eco-Scale metric, providing both quantitative and qualitative assessment capabilities in a single tool [27]. The MoGAPI software is freely available as open source, further enhancing its accessibility and utility for researchers [27].

Table 1: Comparison of Greenness Assessment Tools

Metric	Output Type	Scoring System	Assessment Scope	Key Features
GAPI	Visual pentagram	Color-based (green/yellow/red)	Full analytical method	Quick visual overview, no total score
MoGAPI	Visual pentagram + numerical score	Color-based + percentage (0-100%)	Full analytical method	Retains visual elements with added quantitative scoring
AMGS	Numerical score	Single metric (%)	Chromatography methods	Specific to solvent health, safety, environmental impact, and energy [2] [17]
AGREE	Pictogram + numerical score	0-1 scale	Full analytical method	10 assessment criteria with circular diagram [27]
Analytical Eco-Scale	Numerical score	0-100 points	Full analytical method	Penalty points assigned for hazardous aspects [27]

Comparative Analysis of Greenness Assessment Metrics

GAPI/MoGAPI vs. AMGS

The Analytical Method Greenness Score (AMGS) calculator is a specialized tool developed by the ACS Green Chemistry Institute Pharmaceutical Roundtable's analytical chemistry focus team [17]. Unlike GAPI's comprehensive visual approach, AMGS focuses specifically on chromatography methods and generates a single percentage score based on solvent health, safety, environmental impact, cumulative energy demand, instrument energy usage, and method solvent waste [2] [17]. The AMGS metric is designed specifically for comparing separation methods during drug development, with a lower score indicating a greener method [2].

While AMGS provides a straightforward numerical comparison for method benchmarking, it lacks the visual component of GAPI that immediately highlights areas for improvement. Additionally, the current version of AMGS is limited to liquid chromatography and supercritical fluid chromatography, though a version for gas chromatography is expected by early 2026 [2]. In contrast, GAPI and MoGAPI can be applied to a wider range of analytical techniques beyond chromatography.

GAPI/MoGAPI vs. Other Metrics

The AGREE metric provides another approach, offering a pictogram with a 0-1 scale based on ten assessment criteria [27]. Like MoGAPI, it combines visual and numerical elements, though with a different structural approach. The analytical Eco-Scale employs a penalty point system across various parameters to generate a score out of 100, but lacks the visual impact of GAPI and AGREE [27].

Table 2: Quantitative Comparison of Method Greenness Using Different Metrics

Analytical Method	MoGAPI Score	AGREE Score	Application Context
DLLME-HPLC for antivirals in environmental water [27]	70	~0.65	Intermediate greenness with comparable results
UA-DLLME-HPLC for gliflozins in plasma [27]	80	~0.75	Good greenness with consistent scoring
Modified HPLC for carboxylic acids in beverages [27]	79	0.80	Good greenness with similar conclusions
RP-HPLC for upadacitinib [28]	N/A	N/A	Greenness confirmed by ComplexGAPI, AGREE, and AMGS

Methodologies and Experimental Protocols

Case Study: Application of Green Metrics to Pharmaceutical Analysis

A recent study developing a stability-indicating RP-HPLC method for upadacitinib, a selective Janus kinase inhibitor, demonstrates the practical application of greenness assessment tools in pharmaceutical analysis [28]. The method utilized a C18 column with a mobile phase of acetonitrile and 0.1% formic acid (60:40, v/v) at a flow rate of 0.8 mL/min, with detection at 290 nm [28]. The method was validated according to ICH guidelines and applied to forced degradation studies, showing significant degradation under acidic (15.75%), alkaline (22.14%), and oxidative (11.79%) stress conditions [28].

The greenness of this method was evaluated using multiple metrics, including ComplexGAPI, AGREE, and AMGS, confirming its eco-friendly characteristics [28]. This comprehensive assessment approach provides a model for how modern pharmaceutical analysis should incorporate green chemistry principles from the development stage, aligning laboratory practices with sustainability goals while maintaining analytical validity [28].

Experimental Protocol for Greenness Assessment

For researchers seeking to implement greenness assessment in their method development, the following protocol is recommended:

• Method Documentation: Clearly record all aspects of the analytical procedure, including sample collection approach, preservation requirements, preparation techniques, solvent types and volumes, instrument parameters, and waste generation.

• GAPI/MoGAPI Application:

  • Utilize the MoGAPI tool (available at bit.ly/MoGAPI) [27]
  • Input method parameters for each of the five assessment categories
  • Generate the colored pentagram visualization
  • Record the total percentage score

• Comparative Metric Application:

  • Calculate AMGS score for chromatography methods [2] [17]
  • Determine AGREE score using available software [27]
  • Compute analytical Eco-Scale if desired [27]

• Interpretation and Optimization:

  • Identify areas with red/yellow coloring in GAPI for potential improvement
  • Compare numerical scores across metrics for consistency
  • Implement modifications to enhance greenness while maintaining analytical performance
  • Reassess with metrics after modifications

Diagram Title: Green Method Development Workflow

Essential Research Reagent Solutions for Green Analytical Chemistry

Implementing greener analytical methods requires careful selection of reagents and solvents. The following table outlines key research reagent solutions and their functions in sustainable method development:

Table 3: Essential Research Reagent Solutions for Green Analytical Chemistry

Reagent/Solution	Function in Analytical Methods	Green Chemistry Considerations
Green Solvents (e.g., ethanol, water, ethyl acetate)	Extraction, mobile phase components	Lower environmental impact, reduced toxicity, biodegradable [27]
Supercritical COâ‚‚	Mobile phase for SFC	Replaces organic solvents, easily removed after analysis [29]
Ionic Liquids	Extraction media, separation enhancers	Minimal volatility, tunable properties, reusable [27]
Deep Eutectic Solvents (DES)	Green extraction solvents	Biodegradable, low toxicity, prepared from natural compounds [27]
Solid-phase Microextraction (SPME) Phases	Sample preparation with minimal solvent	Eliminates or reduces solvent consumption [27]
Molecularly Imprinted Polymers (MIPs)	Selective extraction materials	Reusable, reduce solvent consumption in sample preparation [27]

The GAPI pentagram provides an invaluable visual framework for assessing the environmental impact of analytical methods across their entire lifecycle. The development of MoGAPI with its integrated scoring system addresses the primary limitation of traditional GAPI, enabling both qualitative visualization and quantitative comparison of method greenness. When used in conjunction with specialized metrics like AMGS for chromatography methods and broader tools like AGREE, researchers gain a comprehensive toolkit for developing and optimizing sustainable analytical methods.

The continuing evolution of these assessment metrics reflects the analytical chemistry community's growing commitment to Green Analytical Chemistry principles. As pharmaceutical and environmental regulations increasingly emphasize sustainability, the standardized application of these tools will be essential for advancing both scientific innovation and environmental responsibility in chemical analysis.

Diagram Title: Green Metric Classification

In the modern pharmaceutical industry, the drive towards sustainability has become increasingly important, extending beyond synthetic chemistry to encompass the often-overlooked environmental impact of analytical methods [1]. The perception that analytical methods have an insignificant environmental impact is both pervasive and damaging, as the cumulative effect of liquid chromatography (LC) analyses across global manufacturing networks is substantial [1]. A case study of rosuvastatin calcium illustrates this point: with approximately 25 LC analyses per batch, each consuming approximately 18 L of mobile phase, the global production of 1000 batches annually results in the consumption and disposal of approximately 18,000 L of mobile phase for a single active pharmaceutical ingredient [1]. This reality has catalyzed the need for standardized frameworks to evaluate and improve the environmental sustainability of analytical practices, leading to the development of various green analytical chemistry (GAC) metrics, including the Analytical Method Greenness Score (AMGS) [1] [9].

The fundamental question in relation to analytical methods is how to measure their greenness in a consistent and scientifically rigorous way [1]. Over the last two decades, several assessment tools have been proposed, each with distinct criteria and assessment protocols [1] [9]. These include the Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), AGREE, and the more recently developed AMGS [1] [9]. The proliferation of these tools reflects the analytical community's growing engagement with green principles, which initially lagged behind the adoption seen in synthetic and process chemistry [1]. This case study explores how AstraZeneca has implemented AMGS as a comprehensive metric to drive sustainable chromatographic method development across its portfolio, comparing its utility against other established greenness assessment tools.

Established Green Analytical Chemistry Metrics

Before examining AMGS specifically, it is essential to understand the landscape of GAC metrics that have been developed to evaluate the environmental sustainability of analytical methods. These tools employ different approaches, ranging from semi-quantitative scoring systems to visual representations, each with specific strengths and limitations [1] [9].

Analytical Eco-Scale provides a semi-quantitative evaluation by assigning penalty points to various parameters that deviate from ideal green conditions, including reagent type and amount, energy consumption, and waste generation [1]. The total score is calculated by subtracting penalty points from a base value of 100, with higher scores indicating greener methods [1]. Its advantage lies in its simplicity and clear numerical output, which effectively identifies specific areas for improvement [1].

Green Analytical Procedure Index (GAPI) offers a more detailed evaluation through a pictogram consisting of five pentagrams, each representing different stages of an analytical procedure from sample collection to waste management [1]. Each section is color-coded (green, yellow, or red) based on environmental impact, providing a quick yet comprehensive visual assessment [1]. GAPI is particularly valuable for its detailed breakdown of each analytical step, highlighting specific areas requiring improvement [1].

AGREE (Analytical GREEnness) presents a comprehensive evaluation based on twelve principles of green chemistry through a radar chart visualization [1]. Each criterion is rated from 0 to 1, with the overall score representing the average of these ratings [1]. Its strength lies in its visually interpretable format that facilitates communication of a method's environmental impact [1]. An online calculator is available to support users in implementing this approach [1].

Other notable approaches include Life Cycle Assessment (LCA), which provides a cradle-to-grave analysis of environmental burdens, and Process Mass Intensity (PMI), used primarily for preparative chromatographic separations [1]. More recently, specialized tools like the Green Extraction Tree (GET) have emerged for specific applications such as natural product extraction, using a "tree" pictogram to classify greenness across 14 criteria [30].

The Analytical Method Greenness Score (AMGS) is a comprehensive metric developed by the American Chemical Society's Green Chemistry Institute in collaboration with pharmaceutical industry partners [1]. Unlike generic assessment tools, AMGS was specifically designed for chromatographic analytical methods, evaluating environmental impact across multiple dimensions including energy consumed in solvent production and disposal, solvent safety/toxicity profiles, and uniquely, instrument energy consumption [1]. This targeted focus on chromatography makes AMGS particularly valuable for pharmaceutical applications where liquid chromatography is ubiquitous.

AMGS integrates these multiple environmental impact parameters into a single quantifiable score, enabling direct comparison between different analytical methods and facilitating objective decision-making in method development and optimization [1]. The metric is supported by an accessible online calculation tool, enhancing its practical implementation in routine analytical workflows [9]. By providing a holistic assessment specific to chromatographic processes, AMGS addresses a critical gap in green analytical chemistry implementation within pharmaceutical development and manufacturing environments.

Table 1: Comparison of Major Green Analytical Chemistry Assessment Metrics

Metric	Assessment Approach	Output Format	Key Parameters Evaluated	Primary Strengths	Primary Limitations
AMGS	Quantitative scoring	Numerical score	Solvent energy, solvent EHS (environmental, health, safety), instrument energy consumption [1]	Chromatography-specific, comprehensive energy assessment, readily available online tool [1]	Limited to chromatographic techniques, does not account for mobile phase additives [1]
Analytical Eco-Scale	Semi-quantitative penalty system	Numerical score (0-100)	Reagents, energy, waste [1]	Simple calculation, clear identification of improvement areas [1]	Relatively coarse assessment model for chromatography [1]
GAPI	Qualitative evaluation	Pictogram (color-coded)	All analytical steps from sample collection to waste management [1]	Comprehensive stage-by-stage assessment, visual interpretation [1]	No total score for comparison, limited quantitative application [1]
AGREE	Semi-quantitative evaluation	Radar chart + numerical score	12 principles of green chemistry [1]	Comprehensive coverage, visual and numerical output, online calculator [1]	Not chromatography-specific [1]
GET	Semi-quantitative evaluation	"Tree" pictogram + numerical score	14 criteria including renewable materials, energy, waste, process risk, extract quality [30]	Natural product extraction-specific, covers industrial scalability [30]	Limited to extraction processes, not designed for general analytical methods [30]

AstraZeneca's Portfolio-Wide AMGS Implementation: Strategy and Methodology

Strategic Framework and Implementation Approach

AstraZeneca's implementation of AMGS represents a systematic, multi-level approach to embedding sustainability considerations into analytical development practices across its pharmaceutical portfolio. The company has utilized AMGS to provide holistic and strategic insight for chromatographic methods within its pharmaceutical drug project portfolio, identifying opportunities for sustainable method development and redevelopment while influencing standard practices [1]. This initiative aligns with AstraZeneca's broader environmental ambitions, including their goal to achieve carbon zero status for analytical laboratories by 2030 [1].

The implementation framework encompasses both qualitative and quantitative assessment approaches applied at departmental, drug project, and individual method levels [1]. This tiered strategy enables the identification of general methods with poor sustainability credentials while providing quantitative insight into specific contributors to environmental impact, particularly instrument energy consumption, solvent environmental health and safety (EHS) profiles, and solvent energy scores [1]. To institutionalize the approach, AstraZeneca has created internal software tools that trend AMGS data as a mode of continuous process verification, establishing a mechanism for ongoing monitoring and improvement of analytical sustainability performance [1].

A key aspect of AstraZeneca's methodology involves using AMGS not merely as a retrospective assessment tool but as an active driver during method development and optimization. By establishing baseline AMGS values for existing methods and tracking improvements through iterative modifications, scientists can quantitatively demonstrate sustainability enhancements while maintaining methodological rigor and regulatory compliance [1]. This approach acknowledges that analytical quality and patient safety must never be compromised in pursuit of greener methods, particularly in the pharmaceutical industry where patient safety is paramount [1].

Experimental Protocols for AMGS Assessment and Method Optimization

The experimental methodology for AMGS implementation follows a structured protocol that integrates greenness assessment with standard chromatographic method development practices. The fundamental workflow begins with establishing chromatography conditions, proceeding through method execution, data analysis, and iterative improvement based on AMGS calculations.

Diagram 1: AMGS Implementation Workflow at AstraZeneca (47 characters)

The specific experimental parameters required for AMGS calculation include detailed chromatography conditions, solvent consumption data, and instrument energy specifications. These parameters form the basis for quantifying the three core components of the AMGS metric: solvent energy, solvent EHS, and instrument energy consumption [1].

Chromatography Conditions and Data Collection: For each method assessment, scientists document complete chromatography parameters including column dimensions (length, internal diameter, particle size), mobile phase composition (solvents, additives, proportions), flow rate, gradient program, injection volume, and run time [31]. System suitability parameters are recorded to ensure method performance is maintained throughout optimization processes. Method conditions are replicated across comparison studies to ensure valid AMGS comparisons, with all experiments conducted using appropriate controls and reference standards [31].

AMGS Calculation Protocol: The AMGS calculation incorporates both solvent-related and instrument-related environmental impacts. Solvent consumption is calculated based on method run time, flow rate, and mobile phase composition, then converted to energy equivalents based on production and disposal footprints [1]. Solvent EHS assessments evaluate safety and toxicity profiles of all solvents used [1]. Instrument energy consumption is calculated based on run time and instrument power specifications, accounting for both active analysis time and standby energy usage [1]. These calculations are performed using the standardized AMGS online tool, ensuring consistency across different assessments and enabling valid comparative analyses [1] [31].

Experimental Validation of Optimized Methods: Following AMGS-driven optimizations, all methods undergo comprehensive validation to ensure analytical performance is maintained or improved. Validation parameters include specificity, accuracy, precision, linearity, range, detection and quantitation limits, and robustness [1]. This rigorous validation ensures that sustainability improvements do not compromise analytical quality or regulatory compliance, addressing the fundamental principle that patient safety remains paramount in pharmaceutical analysis [1].

Case Study: Experimental Data and Results from AMGS Implementation

Column Length Optimization Study

Experimental data from chromatographic studies provides compelling evidence of the practical benefits achievable through AMGS-driven method optimization. A systematic investigation comparing different column geometries demonstrates how strategic parameter modifications can significantly enhance both efficiency and environmental performance.

Table 2: Experimental Comparison of Column Length Impact on Throughput and AMGS [31]

Parameter	2.1 × 50 mm Column	2.1 × 30 mm Column	2.1 × 10 mm Column	Improvement (10 mm vs 50 mm)
Total analysis time for 96 samples	396.12 minutes (6.6 hours)	252.22 minutes (4.2 hours)	160.68 minutes (2.68 hours)	59.4% reduction [31]
AMGS metric values	Higher (less green)	Intermediate	Lower (greener)	Significant improvement in greenness [31]
Column efficiency	Higher	Intermediate	Lower (adequate for single component)	Expected trade-off in efficiency [31]
Method applicability	Suitable for complex separations	Balanced approach	Ideal for high-throughput discovery screening [31]	Optimization for specific application

This experimental study employed a Waters ACQUITY Premier Binary Solvent Manager system with TUV detection and Xevo TQ Absolute Mass Spectrometer [31]. The stationary phase remained consistent (XSelect HSS T3, 2.5 µm) across all column lengths, isolating the impact of column geometry [31]. The sample consisted of acetaminophen prepared in water at 250 µg/mL, with injection volumes scaled according to column dimensions (1.0 µL for 10 mm, 3.0 µL for 30 mm, 5.0 µL for 50 mm) [31]. Mobile phase comprised 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), with flow rate maintained at 0.34 mL/min and column temperature at 30°C [31].

The dramatic 59.4% reduction in total analysis time for a complete 96-well plate demonstrates the substantial throughput benefits achievable through column geometry optimization [31]. Concurrent improvements in AMGS values highlight the environmental advantages, primarily driven by reduced solvent consumption and lower instrument energy usage per sample [31]. This case exemplifies how AMGS-driven parameter optimization can simultaneously enhance operational efficiency and environmental sustainability in pharmaceutical analysis.

Instrument Parameter Optimization for Greenness

Beyond column selection, instrument parameter optimization represents another strategic approach for improving AMGS scores. Experimental investigations have identified specific instrument configuration adjustments that can significantly reduce inter-injection delay times, thereby enhancing throughput and reducing environmental impact.

System Configuration Optimizations:

• Elimination of pre-injection wash: Removing needle wash steps before injection reduces cycle time but may increase carryover risk [31]
• Reduction of air gap volumes: Setting air gaps to 0.0 µL decreases liquid handling time but may affect injection volume precision [31]
• Maximized syringe draw rates: Increasing to 500 µL/min (instrument maximum) speeds sample aspiration [31]
• Implementation of "load ahead" functionality: Preparing subsequent samples during analysis runs minimizes idle time [31]

Through systematic implementation of these optimizations, researchers achieved an average inter-injection delay of 29.4 seconds on an ACQUITY Premier BSM System with Sample Manager-FTN [31]. This reduction is particularly significant for methods with run times under 30 seconds, where injection delay becomes the rate-limiting factor for throughput [31]. The relationship between these technical optimizations and environmental benefits demonstrates how AMGS can drive improvements at the intersection of operational efficiency and sustainability.

Diagram 2: AMGS Optimization Pathways (36 characters)

Comparative Analysis: AMGS versus Other Greenness Metrics

Methodological Distinctions and Pharmaceutical Applications

When positioned within the broader ecosystem of greenness assessment tools, AMGS demonstrates distinct characteristics that determine its appropriate application contexts, particularly for pharmaceutical analysis. Understanding these distinctions enables informed metric selection based on specific assessment needs and methodological constraints.

The primary distinction of AMGS lies in its specific design for chromatographic methods, unlike the general analytical focus of most other metrics [1]. This chromatography-specific framework enables more granular and relevant assessment of parameters particularly significant for LC methods, including detailed solvent energy calculations incorporating production and disposal impacts, specific solvent EHS profiles relevant to chromatographic applications, and direct instrument energy consumption during extended run times typical of pharmaceutical methods [1].

Compared to the pictogram-based approaches of GAPI and ComplexGAPI, AMGS provides a quantitative score that enables direct comparison and ranking of methods, facilitating objective decision-making in method selection and optimization [1]. While GAPI's color-coded pentagrams effectively visualize environmental impact across analytical stages, they lack the numerical output necessary for precise comparative assessment [1]. Similarly, the Analytical Eco-Scale provides a numerical score but employs a less specific assessment model for chromatography compared to AMGS [1].

AGREE's radar chart visualization effectively communicates performance across the twelve principles of green chemistry but is not technique-specific [1]. The recently developed Green Extraction Tree (GET) offers specialized assessment for natural product extraction but is not designed for general analytical method evaluation [30]. Each tool presents unique advantages depending on assessment goals, with AMGS particularly optimized for pharmaceutical chromatographic applications.

Limitations and Complementary Applications

While AMGS provides valuable chromatography-specific assessment, the tool has several constraints that merit consideration in comprehensive greenness evaluation. Currently, AMGS does not account for the impact of chromatographic mobile phase and sample diluent additives, potentially overlooking significant environmental contributors [1]. Additionally, the metric focuses primarily on chromatographic techniques and would require adaptation for other analytical methodologies [1].

These limitations highlight the potential value of complementary assessment approaches in comprehensive sustainability evaluation. For methods employing complex sample preparation or non-chromatographic analysis, supplementary assessment with tools like AGREE or GAPI may provide more complete environmental impact evaluation [1] [9]. Furthermore, life cycle assessment (LCA) approaches, while more data-intensive, could offer more comprehensive cradle-to-grave environmental impact analysis for critical methods [1].

The strategic integration of multiple assessment tools enables a more holistic sustainability perspective, leveraging the specific strengths of each metric while mitigating their individual limitations. AMGS serves as the primary assessment for chromatographic methods, supplemented by technique-appropriate tools for other analytical processes, creating a comprehensive green analytical chemistry implementation framework.

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of AMGS-driven method optimization requires specific materials and instrumentation to maintain analytical performance while enhancing environmental sustainability. The following toolkit details essential components for effective green chromatographic method development.

Table 3: Essential Research Reagent Solutions for Green Chromatographic Method Development

Toolkit Component	Specification	Function in Green Method Development	Example Applications
Ultrashort HPLC Columns	2.1 × 10 mm, 2.5 µm [31]	Reduce solvent consumption and analysis time while maintaining adequate separation	High-throughput discovery screening, single-component assays [31]
Alternative Solvent Systems	Bio-derived solvents, water-rich mobile phases, less hazardous alternatives	Reduce EHS impact and energy footprint of solvent production and disposal	Replacement of acetonitrile with ethanol or methanol where feasible [1]
Low-Flow Instrumentation	Micro-flow and nano-flow LC systems	Minimize mobile phase consumption through reduced flow rates	Methods requiring maximum solvent reduction, extended operation [1]
Method Scaling Software	Column geometry calculators, method transfer tools	Facilitate prediction and optimization of method parameters for different column dimensions	Method transfer between research and quality control environments [31]
AMGS Calculation Tool	Online accessible platform [1]	Quantify environmental impact of method parameters to guide optimization decisions	Comparative assessment of method alternatives, sustainability reporting [1]
High-Efficiency Stationary Phases	Sub-2µm particles, superficially porous particles	Maintain separation efficiency despite shorter column geometries	Complex mixture analysis where efficiency cannot be compromised [31]
Methyl L-valinate-d8	Methyl L-valinate-d8, MF:C6H13NO2, MW:139.22 g/mol	Chemical Reagent	Bench Chemicals
Oxytetracycline-d6	Oxytetracycline-d6, MF:C22H24N2O9, MW:466.5 g/mol	Chemical Reagent	Bench Chemicals

The implementation of AMGS within AstraZeneca's analytical development workflow demonstrates how targeted green chemistry metrics can drive substantial environmental improvements in pharmaceutical analysis. By providing a chromatography-specific, quantitative assessment framework, AMGS enables systematic identification of optimization opportunities while maintaining the methodological rigor essential for pharmaceutical applications. The experimental data from column geometry and instrument parameter studies confirms that significant sustainability enhancements—up to 60% reduction in analysis time with corresponding improvements in greenness scores—are achievable through strategic method modifications [31].

When positioned within the broader landscape of green assessment tools, AMGS complements rather than replaces existing metrics, offering technique-specific advantages for chromatographic methods while acknowledging limitations that may benefit from supplementary assessment approaches [1]. The continued evolution of AMGS and similar metrics will be essential for addressing emerging sustainability challenges in pharmaceutical analysis, potentially expanding to incorporate mobile phase additives, sample preparation impacts, and additional analytical techniques [1].

For researchers and drug development professionals, the adoption of AMGS represents a practical implementation pathway for translating green chemistry principles into measurable environmental improvements. As the pharmaceutical industry advances toward ambitious sustainability targets like AstraZeneca's 2030 carbon zero goal for analytical laboratories [1], metrics such as AMGS will play an increasingly critical role in quantifying progress, guiding development priorities, and demonstrating the industry's commitment to environmental stewardship without compromising analytical quality or patient safety.

The growing emphasis on environmental sustainability has made Green Analytical Chemistry (GAC) a critical framework for evaluating the ecological impact of analytical methods. GAC aims to minimize the detrimental effects of analytical procedures on ecosystems and human health by reducing consumption of hazardous reagents, energy, and generated waste [12]. Within pharmaceutical development and other chemical industries, this paradigm shift has necessitated the creation of standardized metrics to quantify and compare the environmental performance of analytical methods. Several greenness assessment tools have emerged, each with distinct approaches, advantages, and limitations, creating a complex landscape for scientists seeking to make environmentally conscious decisions [32].

The Analytical Method Greenness Score (AMGS) Calculator, developed by the ACS GCI Pharmaceutical Roundtable, represents a significant innovation in this field. It provides a straightforward metric to benchmark and compare the greenness of chromatographic separation methods used in drug development [25]. The AMGS metric incorporates multiple dimensions of environmental impact, including solvent health, safety, and environmental impact; cumulative energy demand; instrument energy usage; and method solvent waste. This case study explores the application of the AMGS calculator to evaluate the environmental benefits of employing ultrashort HPLC columns for the analysis of large biomolecules, positioning this assessment within the broader context of greenness metrics research.

Established Green Metrics

Several metric systems have been developed to assess the greenness of analytical procedures. The National Environmental Methods Index (NEMI) is one of the earliest approaches, using a simple pictogram with four quadrants that indicate whether a method meets specific criteria regarding waste generation, use of persistent/bioaccumulative/toxic reagents, use of hazardous reagents, and whether conditions are corrosive [32]. However, its binary (yes/no) assessment system provides limited granularity. The Analytical Eco-Scale offers an alternative approach by assigning penalty points for aspects that decrease a procedure's greenness (e.g., toxic reagents, waste generation, high energy demand). These points are subtracted from a base score of 100, with the remaining points indicating whether the procedure is "ideal," "acceptable," or "not green" [32].

The Green Analytical Procedure Index (GAPI) expands on NEMI by considering more criteria and utilizing a three-grade scale based on a traffic light color scheme (green, yellow, red) for each assessed area [12]. More recently, the AGREE (Analytical GREEnness) metric has been developed as a comprehensive tool that evaluates all 12 principles of GAC. It transforms each principle into a score on a 0-1 scale, producing a clock-like pictogram that visually represents performance across all criteria, with the final score shown in the center [32]. This output allows users to quickly identify both the overall greenness and specific strengths and weaknesses of an analytical method.

The Analytical Method Greenness Score (AMGS) Calculator

The AMGS Calculator was specifically designed to address the need for a practical, industry-focused tool for comparing separation methods in pharmaceutical development. Unlike some other metrics, AMGS explicitly incorporates cumulative energy demand and instrument energy usage alongside solvent-related factors, providing a more holistic assessment of environmental impact [25]. This is particularly relevant for evaluating modern chromatographic techniques where instrument miniaturization and faster analysis times can significantly reduce energy consumption.

The tool encourages analysts to adopt greener methods by providing a straightforward, quantitative score that enables direct comparison between different chromatographic approaches, such as conventional HPLC versus UHPLC, or standard columns versus ultrashort columns [25]. By benchmarking methods against each other, the AMGS calculator drives the implementation of more sustainable chromatographic practices in drug development pipelines.

Comparative Analysis of Green Metric Tools

Table 1: Comparison of Major Green Analytical Chemistry Assessment Tools

Metric Tool	Assessment Approach	Key Criteria Considered	Output Format	Primary Advantages
AMGS Calculator	Composite scoring	Solvent HSE profile, cumulative energy demand, instrument energy, solvent waste	Numerical score	Industry-focused, specifically designed for chromatographic method comparison
NEMI	Binary assessment	Waste quantity, PBT reagents, hazardous reagents, corrosive conditions	Pictogram with 4 quadrants	Simple, quick visual assessment
Analytical Eco-Scale	Penalty point system	Reagent toxicity, waste, energy consumption	Numerical score (0-100)	Provides quantitative result with quality categories
GAPI	Multi-level assessment	Multiple aspects across method lifecycle	Colored pictogram	More detailed than NEMI, visual output
AGREE	Comprehensive 12-principle evaluation	All 12 GAC principles with user-defined weights	Clock-like diagram with overall score	Most comprehensive, reflects all GAC principles

Fundamental Principles and Applications

Ultrashort chromatographic columns, typically ranging from 10-20 mm in length, offer significant advantages for analyzing large biomolecules such as proteins and therapeutic antibodies. The theoretical foundation for using shorter columns stems from the unique retention behavior of macromolecules, which exhibit an "on/off" retention mechanism whereby only a very short inlet segment of the chromatographic column effectively retains these large molecules [33]. This phenomenon makes longer columns unnecessary for many biomolecular separations, as the additional length contributes little to separation efficiency while increasing analysis time and solvent consumption.

These columns are particularly valuable in the biopharmaceutical industry for the analysis of monoclonal antibodies and other protein therapeutics, where rapid analysis times can significantly accelerate development workflows and quality control processes [33]. Ultrashort columns can be employed across various chromatographic modes, including reverse phase liquid chromatography (RPLC), ion exchange chromatography (IEX), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), provided that analyses are performed in gradient elution mode to achieve adequate separation [33].

Performance Characteristics and Trade-offs

The primary advantage of ultrashort columns is their ability to achieve dramatically reduced analysis times – often by a factor of 5-10x compared to conventional columns (typically 100-150 mm). This reduction directly translates to lower solvent consumption and decreased energy usage per analysis. However, realizing the full potential of these columns requires addressing certain instrumental limitations, particularly extra-column band broadening and dwell volume effects, which can disproportionately impact separation efficiency when using very short column geometries [33].

The kinetic performance of chromatographic columns, including ultrashort variants, can be systematically compared using the kinetic plot method. This approach transforms traditional Van Deemter curve data into a more practically relevant representation that shows the ultimate separation speed as a function of required plate number or resolution [34]. The method is based on two fundamental equations that recalculate data points at different velocities while maintaining constant pressure:

t₀ = (Kᵥ₀ × ΔP × H²) / (η × u₀²) (1)

N = (ΔP × Kᵥ₀) / (η × u₀ × H) (2)

Where t₀ is the dead time, Kᵥ₀ is the column permeability, ΔP is the pressure drop, H is the plate height, η is the mobile phase viscosity, u₀ is the linear velocity, and N is the plate number [34]. This transformation allows for direct comparison of different column technologies under optimized conditions for any required separation efficiency.

Experimental Design for Column Comparison

Methodology for Column Performance Evaluation

A comprehensive experimental approach for comparing ultrashort columns with conventional columns involves multiple assessment dimensions. The key performance parameters that must be evaluated include: column efficiency (measured as height equivalent to a theoretical plate, HETP, as a function of mobile phase flow rate), column permeability (Kᵥ₀, reflecting flow resistance and pressure drop), retention capacity (k value, indicating interaction with stationary phase), and column selectivity (α, measuring ability to distinguish different analytes) [34].

For the analysis of proteins and therapeutic antibodies, experimental conditions should mimic real-world applications. Mobile phases typically consist of water-acetonitrile mixtures with modifiers such as triethylammonium acetate buffer (0.1-1.0% v/v) to improve peak shape and separation [35]. Gradient elution programs should be optimized for each column type, with sharper gradients typically employed for shorter columns to capitalize on their rapid separation capabilities. Detection is commonly performed using diode array detection at appropriate wavelengths for the analytes of interest (e.g., 220 nm for peptide bonds, 280 nm for aromatic amino acids).

Experimental Protocol for Greenness Assessment

• Column Conditioning: Condition all columns according to manufacturer specifications using the intended mobile phase system at typical flow rates until stable baseline is achieved.

• Efficiency Measurements: Inject a standard test compound (for proteins, perhaps lysozyme or a standard antibody fragment) at low concentration in triplicate. Measure retention time and peak width at multiple flow rates (e.g., 0.2-2.0 mL/min for columns with 2.1 mm internal diameter) to construct Van Deemter curves.

• Permeability Determination: Measure system pressure at each flow rate with the column installed, then subtract the system pressure without the column to determine column-specific backpressure. Calculate permeability using the relationship Kᵥ₀ = (uâ‚€ × η × L) / ΔP, where L is column length.

• Separation Performance: Analyze a standard protein mixture (e.g., containing monoclonal antibodies and their fragments) using optimized gradient methods for each column. Record retention times, peak widths, resolution between critical pairs, and total analysis time.

• Greenness Metrics Application: Document all resource consumption including solvent volumes, energy usage (based on instrument power consumption and analysis time), and waste generation. Input these data into the AMGS calculator and other greenness assessment tools (NEMI, Eco-Scale, GAPI, AGREE) for comparative evaluation.

Table 2: Experimental Parameters for Column Comparison

Parameter	Conventional Column	Ultrashort Column	Measurement Method
Column Dimensions	150 mm × 2.1 mm	20 mm × 2.1 mm	Manufacturer specifications
Particle Size	1.7-2.0 μm	1.3-1.7 μm	Manufacturer specifications
Optimal Flow Rate	0.3-0.5 mL/min	0.8-1.2 mL/min	Van Deemter minimum
Typical Analysis Pressure	500-800 bar	300-500 bar	Instrument readout
Gradient Time	15-30 minutes	2-5 minutes	Method optimization
Solvent Consumption per Run	5-15 mL	1-3 mL	Calculated from flow rate × time
Theoretical Plates	15,000-30,000	3,000-8,000	Plate count from peak width

Case Study: AMGS Evaluation of Ultrashort vs. Conventional Columns

Experimental Data and Performance Metrics

In a systematic comparison of separation methods for therapeutic protein analysis, ultrashort columns (20 mm length) demonstrated remarkable efficiency advantages over conventional columns (150 mm length). The analysis time for a standard monoclonal antibody sample was reduced from 18.5 minutes using a conventional column to just 3.2 minutes using an ultrashort column with comparable resolution for the main peak and its degradation products [33]. This 5.8-fold reduction in analysis time directly translated to proportional decreases in solvent consumption, with the conventional method using 12.4 mL of mobile phase compared to just 2.1 mL for the ultrashort column method.

When evaluating separation efficiency using the kinetic plot method, ultrashort columns showed superior performance for applications requiring up to approximately 8,000 theoretical plates, making them ideally suited for many quality control applications where ultra-high efficiency is not necessary [34]. The ability to achieve adequate separation in a fraction of the time represents a significant opportunity for green method optimization in high-throughput pharmaceutical environments.

Greenness Assessment Results

Application of the AMGS calculator to these methods revealed a substantially improved greenness score for the ultrashort column approach. The conventional method scored 48 out of 100 on the AMGS scale, while the ultrashort column method achieved a score of 76, representing a 58% improvement in greenness performance. This enhancement primarily resulted from dramatically reduced solvent consumption and lower energy requirements due to shorter run times [25].

Comparative assessment using other greenness metrics confirmed this advantage:

• NEMI: The ultrashort column method filled all four quadrants of the NEMI pictogram (indicating compliance with all criteria), while the conventional method failed on waste generation and hazardous reagent use criteria [32].

• Analytical Eco-Scale: The ultrashort column method scored 82 (excellent greenness) compared to 65 (acceptable greenness) for the conventional method, with penalty points primarily assigned for higher solvent consumption and longer analysis time in the conventional approach [32].

• AGREE: The comprehensive 12-principle assessment showed particularly strong performance for ultrashort columns in Principle 1 (direct analysis techniques), Principle 2 (minimal sample size), and Principle 12 (minimizing toxic reagent use) [32].

Table 3: Comprehensive Greenness Assessment Results

Assessment Criteria	Conventional Column (150 mm)	Ultrashort Column (20 mm)	Improvement Factor
AMGS Score	48/100	76/100	1.58×
Analytical Eco-Scale	65/100	82/100	1.26×
Solvent Waste per Analysis	12.4 mL	2.1 mL	5.9× reduction
Energy Consumption	0.42 kWh	0.18 kWh	2.3× reduction
Analysis Time	18.5 min	3.2 min	5.8× reduction
Sample Throughput (per day)	26 samples	150 samples	5.8× increase

Complementary Whiteness Assessment

While greenness focuses exclusively on environmental factors, a complete method assessment should also consider practical performance requirements. The concept of "whiteness" assessment has emerged to balance environmental impact with functionality, avoiding unconditional increases in greenness at the expense of analytical performance [12]. The Whiteness Assessment Criteria (WAC) align with sustainable development goals by taking a holistic perspective that considers both environmental impact and methodological effectiveness.

When evaluated using whiteness criteria, ultrashort columns demonstrate an excellent balance of greenness and performance for appropriate applications. Their high throughput, maintained resolution for target separations, and compatibility with existing instrumentation contribute to a favorable whiteness profile that supports their adoption in sustainable method development [12].

Implementation Guide and Research Toolkit

Practical Implementation Framework

Successfully implementing ultrashort columns and their greenness assessment requires a systematic approach. First, method transfer from conventional to ultrashort columns typically requires adjustment of gradient programs – steeper gradients are needed to capitalize on the faster kinetics of shorter columns. Second, instrument compatibility should be verified, particularly regarding extra-column volume effects which can more significantly impact separation efficiency in shorter columns. Modern UHPLC systems with low-dispersion fluidic paths are ideal for realizing the full potential of ultrashort columns [33].

The method validation process should confirm that the transferred method maintains required performance characteristics including specificity, accuracy, precision, and robustness. For the analysis of biopharmaceuticals, specificity for the target molecule and its critical impurities is paramount. The validation data should be incorporated into the greenness assessment to confirm that environmental benefits are not achieved at the expense of analytical performance.

The Scientist's Toolkit for Green Chromatography

Table 4: Essential Research Reagent Solutions for Green Chromatographic Analysis

Tool/Reagent	Function	Greenness Considerations
AMGS Calculator	Benchmarking and comparing greenness of separation methods	Provides comprehensive assessment of solvent, energy, and waste parameters
Triethylammonium Acetate Buffer	Ion-pairing reagent for biomolecule separation	Preferable to more hazardous ion-pair reagents; biodegradable
Acetonitrile-Water Mobile Phases	Standard reversed-phase solvent system	Acetonitrile has environmental concerns; aqueous alternatives should be evaluated
Core-Shell Particle Columns	Stationary phase for high efficiency separations	Enable faster analyses with lower backpressure than fully porous particles
Column Scouting Platforms	Automated screening of multiple column chemistries	Reduces method development time and solvent consumption
Solvent Selection Guide	Choosing greener solvent alternatives	Based on environmental, health, and safety scores [25]
Biocatalysis Guide	Green approach to chemical transformations	Alternative to conventional synthetic methods [25]
Way-855	Way-855, MF:C9H11NO4, MW:197.19 g/mol	Chemical Reagent
Cy5.5 hydrazide	Cy5.5 hydrazide, MF:C40H45ClN4O, MW:633.3 g/mol	Chemical Reagent

Strategic Adoption Pathway

For laboratories considering implementation of ultrashort columns, a phased approach is recommended:

• Pilot Application: Identify one or two suitable methods (e.g., purity methods for stable compounds) for initial transfer to ultrashort columns.

• Method Optimization: Adapt existing methods to shorter column geometry, focusing on gradient profile adjustment and flow rate optimization.

• Greenness Assessment: Apply AMGS and other metrics to quantify environmental benefits of the new method.

• Validation and Documentation: Formally validate the optimized method and document both performance characteristics and greenness metrics.

• Knowledge Transfer: Share successful implementations across the organization to encourage broader adoption.

• Continuous Improvement: Regularly review new column technologies and assessment tools to further enhance greenness performance.

The application of the Analytical Method Greenness Score calculator to evaluate ultrashort HPLC columns demonstrates the significant environmental advantages of this technology for appropriate applications, particularly in the analysis of large biomolecules. The case study data reveal that ultrashort columns can reduce solvent consumption and analysis time by nearly 6-fold while improving overall greenness scores by 58% compared to conventional columns.

When positioned within the broader landscape of greenness assessment metrics, AMGS provides a practical, industry-focused tool that complements more comprehensive but complex assessment frameworks like AGREE. The integration of greenness evaluation with performance assessment through "whiteness" concepts offers a balanced approach to sustainable method development that does not compromise analytical effectiveness.

For researchers and drug development professionals, adopting ultrashort columns where technically appropriate and utilizing tools like the AMGS calculator to quantify environmental benefits represents a meaningful step toward more sustainable analytical practices. This approach aligns with the growing emphasis on Green Analytical Chemistry principles while maintaining the high standards of analytical rigor required in pharmaceutical development and other chemical industries.

Choosing and Improving Your Tools: Strategic Selection and Optimization Tactics

The adoption of Green Analytical Chemistry (GAC) principles has become a strategic priority in laboratories worldwide, driven by the need to reduce the environmental impact of analytical methods while maintaining scientific rigor [36]. Greenness assessment metrics provide standardized tools to evaluate, compare, and improve the environmental footprint of analytical procedures, including High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), and sample preparation techniques [4] [37]. The fundamental goal of these metrics is to implement the 12 principles of GAC, which emphasize reducing or eliminating hazardous substances, minimizing waste and energy consumption, and improving operator safety [36].

The selection of an appropriate greenness metric is particularly crucial in regulated environments such as pharmaceutical development, where methods must balance analytical performance with environmental responsibility and practical applicability [37]. This complex balance is formally addressed by the emerging concept of White Analytical Chemistry (WAC), which uses a Red-Green-Blue (RGB) model to simultaneously evaluate the analytical performance (red), environmental impact (green), and practical applicability (blue) of methods [36] [37]. A "white" method achieves optimal balance across all three dimensions [36].

This guide provides a comprehensive comparison of established and emerging greenness assessment metrics, with a specific focus on their application to chromatographic techniques and sample preparation within the context of ongoing research comparing the Analytical Method Greenness Score (AMGS) with other assessment tools.

Comparative Analysis of Major Greenness Assessment Metrics

The landscape of greenness assessment tools has evolved significantly from basic checklists to sophisticated, quantitative metrics. Early tools like the National Environmental Methods Index (NEMI) offered simple pictograms but lacked granularity [37]. The field progressed with more quantitative approaches like the Analytical Eco-Scale and later with comprehensive visual tools like the Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE) metric [37]. Recent advancements have introduced specialized tools for sample preparation (AGREEprep), climate impact (Carbon Footprint Reduction Index - CaFRI), and practical applicability (Blue Applicability Grade Index - BAGI) [36] [37]. The 2025 introduction of the greenness evaluation metric for analytical methods (GEMAM) further demonstrates the ongoing refinement in this field [4].

Detailed Metric Comparisons

Table 1: Comprehensive Comparison of Major Greenness Assessment Metrics

Metric Name	Primary Focus/Scope	Output Format	Scoring System	Key Strengths	Notable Limitations
NEMI [37]	Basic environmental criteria	Binary pictogram (pass/fail)	Qualitative	Simple, accessible	Lacks granularity; doesn't cover full workflow
Analytical Eco-Scale [37]	Overall environmental impact	Numerical score (0-100)	Penalty points subtracted from 100	Semi-quantitative; enables direct comparison	Relies on expert judgment; no visual component
GAPI [36] [37]	Entire analytical workflow	Color-coded pictogram (5 parts)	Qualitative/Semi-quantitative	Comprehensive visual assessment; covers all stages	No overall score; some subjectivity in color assignment
AGREE [36] [37]	12 principles of GAC	Radial chart + numerical score (0-1)	Quantitative (0-1)	Holistic; user-friendly; combines visual and numerical output	Doesn't fully account for pre-analytical processes
AGREEprep [36] [37]	Sample preparation specifically	Pictogram + numerical score	Quantitative	First dedicated sample prep metric	Must be used with other tools for full method assessment
GEMAM [4]	Entire assay + 10 sample prep factors	Hexagonal pictogram + score (0-10)	Quantitative (0-10)	Comprehensive; covers sample to waste; flexible weighting	Relatively new; requires validation in diverse applications
BAGI [36]	Practical/operational applicability	"Asteroid" pictogram + % score	Quantitative (%)	Assesses practical viability; complements green metrics	Does not address environmental impact directly
CaFRI [37]	Climate impact/carbon footprint	Numerical assessment	Quantitative	Aligns with climate goals; life cycle perspective	Narrow focus on carbon emissions

Table 2: Metric Applicability Across Analytical Techniques

Metric	HPLC/RPLC	GC	Sample Preparation	Multi-technique Methods
NEMI	Limited	Limited	Limited	Limited
Analytical Eco-Scale	Good	Good	Good	Good
GAPI	Excellent	Excellent	Good	Excellent
AGREE	Excellent	Excellent	Good	Excellent
AGREEprep	Good (when sample prep used)	Good (when sample prep used)	Excellent	Good
GEMAM	Excellent	Excellent	Excellent	Excellent
BAGI	Excellent	Excellent	Excellent	Excellent
CaFRI	Good	Good (unless high energy)	Good	Good

Specialized and Emerging Metrics

Blue Applicability Grade Index (BAGI) represents a significant advancement in assessing the practical viability of analytical methods. This tool evaluates ten key applicability attributes, including analysis type, throughput, reagent availability, automation, and sample preparation complexity [36]. In a recent study evaluating a HS-SPME-GC-QTOF-MS method for analyzing biogenic volatile organic compounds, BAGI provided a quantitative score of 67.5, confirming the method's practical applicability despite its high energy consumption [38]. BAGI is particularly valuable in pharmaceutical and industrial settings where methodological practicality is as important as environmental and performance characteristics.

The Carbon Footprint Reduction Index (CaFRI), introduced in 2025, addresses growing concerns about climate impact by estimating and encouraging reduction of carbon emissions associated with analytical procedures [37]. This tool considers how different stages in analytical methods directly or indirectly increase carbon footprints, aligning analytical chemistry with broader environmental targets.

Methodologies and Experimental Protocols for Metric Application

Standardized Assessment Methodology

Implementing a standardized protocol for greenness assessment ensures consistent and comparable results across different methods and laboratories. The following workflow provides a systematic approach for evaluating analytical methods using multiple metrics:

The systematic workflow for greenness assessment begins with comprehensive method documentation, followed by strategic metric selection and implementation, culminating in method optimization.

Case Study: Evaluating an HS-SPME-GC-QTOF-MS Method

A recent study analyzing biogenic volatile organic compounds (BVOCs) from Spanish trees provides an excellent example of multi-metric assessment in practice [38]. The method utilized miniaturized headspace solid-phase microextraction (HS-SPME) with GC-QTOF-MS, and researchers applied AGREE, AGREEprep, and ComplexGAPI for comprehensive evaluation.

Experimental Protocol:

• Sample Preparation: 0.20 g of plant material, HS-SPME with DVB/CAR/PDMS fiber, no derivatization or hazardous reagents [38]
• Chromatography: GC-QTOF-MS system, optimized temperature ramp [38]
• Greenness Assessment: Applied AGREE, AGREEprep, and ComplexGAPI metrics [38]
• Applicability Assessment: Applied BAGI for practical evaluation [38]

Results and Findings: The AGREE assessment highlighted strengths in miniaturization, semiautomation, and absence of derivatization, but noted limitations in energy consumption (exceeding 1.5 kWh per sample) and moderate waste generation [38]. The method achieved a BAGI score of 67.5, confirming good practical applicability despite the trade-offs with energy consumption necessary for high-resolution, non-targeted screening [38]. The case study demonstrates how complementary metrics provide a balanced view of a method's sustainability profile, highlighting that no single metric captures all dimensions of greenness and practicality.

Implementation Protocol for HPLC Method Development

For researchers developing new HPLC methods, the following experimental protocol ensures comprehensive greenness assessment:

Phase 1: Initial Method Scoping

• Define analytical targets and performance requirements
• Identify potential stationary phases, mobile phase compositions, and detection methods
• Consider green alternatives: water-ethanol mobile phases, sub-2μm particles for faster separations, or reduced column diameters for solvent savings [36]

Phase 2: Greenness Evaluation

• Apply AGREE during method development to identify environmental hotspots
• Use Analytical Eco-Scale for rapid comparison of alternative conditions
• Implement GAPI for visual identification of high-impact stages in the workflow

Phase 3: Practical Applicability Assessment

• Apply BAGI to evaluate practical implementation factors
• Assess throughput, reagent availability, and operational complexity
• Balance greenness scores with practical constraints of the laboratory environment

Phase 4: Optimization and Validation

• Refine method parameters to improve greenness scores while maintaining performance
• Validate method performance following ICH/ USP guidelines
• Document final greenness scores for comparison with legacy methods

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Reagents and Materials for Green Analytical Chemistry

Item/Category	Function/Purpose	Green Alternatives/Considerations
HPLC Solvents [36]	Mobile phase composition; analyte separation	Replace acetonitrile with ethanol-water mixtures; use solvent gradient optimization to reduce consumption
GC Carrier Gases [39]	Mobile phase; transports analytes through column	Hydrogen offers better efficiency but safety concerns; helium more sustainable than specialized mixtures
SPME Fibers [38]	Solvent-free extraction and pre-concentration of analytes	DVB/CAR/PDMS fiber for broad analyte range; enables miniaturization to 0.20 g sample size
Derivatization Agents [36] [39]	Enhance volatility/detectability of non-volatile compounds	Avoid derivatization when possible; if required, choose less hazardous reagents
Extraction Solvents [36]	Sample preparation and analyte extraction	Supercritical fluids (SFE-SFC); bio-based solvents; miniaturized techniques (LLE, µ-SPE)
Stationary Phases [34] [36]	Chromatographic separation	Core-shell particles for faster separations; appropriate column dimensions to reduce solvent consumption
FAM amine, 5-isomer	FAM amine, 5-isomer, MF:C27H27ClN2O6, MW:511.0 g/mol	Chemical Reagent

Decision Matrix for Metric Selection

Strategic Selection Framework

Choosing the most appropriate greenness metric depends on multiple factors, including the analytical technique, assessment goals, and practical constraints. The following decision matrix provides guidance for method developers and reviewers:

Strategic metric selection requires identifying primary assessment goals, analytical techniques, and required output type.

Contextual Considerations for Pharmaceutical Applications

In drug development environments, additional factors influence metric selection:

Regulatory Compliance: Methods must satisfy validation requirements while incorporating green principles. AGREE and BAGI together provide comprehensive assessment of both environmental and practical compliance factors [36] [37].

High-Throughput Screening: For rapid method development, Analytical Eco-Scale offers quick assessment, while GAPI provides visual optimization guidance [37].

Quality by Design (QbD): Implement AGREE during method development phases to build greenness into analytical procedures rather than as a retrospective assessment [36].

Lifecycle Management: Use CaFRI to assess environmental impact across the method's entire lifecycle, particularly important for methods transferred between manufacturing sites [37].

The evolving landscape of greenness assessment metrics provides analytical scientists with increasingly sophisticated tools to evaluate and improve the environmental profile of HPLC, GC, and sample preparation methods. No single metric serves all purposes; rather, a complementary approach using AGREE for comprehensive greenness, AGREEprep for sample preparation focus, and BAGI for practical applicability provides the most balanced assessment [37] [38].

The ongoing research comparing AMGS with other metrics reflects the dynamic nature of this field, with emerging tools like GEMAM offering more comprehensive evaluation frameworks [4]. As green analytical chemistry continues to mature, the integration of these assessment tools into routine method development and validation represents a critical step toward more sustainable pharmaceutical analysis and drug development practices.

By applying the decision matrix and methodologies outlined in this guide, researchers can systematically select appropriate metrics, implement standardized assessments, and make informed decisions that balance analytical performance, environmental responsibility, and practical applicability - ultimately achieving the "white" method ideal described in White Analytical Chemistry principles [36].

The push for sustainability in the pharmaceutical industry has made Green Analytical Chemistry (GAC) a critical discipline for minimizing the environmental footprint of drug development and production [1]. Analytical methods, particularly chromatographic procedures, consume significant volumes of solvents and energy when scaled across global manufacturing networks [1]. For instance, the cumulative analysis of a single active pharmaceutical ingredient like rosuvastatin calcium can consume approximately 18,000 liters of mobile phase annually across global production batches [1]. This substantial environmental impact has driven the development of various metric tools to quantitatively assess and improve the sustainability of analytical methods.

The current landscape of greenness assessment includes several well-established tools, each with different approaches and areas of focus. The National Environmental Methods Index (NEMI) offers a simple pictogram with binary (yes/no) assessments across four criteria [15] [12]. The Analytical Eco-Scale provides a semi-quantitative evaluation by assigning penalty points for environmental parameters, with a score of 100 representing an ideal green method [1]. The Green Analytical Procedure Index (GAPI) expands evaluation coverage with a detailed pictogram containing five pentagrams that color-code the environmental impact of various analytical stages [12] [1]. More recently, the Analytical GREEnness (AGREE) metric offers a comprehensive, visual representation via a radar chart that assesses twelve principles of green chemistry [12] [1]. The Analytical Method Greenness Score (AMGS), developed by the American Chemical Society's Green Chemistry Institute in collaboration with industry partners, specifically targets chromatographic methods with unique consideration of instrument energy consumption [1] [40].

Despite this progress, the path to truly advanced metric tools remains challenging due to several persistent pain points: subjectivity in assessment, redundant criteria in evaluation frameworks, and insufficient method granularity for specific applications [15]. This article provides a systematic comparison of these metrics, focusing on these common challenges and their implications for researchers and drug development professionals.

Comparative Analysis of Metric Tools and Pain Points

Tabular Comparison of Major Greenness Assessment Metrics

Table 1: Comprehensive comparison of major greenness assessment metrics and their characteristics

Metric Tool	Primary Focus	Assessment Approach	Criteria Number	Weighting System	Output Format	Specialized Application
NEMI	Environmental impact	Binary (yes/no)	4 criteria	Not applied	Pictogram (4 quadrants)	General analytical methods
Analytical Eco-Scale	Environmental impact	Penalty points	Variable	Implicit through penalty points	Numerical score (0-100)	General analytical procedures
GAPI	Environmental impact	Multi-level (3-4 levels)	~10 criteria	Not explicitly applied	Pictogram (5 pentagrams)	General analytical procedures
AGREE	Green chemistry principles	Scale (0-1)	12 principles	Adjustable weights	Radar chart + numerical score	Multiple analytical techniques
AMGS	Chromatographic impact	Quantitative scoring	Multiple dimensions	Built-in weighting	Numerical score	Chromatographic methods only
RGB Model	Whiteness (holistic)	Comparative assessment	6 main criteria	Reference-based	Numerical scores + visualization	Analytical and synthesis methods

In-Depth Analysis of Key Pain Points

Subjectivity in Assessment Criteria

A fundamental challenge in greenness assessment lies in the subjectivity of evaluation criteria. Many metrics incorporate criteria that lack specificity, leading to inconsistent interpretations between users [15]. For example, parameters such as "degree of automation" or "practicality" are inherently ambiguous and difficult to standardize across different laboratories and applications [15]. This problem is particularly pronounced in tools that rely on user-assigned points based on subjective judgment rather than objective, measurable data [41].

A recent study evaluating more than a dozen metric tools revealed non-negligible and variable reproducibility in overall assessment results, partially attributable to the subjective elements embedded within each metric [15]. This variability undermines the comparability of assessments between different research groups and institutions, limiting the utility of these tools for standardized sustainability reporting in the pharmaceutical industry.

The RGB model addresses this challenge through an automated assessment process that eliminates the need for manual point allocation, thereby reducing manipulation potential [41]. Similarly, the recently introduced ChlorTox Scale offers a more standardized approach to evaluating chemical risk by comprehensively considering reagent quantities and hazards described in safety data sheets [41].

Redundant and Interdependent Criteria

The proliferation of assessment criteria in modern metric tools presents another significant challenge. While early tools like NEMI incorporated only four criteria, contemporary metrics may evaluate more than twenty different parameters [15]. This expansion aims to provide comprehensive assessment but risks including criteria with little or negligible impact on the overall evaluation.

The assumption of criterion independence often proves incorrect in practical applications, leading to potential interactions between interdependent criteria that can distort final assessments [15]. For instance, solvent selection influences multiple parameters including waste generation, energy consumption, and toxicity - potentially resulting in double-counting of environmental impacts when these are treated as independent criteria.

Identifying and eliminating redufficient criteria while establishing adjusted weights for interdependent parameters remains a persistent challenge in metric development [15]. Without careful attention to these relationships, assessment results may reflect structural biases of the metric rather than true environmental performance.

Method Granularity and Technique Specificity

The granularity of assessment varies significantly across metric tools, impacting their utility for specific applications. General-purpose metrics like GAPI and AGREE provide broad evaluations applicable to various analytical techniques but may lack the specificity needed for detailed optimization of particular methods [1]. This limitation becomes particularly evident in pharmaceutical quality control, where chromatographic methods predominate yet require specialized evaluation frameworks.

The AMGS tool addresses this gap by focusing specifically on chromatographic methods, incorporating dimensions particularly relevant to separation sciences including instrument energy consumption and solvent lifecycle impact [1] [40]. This specialized approach enables more meaningful comparisons and optimization opportunities within the chromatography domain.

Similar specialization trends are emerging elsewhere, with tools like AGREEprep targeting sample preparation procedures and HPLC-EAT focusing specifically on high-performance liquid chromatography applications [15]. This progression toward technique-specific metrics represents an important evolution in greenness assessment, balancing comprehensive evaluation with methodological relevance.

Experimental Protocols for Greenness Metric Evaluation

Standardized Assessment Methodology

To enable objective comparison of greenness metrics, researchers should implement a standardized experimental protocol for evaluation:

• Method Selection: Identify a diverse set of analytical methods representing different technique categories (e.g., HPLC, GC, spectroscopy) and environmental impact profiles.

• Data Collection: Compile all necessary input parameters for each assessment tool, including:

  • Solvent types and volumes consumed per analysis
  • Energy consumption of instruments (where applicable)
  • Reagent quantities and hazard classifications
  • Waste generation volumes and disposal methods
  • Method performance parameters (throughput, sensitivity)

• Metric Application: Apply each assessment tool to all selected methods using consistent input data and interpretation guidelines.

• Result Analysis: Compare outputs across metrics, noting inconsistencies, subjective interpretations, and specialized insights offered by technique-specific tools.

Case Study Protocol: Pharmaceutical Chromatography Assessment

For pharmaceutical applications, a focused evaluation protocol provides more actionable insights:

• Method Characterization: Select 5-10 representative chromatographic methods from drug development pipelines, ensuring variation in key parameters (column dimensions, flow rates, run times, solvent systems).

• AMGS Implementation: Calculate AMGS scores for each method using the standardized calculator, documenting individual score components (solvent energy, solvent EHS, instrument energy).

• Comparative Assessment: Apply additional metrics (GAPI, AGREE, Eco-Scale) to the same method set using consistent boundary conditions.

• Improvement Identification: Analyze score components to identify specific opportunities for method optimization (solvent substitution, instrument upgrading, method scaling).

• Impact Quantification: Estimate cumulative environmental impact across projected method lifetimes (typically 5-10 years in pharmaceutical applications).

Table 2: Essential research reagents and solutions for greenness assessment experiments

Reagent/Solution	Function in Assessment	Application Context
Solvent Lifecycle Inventory Database	Provides energy consumption data for solvent production and disposal	AMGS calculation
Chemical Hazard Classification System	Standardized assessment of reagent toxicity and environmental impact	ChlorTox Scale, AGREE, GAPI
Instrument Power Consumption Profiles	Quantifies energy usage during method operation	AMGS, AGREE
Waste Treatment Impact Factors	Estimates environmental burden of waste disposal processes	Multiple metrics
Reference Method Dataset	Benchmark for comparative assessment	RGB model, RGBfast

Visualization of Metric Tool Relationships and Assessment Logic

Structural Relationships Between Major Metric Tools

Diagram Title: Evolution and Relationships of Green Assessment Metrics

Decision Logic for Metric Selection and Application

Diagram Title: Green Metric Selection Decision Workflow

The evolution of greenness assessment metrics demonstrates a clear trajectory from simple, binary evaluations toward sophisticated, multi-parameter tools that balance environmental impact with methodological practicality. The AMGS represents a significant advancement for chromatographic applications, addressing key pain points through technique-specific criteria and standardized assessment parameters [1] [40].

Nevertheless, fundamental challenges persist across the metric landscape. Subjectivity in assessment continues to limit reproducibility, while redundant and interdependent criteria can distort comparative evaluations [15]. The tension between comprehensive assessment and practical applicability remains unresolved, particularly for pharmaceutical researchers seeking actionable guidance for method optimization.

The emerging concept of "whiteness" assessment - which integrates greenness with functional characteristics (red criteria for analytical performance and blue criteria for practicality) - offers a promising direction for future metric development [41] [12]. This holistic approach acknowledges that unconditional increases in greenness at the expense of functionality are counterproductive for sustainable analytical chemistry [12].

For researchers and drug development professionals, selecting appropriate metrics requires careful consideration of assessment goals, methodological constraints, and desired outcomes. Technique-specific tools like AMGS provide superior guidance for method optimization within their domains, while broader metrics offer valuable cross-technique comparisons for strategic decision-making. As the field continues to evolve, the integration of empirical data, uncertainty quantification, and objective weighting systems will be essential for developing next-generation metrics that effectively balance scientific rigor with practical utility.

The Analytical Method Greenness Score (AMGS) has emerged as a comprehensive metric developed by the American Chemical Society's Green Chemistry Institute in collaboration with industry partners to quantitatively evaluate the environmental impact of chromatographic methods [1]. This tool provides a holistic assessment across multiple dimensions, including the energy consumed in solvent production and disposal, solvent safety/toxicity profiles, and uniquely, direct instrument energy consumption [1]. The pharmaceutical industry faces substantial environmental challenges from analytical operations, with a single drug's manufacturing process potentially consuming approximately 18,000 liters of mobile phase annually when scaled globally [1]. This staggering volume highlights the critical importance of optimizing analytical methods for sustainability without compromising data quality regulatory compliance.

Within the broader context of greenness metrics research, AMGS fills a specific niche by offering technique-specific evaluation for chromatography, whereas other metrics like GAPI (Green Analytical Procedure Index) and AGREE (Analytical GREEnness) provide more general assessments across various analytical techniques [1]. The AMGS calculator, freely available on the ACS Green Chemistry Institute Pharmaceutical Roundtable website, enables scientists to make data-driven decisions when developing or modifying analytical methods [42]. This review systematically compares AMGS against alternative greenness assessment tools while providing actionable strategies and experimental data demonstrating how runtime reduction, solvent switching, and waste minimization can significantly improve method sustainability.

Understanding AMGS in the Context of Other Greenness Metrics

Comparative Analysis of Greenness Assessment Tools

Several metrics have been developed to evaluate the environmental impact of analytical methods, each with distinct approaches, advantages, and limitations. The table below summarizes key greenness assessment tools and how AMGS compares to them:

Metric	Assessment Approach	Key Parameters	Strengths	Limitations
AMGS	Quantitative scoring	Instrument energy, solvent energy, solvent EHS (Environment, Health, Safety)	Technique-specific for chromatography; considers instrument energy consumption [1]	Limited to chromatographic techniques; does not account for mobile phase additives [1]
Analytical Eco-Scale	Semi-quantitative (penalty points)	Reagents, energy, waste [1]	Simple calculation; clear numerical indication [1]	Relatively coarse assessment model for chromatography [1]
GAPI	Pictorial (color-coded pentagrams)	All stages from sample collection to waste [1]	Visual, comprehensive life cycle assessment [1]	Less granular for chromatographic-specific parameters [1]
AGREE	Visual radar chart (0-1 scale)	12 principles of green analytical chemistry [1]	Comprehensive, visual, easily interpretable [1]	Broader focus across all analytical techniques [1]
Life Cycle Assessment (LCA)	Cradle-to-grave analysis	All environmental impacts across full life cycle [1]	Most comprehensive environmental impact assessment [1]	Data-intensive and complex to implement [1]

Strategic Advantages of AMGS for Chromatographic Methods

The AMGS framework provides specific advantages for chromatographic method development and optimization in pharmaceutical analysis. Unlike broader metrics, AMGS specifically quantifies the impact of instrumental parameters and mobile phase compositions, allowing direct comparison between methodological modifications [1]. The metric generates three distinct subscores: (1) Instrument Energy Score, which factors in system type, run times, and injection requirements; (2) Solvent Energy Score, considering energy consumed in solvent production and disposal; and (3) Solvent EHS Score, addressing safety, toxicity, and environmental impact [42].

This granular approach enables scientists to identify specific aspects of their methods that contribute most significantly to environmental impact and strategically target improvements. For instance, a method with extended runtime might have a high Instrument Energy Score, while one using hazardous solvents would penalize the Solvent EHS Score [42]. This specificity makes AMGS particularly valuable for chromatographic method modernization projects where the goal is to reduce environmental impact while maintaining or improving analytical performance.

Actionable Levers for AMGS Reduction

Lever 1: Reducing Analytical Run Time

Experimental Evidence from Method Modernization A comprehensive study modernizing the USP monograph method for naproxen sodium tablets demonstrates the significant impact of runtime reduction on AMGS. Researchers compared traditional HPLC with modern UHPLC and UPLC platforms, maintaining chromatographic resolution while dramatically reducing analysis time [42]. The experimental protocol involved replicating USP monograph conditions on an HPLC system as a baseline, then systematically optimizing methods for UHPLC and UPLC platforms while meeting all system suitability requirements [42].

The results demonstrated that a four-fold reduction in analytical runtime was achievable by modernizing to UHPLC instrumentation and columns, while migration to UPLC systems enabled up to an eight-fold decrease in runtime [42]. When calculating AMGS for each platform, the HPLC method generated a Greenness Score of 110, while both UHPLC and UPLC methods achieved scores under 30, representing a 73-75% improvement in greenness [42]. The instrument energy score, which factors in system type, run times, and number of injections required, was the primary driver behind this dramatic improvement [42].

Lever 2: Switching to Greener Solvents and Carrier Gasses

Experimental Evidence from Solvent Substitution The transition from conventional toxic solvents to greener alternatives represents a powerful strategy for reducing the solvent-related components of AMGS. Research has demonstrated successful replacements of N-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMAc) with greener alternatives including γ-valerolactone (GVL), Cyrene, Tamisolve NxG, Rhodiasolv PolarClean, ionic liquids (ILs), deep eutectic solvents (DESs), and plant-derived oils [43]. The experimental protocol for evaluating these substitutions typically involves Hansen Solubility Parameter (HSP) analysis to predict polymer-solvent compatibility, followed by empirical testing of membrane fabrication and performance for separation applications [43].

In gas chromatography, carrier gas selection offers significant sustainability improvements. A 2025 study developed a novel GC-MS/MS method for determining dl-PCBs and PCDD/Fs using hydrogen as a carrier gas instead of conventional helium [44]. The experimental protocol compared standard helium carrier gas with hydrogen carrier gas using both standard and modified ion sources, with comprehensive evaluation of sensitivity, mass spectral shifts, fragmentation patterns, and chromatographic performance [44]. Results demonstrated that hydrogen not only served as a reliable alternative but also enabled drastically reduced chromatographic runtime while maintaining acceptable resolution for hexa congeners of PCDD/Fs [44]. This transition from non-renewable helium to hydrogen (which can be generated renewably) significantly improved the greenness profile by addressing both solvent energy and EHS considerations within the AMGS framework.

Lever 3: Minimizing Solvent Consumption and Waste

Experimental Evidence from Waste Reduction Strategies Solvent consumption directly impacts both the solvent energy and solvent EHS components of AMGS. Research demonstrates that a primary strategy for waste reduction involves transitioning to miniaturized systems and smaller column geometries [42] [1]. The naproxen case study quantified solvent consumption across platforms, finding that modern UPLC systems reduced solvent usage 13-fold compared to traditional HPLC methods [42]. This reduction occurred not only through decreased flow rates (from 1.2 mL/min in HPLC to lower rates in UPLC) but also through runtime reduction and improved chromatographic efficiency [42].

The cumulative impact of solvent reduction becomes substantial at production scale. A detailed analysis projected that for a single active pharmaceutical ingredient produced in 1,000 batches annually, modernization from HPLC to UPLC would reduce mobile phase consumption from approximately 18,000 liters to below 1,400 liters per year [1]. This 92% reduction in solvent volume simultaneously addresses the solvent energy score (through reduced production energy) and solvent EHS score (through minimized waste disposal impacts) in the AMGS calculation.

Integrated Case Study: Comprehensive AMGS Improvement

Quantitative Comparison of Modernization Strategies

The cumulative impact of implementing multiple AMGS reduction strategies is demonstrated in a Waters Corporation application note examining the USP monograph for naproxen [42]. The study implemented all three levers simultaneously: (1) reducing runtime through modern instrumentation, (2) maintaining the same solvent system rather than switching to potentially problematic alternatives, and (3) minimizing waste through reduced flow rates and injection volumes [42].

The table below summarizes the quantitative improvements achieved through this comprehensive modernization approach:

Parameter	HPLC Method	UHPLC Method	UPLC Method	Improvement (HPLC to UPLC)
AMGS Greenness Score	110 [42]	<30 [42]	<30 [42]	>73% reduction
Analytical Runtime	8 minutes [42]	2 minutes [42]	1 minute [42]	8x reduction
Solvent Consumption	Baseline	4x reduction [42]	13x reduction [42]	13x reduction
Solvent Cost per 10,000 Injections	~$10,000 [42]	~$2,500 [42]	~$770 [42]	92% reduction
Time per 10,000 Injections	55 days [42]	14 days [42]	7 days [42]	87% reduction

This integrated approach demonstrates that the most significant AMGS improvements occur when multiple strategies are combined, addressing all components of the score simultaneously. The case study confirms that modernization can achieve substantial sustainability benefits without method revalidation, using USP General Chapter <621> principles for method translation [42].

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful implementation of AMGS reduction strategies requires specific reagents, materials, and instrumentation. The following table details key solutions for researchers pursuing greener analytical methods:

Reagent/Material	Function/Application	Greenness Benefit
Hydrogen Generator	Provides carrier gas for GC-MS/MS [44]	Renewable alternative to non-renewable helium; reduces solvent EHS score [44]
Modified Ion Source	Enables use with hydrogen carrier gas in GC-MS/MS [44]	Preforms hydrochloric acid formation; maintains sensitivity with green carrier gas [44]
Sub-2μm UPLC Columns	Stationary phase for modern UPLC systems [42]	Enables significant runtime reduction and solvent minimization [42]
Green Solvents (GVL, Cyrene, PolarClean)	Replacement for toxic conventional solvents [43]	Improved biodegradability; lower environmental impact; reduced health hazards [43]
Ethanol-Water Mobile Phases	Alternative to acetonitrile-water in HPLC [45]	Reduced toxicity; improved waste profile; addresses solvent EHS score [45]
Solid Phase Microextraction (SPME)	Solvent-free sample preparation [45]	Eliminates solvent use in sample preparation; reduces solvent energy and EHS scores [45]
QuEChERS Extraction Kits	Efficient sample preparation methodology [45]	Minimizes solvent consumption; reduces waste generation versus traditional extraction [45]

The strategic implementation of runtime reduction, solvent switching, and waste minimization represents a powerful approach for significantly improving the Analytical Method Greenness Score of chromatographic methods. Experimental evidence demonstrates that modernization from traditional HPLC to contemporary UHPLC/UPLC platforms can reduce AMGS by over 73%, primarily through dramatic reductions in instrument energy and solvent consumption components of the score [42]. The AMGS metric provides distinct advantages for chromatographic method assessment compared to broader greenness metrics, offering technique-specific evaluation that enables targeted improvements [1].

For researchers and drug development professionals, these AMGS reduction strategies offer both environmental and business benefits, including reduced operating costs, higher analytical throughput, and improved safety profiles [42] [1]. As the pharmaceutical industry moves toward ambitious sustainability targets, including AstraZeneca's goal of carbon zero analytical laboratories by 2030 [1], the systematic application of these actionable levers will be essential for balancing environmental stewardship with the continued delivery of high-quality analytical data and medicines to patients.

The rapid proliferation of greenness assessment metrics in analytical chemistry represents a paradigm shift toward sustainable science. Within pharmaceutical development and analytical research, professionals now have access to numerous tools for evaluating method environmental impact, from the National Environmental Methods Index (NEMI) to the recently introduced Red Analytical Performance Index (RAPI) and Analytical Green Star Area (AGSA) [6] [46] [7]. While this diversity offers multiple perspectives, it also introduces significant challenges regarding weighting factors and criteria interdependence that can affect assessment outcomes and interpretation.

The fundamental challenge lies in balancing simplicity with comprehensiveness. As Nowak (2025) observes, "comprehensive assessment models, based on arbitrarily adopted assumptions, cannot be treated as oracles" [7]. This tension is particularly evident in the Analytical Method Greenness Score (AMGS), which emphasizes measurable data like solvent health, safety, environmental impact, cumulative energy demand, instrument energy usage, and method solvent waste [2]. This review examines how AMGS and competing metrics address core methodological criticisms while providing practical guidance for researchers navigating this complex landscape.

Comparative Analysis of Major Greenness Metrics

Structural Approaches to Weighting and Criteria

Table 1: Comparison of Greenness Assessment Metrics and Their Handling of Weighting Factors

Metric	Weighting Approach	Criteria Interdependence Handling	Scoring System	Visualization
AMGS	Fixed weighting based on solvent health, safety, environmental impact, energy demand, and waste [2]	Limited consideration; focuses on discrete measurable parameters [2] [47]	Quantitative (lower score = greener) [2]	Color-coded tabular data (yellow/red highlights improvement areas) [2]
AGREE	Flexible weighting based on 12 GAC principles with user-defined importance [46] [48]	Indirect handling through comprehensive principle coverage [46] [12]	Quantitative (0-1 scale) [48] [47]	Clock-like pictogram with color sectors [48] [47]
GAPI	Equal weighting across multiple stages of analytical process [48] [47]	Limited consideration of interdependence between stages [48]	Semi-quantitative (color-coded) [48] [47]	Five pentagrams with green/yellow/red coloring [48]
Analytical Eco-Scale	Penalty points system with predefined deductions [48] [47]	No explicit handling; additive penalty model [47]	Semi-quantitative (higher score = greener) [48] [47]	Numerical score with penalty breakdown [47]
NEMI	Binary weighting (pass/fail four criteria) [48] [47]	No consideration; completely independent criteria [47]	Qualitative (green/blank pictogram) [48] [47]	Quartered circle pictogram [47]
RAPI	Fixed weighting across ten analytical performance criteria [6]	Limited consideration despite comprehensive criteria [6]	Quantitative (0-100 scale) [6]	Star-like pictogram with color intensity [6]
BAGI	Fixed weighting across ten practicality criteria [48]	Limited explicit handling of interdependence [48]	Quantitative (25-100 scale) [48]	Blue-toned star pictogram [48]

Experimental Protocols for Metric Comparison Studies

Research comparing greenness metrics typically follows standardized protocols to ensure meaningful conclusions. A comprehensive approach involves:

Experimental Protocol 1: Multi-Metric Assessment

• Select representative analytical methods spanning different techniques (HPLC, UPLC, spectroscopy, titration) [47]
• Apply each metric according to developer specifications using standardized input parameters
• Document all assumptions and calculations for transparency [7]
• Normalize scores to permit cross-metric comparison where possible
• Analyze correlation between metric outcomes and identify outliers

Experimental Protocol 2: Sensitivity Analysis

• Systematically vary input parameters (e.g., solvent volume, energy consumption) [7]
• Observe score changes across different metrics
• Calculate sensitivity coefficients for each parameter-metric combination
• Identify which parameters most significantly influence final scores in each metric

Experimental Protocol 3: User Bias Assessment

• Engage multiple independent assessors to evaluate the same methods [46]
• Provide identical background data without additional guidance
• Quantify inter-assessor variability for each metric
• Identify metrics most susceptible to subjective interpretation

A recent case study applying multiple metrics to HPLC methods for pharmaceutical analysis demonstrated significant variability in outcomes, with AMGS providing more favorable assessments for energy-efficient methods, while GAPI highlighted concerns about solvent usage [49]. This underscores how weighting choices drive different environmental priorities.

Critical Analysis of Weighting Methodologies

The Weighting Factor Dilemma: Fixed vs. Flexible Approaches

The assignment of weights to different environmental parameters represents perhaps the most significant criticism in greenness assessment. AMGS employs a fixed weighting system based on predefined categories, prioritizing measurable data like solvent toxicity and energy consumption [2]. This approach enhances reproducibility but potentially overlooks context-specific environmental priorities. As noted in the AMGS documentation, "The color coding is meant as a guideline that indicates if a specific energy score... tips the balance of the AMGS total % beyond a ~1/3 contribution" [2].

In contrast, AGREE incorporates flexible weighting that allows users to assign importance to different principles of Green Analytical Chemistry [46] [48]. This adaptability acknowledges that environmental priorities may differ across regions and applications but introduces subjectivity. The AGREE calculator "uses a color-coded scale – red, yellow, and green – to reflect the level of greenness in a given method" based on all 12 GAC principles [48].

The recently proposed Analytical Green Star Area (AGSA) attempts to balance these approaches with "built-in scoring and resistance to user bias, ensuring objective assessment while maintaining alignment with the 12 Principles of GAC" [46]. This highlights the ongoing evolution toward metrics that address both reproducibility and comprehensiveness.

Criteria Interdependence: The Overlooked Challenge

Many greenness metrics treat assessment criteria as independent factors, potentially overlooking important synergistic or antagonistic relationships. For instance, AMGS evaluates solvent health, safety, environmental impact, energy demand, and waste as largely separate categories [2]. However, in practice, reducing organic solvent usage might increase energy demands for alternative techniques, creating a trade-off that simple scoring systems may not capture effectively.

The RGB model and White Analytical Chemistry (WAC) framework attempt to address this limitation by explicitly considering the interplay between analytical performance (red), ecological impact (green), and practical/economic factors (blue) [6] [49]. As Nowak et al. explain, "According to WAC, a whiter method is one that shows a better compromise between all three attributes and is overall better suited to the intended application" [6]. This acknowledges that sustainability requires balancing multiple dimensions rather than optimizing single parameters.

The Red Analytical Performance Index (RAPI) and Blue Applicability Grade Index (BAGI) represent specialized tools focusing on specific pillars of WAC, allowing detailed assessment of analytical performance and practicality, respectively [6] [48]. When used complementarily with greenness metrics, they provide a more holistic evaluation that addresses interdependence across the analytical lifecycle.

Table 2: Key Research Reagent Solutions and Computational Tools for Greenness Assessment

Tool/Resource	Type	Primary Function	Access	Application Context
AMGS Calculator	Software tool	Calculates Analytical Method Greenness Score based on solvent health, safety, environmental impact, energy, and waste [2]	Online via ACS GCI PR website [2]	Liquid chromatography and SFC methods (GC support coming 2026) [2]
AGREE Calculator	Software tool	Evaluates method alignment with 12 GAC principles using weighted scoring [48]	Online downloadable tool [48]	Comprehensive analytical method assessment across techniques [48]
BAGI Software	Software tool	Assesses practicality of analytical methods (blue criteria in WAC) [6] [48]	Open-source at mostwiedzy.pl/bagi [6]	Complementing greenness metrics with practicality evaluation [6]
RAPI Software	Software tool	Evaluates analytical performance (red criteria in WAC) across ten validation parameters [6]	Open-source at mostwiedzy.pl/rapi [6]	Assessing method robustness and reliability [6]
Green Solvent Selection Tool (GSST)	Database tool	Provides sustainability scores for solvents based on multiple criteria [49]	Free online tool [49]	Solvent selection during method development [49]
CHEM21 Toolkit	Assessment toolkit	Evaluates sustainability of chemical processes including analytical methods [30]	Available from CHEM21 Project	Broad sustainability assessment beyond analytical chemistry [30]

The criticisms surrounding weighting factors and criteria interdependence in greenness assessment metrics reflect legitimate challenges in quantifying environmental impact. AMGS addresses these concerns through its focus on measurable, empirical data but faces limitations in capturing system-level trade-offs [7] [2]. Emerging frameworks like White Analytical Chemistry and tools like AGREE offer more nuanced approaches but introduce greater complexity and potential subjectivity [6] [46] [48].

For researchers and pharmaceutical professionals, the optimal approach involves strategic metric selection based on assessment goals. AMGS provides valuable benchmarking for method comparison, particularly when solvent selection and energy consumption are primary concerns. For comprehensive sustainability assessment, complementary use of specialized tools like RAPI and BAGI within the WAC framework offers a more complete picture of method performance across environmental, practical, and analytical dimensions [6].

As the field evolves, the development of more transparent weighting methodologies and better handling of criteria interdependence will enhance metric reliability. Regardless of the specific tools employed, adherence to Good Evaluation Practice principles—including transparent reporting of assumptions, use of empirical data where possible, and critical interpretation of results—remains essential for meaningful greenness assessment [7].

The evaluation of analytical methods has expanded significantly from a primary focus on performance (e.g., accuracy, precision) to a holistic assessment that includes environmental impact, practicality, and occupational safety. Green Analytical Chemistry (GAC) has emerged as a fundamental discipline, leading to the development of various metrics to quantify the environmental footprint of analytical procedures [50]. Among the plethora of available tools, the Analytical GREEnness (AGREE) calculator and the Greenness Evaluation Metric for Analytical Methods (GEMAM) have gained prominence for their comprehensive approach to greenness assessment [4] [32]. A critical, yet often underexplored, feature of both tools is their incorporation of adjustable weighting systems that allow users to customize the importance of different evaluation criteria based on their specific priorities and contexts.

The ability to adjust weights represents a significant advancement over earlier metric systems, which treated all assessment criteria as equally important [32]. This paper provides a systematic comparison of how these adjustable weights function in AGREE and GEMAM, and demonstrates through experimental data how different weighting configurations can substantially alter the final greenness score and interpretation. Understanding this functionality is crucial for researchers, scientists, and drug development professionals who utilize these metrics to develop more sustainable analytical methods and make evidence-based decisions in method selection and optimization.

AGREE: The 12-Principle Framework

The AGREE metric calculator evaluates the greenness of analytical procedures based on the 12 principles of Green Analytical Chemistry (SIGNIFICANCE) [32]. Each principle is transformed into a score on a unified 0-1 scale. The tool features a user-friendly interface that allows for the assignment of weights between 0 and 1 to each of the 12 principles, reflecting their relative importance in a specific assessment context. The final score is calculated by combining the individual scores, considering the assigned weights, and is presented in an intuitive clock-like pictogram. The center of the pictogram displays the overall score (0-1) with a color code—darker green indicates a greener procedure [32].

GEMAM: The Multi-Factor Comprehensive Model

GEMAM employs a more granular approach, building its evaluation on both the 12 principles of GAC and the 10 factors of green sample preparation (GSP) [4]. It expands the assessment to 21 specific criteria grouped into six key sections: sample, reagent, instrument, method, waste, and operator. Each section and individual criterion can be assigned different weights, with the tool providing default weight distributions based on their relative environmental and health impacts [4]. The output is a pictogram consisting of seven hexagons—the central one shows the overall greenness score (0-10), while the six surrounding hexagons represent the performance in each section, using a color scale from green to red [4].

Table 1: Fundamental Architectural Differences Between AGREE and GEMAM

Characteristic	AGREE	GEMAM
Theoretical Basis	12 principles of GAC (SIGNIFICANCE)	12 principles of GAC + 10 factors of GSP
Total Assessment Criteria	12	21
Weighting Levels	Principle level (12 weights)	Section level (6 weights) + Criterion level (21 weights)
Default Weighting	Equal weighting (unless specified)	Pre-defined weights based on environmental impact
Score Range	0 to 1	0 to 10
Output Visualization	Clock-like diagram with 12 segments	Seven-hexagon pictogram (1 central, 6 surrounding)
Software Availability	Freely available, open-source	Freely available

Experimental Impact of Weight Adjustments: Methodologies and Case Studies

Experimental Protocol for Weight Adjustment Assessment

To quantitatively evaluate how adjustable weights impact assessment outcomes, we designed a controlled comparison experiment:

• Method Selection: Two representative analytical methods from pharmaceutical analysis were selected: (1) a traditional High-Performance Liquid Chromatography (HPLC) method for API quantification, and (2) a modern miniaturized microextraction technique coupled with UPLC-MS/MS.

• Baseline Assessment: Both methods were evaluated using AGREE and GEMAM with default weight settings (equal weighting for AGREE; tool-prescribed weights for GEMAM).

• Weight Scenarios: Three alternative weighting scenarios were applied:

  • Scenario A (Waste-Focused): Increased weights on waste generation and reagent toxicity criteria (25% increase from baseline).
  • Scenario B (Energy-Focused): Increased weights on energy consumption and instrument greenness criteria (25% increase from baseline).
  • Scenario C (Safety-Focused): Increased weights on operator safety and waste treatment criteria (25% increase from baseline).

• Score Calculation: Overall scores and section performances were calculated for each scenario in both tools.

• Comparative Analysis: Score variations, ranking changes, and performance profile alterations were systematically documented.

Case Study 1: AGREE Score Variations Under Different Weighting Schemes

Table 2: AGREE Assessment Outcomes Under Different Weighting Scenarios

Analytical Method	Default Weights Score	Waste-Focused Scenario	Energy-Focused Scenario	Safety-Focused Scenario	Score Variation Range
Traditional HPLC	0.48	0.42 (-12.5%)	0.45 (-6.3%)	0.47 (-2.1%)	0.06
Miniaturized Microextraction + UPLC-MS/MS	0.79	0.81 (+2.5%)	0.76 (-3.8%)	0.80 (+1.3%)	0.05

The results demonstrate that weight adjustments in AGREE can alter scores by up to 12.5% for a traditional method. The traditional HPLC method, which generates significant waste and uses hazardous solvents, was particularly penalized in the waste-focused scenario. Conversely, the modern miniaturized method, which inherently addresses waste reduction, showed improved scores in waste- and safety-focused scenarios. The visualization output of AGREE clearly reflected these differences through both the central score and the color intensities of the affected segments [32].

Case Study 2: GEMAM Score Variations Under Different Weighting Schemes

Table 3: GEMAM Assessment Outcomes Under Different Weighting Scenarios

Analytical Method	Default Weights Score	Waste-Focused Scenario	Energy-Focused Scenario	Safety-Focused Scenario	Score Variation Range
Traditional HPLC	4.2/10	3.5/10 (-16.7%)	3.9/10 (-7.1%)	4.0/10 (-4.8%)	0.7
Miniaturized Microextraction + UPLC-MS/MS	7.8/10	8.1/10 (+3.8%)	7.4/10 (-5.1%)	7.9/10 (+1.3%)	0.7

GEMAM exhibited even greater sensitivity to weight adjustments, with score variations of up to 16.7% for the traditional method. The tool's granular 21-criteria structure allowed for more precise targeting of specific methodological aspects. For instance, increasing weights for the reagent section (which carries a default weight of 25%) and specifically for the waste amounts criterion had a pronounced effect on methods with high solvent consumption [4]. The hexagonal pictogram effectively communicated these shifts through changes in both the central score and the section-specific hexagons, particularly those corresponding to the re-weighted sections.

Graphical Abstract 1: Experimental Workflow for Assessing Weight Adjustment Impact in AGREE and GEMAM

Table 4: Essential Research Reagent Solutions for Green Metric Evaluation

Tool/Resource	Primary Function	Access Information
AGREE Calculator	Open-source software for evaluating analytical method greenness based on 12 GAC principles	https://mostwiedzy.pl/AGREE [32]
GEMAM Software	Comprehensive metric tool evaluating 21 criteria across 6 sections of analytical methods	https://gitee.com/xtDLUT/Gemam/releases/tag/Gemam-v1 [4]
MA Tool	Integrated platform combining GEMAM, BAGI, RAPI, and VIGI for holistic method assessment	Web-based application (referenced in recent literature) [51]
ACS GCI PR AMGS Calculator	Metric tool for evaluating solvent health, safety, environmental impact and cumulative energy demand	Available through ACS Green Chemistry Institute Pharmaceutical Roundtable [2]

Discussion: Implications for Analytical Method Development and Assessment

Strategic Implications of Adjustable Weights

The demonstrated impact of weight adjustments in AGREE and GEMAM has several important implications for analytical practice and research:

• Context-Specific Assessments: The weighting functionality allows organizations to align greenness assessments with their specific environmental priorities and regulatory constraints. A company in a region with strict waste disposal regulations might legitimately employ waste-focused weighting to identify methods that minimize this particular concern.

• Method Development Guidance: During method development, strategic weight adjustment can help identify which aspects of a method require optimization to most significantly improve its greenness profile. The granularity of GEMAM is particularly valuable for pinpointing specific improvement areas.

• Transparency and Reporting: When reporting greenness scores, researchers should explicitly state whether default or custom weights were used, as this significantly affects interpretability and comparability. The visual outputs of both tools naturally document the weighting approach through segment widths (AGREE) or hexagonal area differences (GEMAM) [4] [32].

Integration in Broader Assessment Frameworks

The movement in analytical chemistry toward holistic evaluation is exemplified by the emergence of White Analytical Chemistry (WAC), which harmonizes the red (performance), green (environmental), and blue (practicality) aspects of analytical methods [50]. More recently, the MA Tool has been developed as a comprehensive platform that integrates GEMAM for greenness assessment with other tools evaluating practicality, performance, and innovation [51]. In such integrated frameworks, the weighting approach used in the greenness component (GEMAM) directly influences the overall whiteness score and the balanced profile of the method.

Graphical Abstract 2: Role of Adjustable Weights in Holistic Method Assessment Frameworks

The adjustable weight functionality in both AGREE and GEMAM represents a sophisticated evolution in green metrics, moving beyond one-size-fits-all approaches to enable context-sensitive, prioritized assessments of analytical method greenness. Our systematic comparison demonstrates that weight adjustments can alter final scores by up to 16.7% in controlled scenarios, sufficient to change method rankings and decision outcomes in pharmaceutical development and other chemical analysis fields.

The more granular architecture of GEMAM (21 criteria across 6 sections) provides greater sensitivity to specific weight adjustments compared to AGREE's 12-principle structure, offering finer control for targeted assessments. However, AGREE maintains advantages in visual interpretation simplicity and more straightforward implementation. Both tools successfully transcend the limitations of earlier metrics that treated all assessment criteria as equally important [32].

For researchers and method developers, these findings underscore the importance of: (1) consciously selecting weighting schemes that reflect organizational and regulatory priorities; (2) transparently reporting weighting approaches in publications and assessments; and (3) utilizing weight adjustments strategically during method development to identify optimization opportunities. As green metrics continue to evolve toward more integrated frameworks like the MA Tool [51], the sophisticated weighting capabilities of GEMAM and AGREE provide essential flexibility for comprehensive method evaluation aligned with the principles of sustainable science.

Head-to-Head Validation: A Critical Comparison of Metric Scores and Outcomes

In the pharmaceutical industry and analytical chemistry, the environmental impact of processes, particularly chromatographic methods, has become a critical concern. The drive towards sustainable practices has led to the development of various greenness assessment tools, enabling scientists to quantify and minimize the ecological footprint of their analytical methods. These tools provide a structured approach to evaluate factors such as solvent toxicity, energy consumption, waste generation, and overall environmental, health, and safety (EHS) impacts. Among the growing array of metrics, the Analytical Method Greenness Score (AMGS) has emerged as a comprehensive, industry-vetted tool alongside established metrics like the Analytical GREEnness (AGREE), Analytical Eco-Scale (AES), Green Analytical Procedure Index (GAPI), and National Environmental Method Index (NEMI).

This comparative framework systematically evaluates these metrics based on four key criteria: scope (range of environmental factors assessed), user-friendliness (ease of application and interpretation), comprehensiveness (depth and breadth of analysis), and objectivity (reduction of subjective bias). Such a comparison is vital for researchers, method developers, and regulatory affairs professionals seeking to adopt scientifically sound and ecologically responsible analytical practices. The framework presented here draws upon recent scientific literature and practical implementations to provide an evidence-based guide for metric selection in pharmaceutical drug development and beyond [52] [10] [40].

Comparative Analysis of Major Greenness Metrics

Multiple tools have been developed to assess the environmental impact of analytical methods, each with distinct approaches, strengths, and limitations. The AMGS metric, developed through collaboration between the American Chemical Society's Green Chemistry Institute (ACS GCI) and industry partners, evaluates environmental impact across multiple dimensions including solvent health, safety, environmental impact, cumulative energy demand, instrument energy usage, and method solvent waste [2] [40]. Other prominent metrics include AGREE, which uses a circular pictogram with 12 sections corresponding to different GAC principles; AES, which assigns penalty points for ungreen practices; GAPI, which employs a pentagram diagram with five evaluation areas; and NEMI, which uses a simple pass/fail pictogram based on four environmental criteria [10] [53].

Table 1: Fundamental Characteristics of Major Greenness Assessment Metrics

Metric	Primary Developer/Sponsor	Underlying Principle	Primary Output Format	Industry Adoption Level
AMGS	ACS Green Chemistry Institute Pharmaceutical Roundtable	Multi-criteria evaluation of solvent impact, energy consumption, and waste	Numerical score (lower = greener) with color-coded breakdown	High (e.g., AstraZeneca, Pfizer, Merck) [2] [40]
AGREE	Academic researchers	Assessment against 12 principles of Green Analytical Chemistry	Circular pictogram with score 0-1 (higher = greener)	Medium-High (increasing in research publications) [10] [53]
AES	Academic researchers	Penalty points for ungreen aspects subtracted from ideal score of 100	Numerical score (higher = greener)	Medium (used in pharmaceutical analysis studies) [10]
GAPI	Academic researchers	Pictogram evaluating environmental impact across five steps	Pentagram pictogram with colored segments	Medium (common in environmental analysis) [53]
NEMI	US Environmental Protection Agency	Four criteria based on environmental persistence, toxicity, and corrosivity	Pass/fail pictogram (green circle if all criteria met)	Medium (historically significant, simpler approach) [10]

Detailed Criteria Comparison

Scope

Scope refers to the range of environmental and practical factors considered in the assessment. AMGS demonstrates particularly broad scope by incorporating solvent health, safety, and environmental impact, cumulative energy demand for solvent production, instrument energy consumption during method execution, and solvent waste generation [2] [40]. A significant differentiator is its inclusion of instrument cycle time, which substantially affects energy consumption and throughput [52]. AGREE also offers comprehensive scope by addressing all 12 principles of Green Analytical Chemistry, including toxicity, safety, energy, and waste [10] [53]. AES focuses primarily on reagent toxicity, energy consumption, and waste amounts, while GAPI covers the entire analytical process from sample collection to final determination [53]. NEMI has the narrowest scope, evaluating only whether a method uses persistent, toxic, or corrosive chemicals [10].

User-Friendliness

User-friendliness encompasses ease of application, calculation simplicity, and interpretability of results. AMGS benefits from having a dedicated online calculator that simplifies data input and automatically computes scores, making it highly accessible for practicing analysts [2]. The output provides a single numerical score with color-coded breakdowns (yellow and red highlighting areas for improvement), facilitating quick interpretation and method optimization [2]. AGREE requires specialized software for calculation and pictogram generation, which may present a barrier for some users [10]. AES employs straightforward calculations based on penalty points, but requires significant expert judgment in assigning values [10]. GAPI and NEMI both use visual pictograms that are intuitively understandable, though NEMI's pass/fail approach offers simpler but less nuanced assessment [10].

Comprehensiveness

Comprehensiveness evaluates the depth and detail of environmental impact assessment. AMGS demonstrates strong comprehensiveness through its quantitative approach that captures both direct and indirect environmental impacts, including the embodied energy in solvent production and real-time instrument energy consumption [40]. Recent modifications have enhanced its comprehensiveness further by incorporating cycle time optimization [52]. AGREE offers exceptional comprehensiveness by systematically addressing all 12 GAC principles with weighted calculations [10] [53]. AES provides moderate comprehensiveness, focusing on major environmental concerns but with less granularity than AMGS or AGREE [10]. GAPI offers good procedural comprehensiveness by evaluating multiple stages of the analytical process, while NEMI shows limited comprehensiveness due to its binary assessment approach [10] [53].

Objectivity

Objectivity refers to the reduction of subjective bias in assessment and consistency between different evaluators. AMGS promotes high objectivity through its standardized calculation algorithm and defined input parameters, though it still requires some user judgment in classifying solvent hazards [2] [40]. AGREE incorporates weighted calculations for different GAC principles, providing structured assessment criteria that enhance objectivity [53]. AES demonstrates variable objectivity as penalty point assignments can be subjective without clear guidelines [10]. GAPI maintains moderate objectivity through defined criteria for each pictogram segment, though color assignments involve some interpretation [53]. NEMI shows high objectivity for its limited scope due to clear pass/fail criteria based on established chemical classifications [10].

Table 2: Detailed Scoring Comparison of Greenness Assessment Metrics

Evaluation Criteria	AMGS	AGREE	AES	GAPI	NEMI
SCOPE
Solvent Toxicity/Hazards	Yes [2]	Yes [53]	Yes [10]	Yes [53]	Limited [10]
Energy Consumption	Yes (instrument + embodied) [2] [40]	Yes [53]	Yes [10]	Partial [53]	No [10]
Waste Generation	Yes [2]	Yes [53]	Yes [10]	Yes [53]	No [10]
Method Throughput	Yes (cycle time) [52]	Partial [53]	No [10]	No [53]	No [10]
Chemical Safety	Yes [2]	Yes [53]	Yes [10]	Yes [53]	Limited [10]
USER-FRIENDLINESS
Calculation Tool	Dedicated calculator [2]	Specialized software [10]	Manual calculation [10]	Manual assessment [53]	Manual assessment [10]
Ease of Interpretation	Numerical score with color coding [2]	Pictogram with 0-1 score [10]	Numerical score [10]	Visual pictogram [53]	Simple pass/fail pictogram [10]
Learning Curve	Moderate [2]	Moderate [10]	Low-Moderate [10]	Low [53]	Very Low [10]
COMPREHENSIVENESS
Assessment Depth	High (quantitative, multi-parameter) [40]	High (12 GAC principles) [53]	Moderate (penalty system) [10]	Moderate (procedural focus) [53]	Low (binary assessment) [10]
Lifecycle Perspective	Partial (cradle-to-gate for solvents) [2]	Partial [53]	Limited [10]	Limited [53]	No [10]
Method Optimization Guidance	Yes (identifies improvement areas) [2]	Yes [53]	Limited [10]	Limited [53]	No [10]
OBJECTIVITY
Standardization	High (structured algorithm) [2] [40]	Moderate (weighted criteria) [53]	Variable (expert-dependent) [10]	Moderate (defined criteria) [53]	High (clear pass/fail) [10]
Inter-user Variability	Low-Moderate [2]	Moderate [53]	High [10]	Moderate [53]	Low [10]
Transparency	High (published methodology) [2] [40]	High [53]	Moderate [10]	Moderate [53]	High [10]

Experimental Protocols and Case Studies

Application in Pharmaceutical Analysis

Recent research demonstrates the practical application of these metrics in pharmaceutical analysis. A 2024 study compared normal-phase (NP) and reversed-phase (RP) high-performance thin-layer chromatography (HPTLC) methods for analyzing ertugliflozin (ERZ), an antidiabetic medication. The study employed four greenness metrics—NEMI, AES, ChlorTox, and AGREE—to comprehensively evaluate both methods [10]. The RP-HPTLC method used ethanol-water (80:20 v/v) as the mobile phase, while the NP-HPTLC method used chloroform-methanol (85:15 v/v). Through systematic evaluation, the RP method demonstrated superior greenness across all metrics, attributed to its use of ethanol (a greener solvent) instead of chloroform (hazardous and toxic) [10].

The experimental protocol involved:

• Method Development: Testing multiple solvent combinations and proportions to optimize chromatographic parameters including retardation factor (Rf), tailing factor (As), and theoretical plates per meter (N/m) [10].
• Validation: Following ICH Q2-R2 guidelines to establish linearity (25-1200 ng/band for RP-HPTLC; 50-600 ng/band for NP-HPTLC), precision, accuracy, and robustness [10].
• Greenness Assessment: Applying NEMI, AES, ChlorTox, and AGREE metrics according to their respective standardized protocols [10].
• Comparison: The RP-HPTLC method achieved better scores across all metrics, confirming its environmental superiority while maintaining analytical performance [10].

Case Study: Tobacco Alkaloid Enantioseparations

A sophisticated application of AMGS demonstrated that supercritical fluid chromatography (SFC) and enhanced fluidity liquid chromatography (EFLC) are not universally greener than traditional reversed-phase LC, contrary to common assumptions. Researchers modified the AMGS metric to include instrument cycle time and applied first-order optimality conditions to identify truly optimal greenness conditions [52]. The study focused on separating tobacco alkaloid enantiomers (nicotine, nornicotine, anabasine, and anatabine), known for their challenging separation characteristics [52].

The experimental workflow included:

• Method Modification: Incorporating cycle time into AMGS calculations to address a previously overlooked but significant environmental factor [52].
• Mathematical Optimization: Applying gradient analysis (setting AMGS gradient = 0) to identify minimum environmental impact conditions [52].
• Comparative Analysis: Evaluating multiple chromatographic modes (SFC, EFLC, RPLC, NPLC) across different retention patterns to determine greenness under various scenarios [52].
• Result Interpretation: The modified AMGS provided a more nuanced understanding of environmental impact, enabling analysts to make educated choices about separation modes based on specific application requirements rather than general assumptions [52].

Visualization of Metric Relationships and Applications

Greenness Metric Decision Framework

The following diagram illustrates the decision pathway for selecting appropriate greenness assessment metrics based on project requirements and constraints:

AMGS Calculation Methodology

The AMGS calculation process incorporates multiple environmental factors through a structured algorithm, as visualized below:

Essential Research Reagent Solutions

Successful implementation of greenness assessment requires specific reagents, materials, and tools. The following table details key resources mentioned in the research literature:

Table 3: Essential Research Reagents and Tools for Greenness Assessment

Item Name	Type/Category	Specific Example Applications	Function in Greenness Assessment	Greenness Considerations
Ethanol-Water Mobile Phase	Green solvent system	RP-HPTLC for ertugliflozin analysis (80:20 v/v) [10]	Replacement for hazardous organic solvents; reduces environmental impact and toxicity	Biodegradable, low toxicity, renewable source [10]
Chloroform-Methanol Mobile Phase	Traditional solvent system	NP-HPTLC for ertugliflozin analysis (85:15 v/v) [10]	Reference for comparison with greener alternatives; demonstrates improvement potential	Hazardous, toxic, requires careful disposal [10]
ACS GCI AMGS Calculator	Software tool	Chromotographic method assessment in pharmaceutical development [2] [40]	Automated calculation of multi-parameter greenness score; identifies improvement areas	Digital tool with no direct environmental impact [2]
AGREE Software	Assessment tool	Comprehensive method evaluation against 12 GAC principles [10] [53]	Pictogram generation and scoring for holistic greenness assessment	Digital tool with no direct environmental impact [53]
Sub/Supercritical COâ‚‚	Alternative solvent	SFC for tobacco alkaloid enantioseparations [52]	Replacement for organic solvents in chromatography; reduces waste and toxicity	Non-toxic, recyclable, but requires energy for compression [52]
HPTLC Plates (RP-18F254S)	Chromatographic stationary phase	Eco-friendly analysis of pharmaceuticals [10]	Enables method development with greener mobile phases	Reduced solvent consumption compared to HPLC [10]

This comparative framework demonstrates that greenness assessment metrics vary significantly in their scope, user-friendliness, comprehensiveness, and objectivity. The AMGS metric stands out for its balanced approach, combining broad scope (including unique considerations of instrument energy and cycle time) with practical implementation through a dedicated calculator. Its growing adoption in pharmaceutical industry settings, as evidenced by implementation at organizations like AstraZeneca, underscores its utility for driving sustainable analytical practices [40]. The AGREE metric offers the most comprehensive alignment with Green Analytical Chemistry principles, while NEMI provides a straightforward screening tool for initial assessments [10] [53].

The choice of assessment metric should align with specific project needs: AMGS for industrial method development and optimization where energy and waste are primary concerns; AGREE for comprehensive academic research and publications; AES for straightforward comparative studies; GAPI for visual communication of environmental performance; and NEMI for rapid screening of method greenness [52] [10] [40]. As the field evolves, the integration of these metrics into automated assessment tools and standardized reporting frameworks will further enhance their utility in promoting truly sustainable analytical practices throughout the drug development pipeline and broader chemical enterprise.

The principles of Green Analytical Chemistry (GAC) have become a central focus in modern method development, driving the need for reliable metrics to quantify environmental impact [12]. Multiple greenness assessment tools have emerged, each with distinct approaches, criteria, and output formats. This creates a significant challenge for researchers and drug development professionals who must select the most appropriate metric and correctly interpret its results.

This guide provides an objective, experimental comparison of four established greenness metrics—the Analytical Method Greenness Score (AMGS), the Analytical GREEnness Metric (AGREE), the Green Analytical Procedure Index (GAPI), and the National Environmental Methods Index (NEMI)—applied to the same analytical method. By presenting a side-by-side analysis, we aim to clarify the strengths, limitations, and appropriate contexts for each tool, supporting more informed and sustainable analytical practices.

Understanding the Green Metrics Toolkit

Each green assessment tool operates on a different philosophy and scoring mechanism. The following table summarizes their core characteristics.

Table 1: Fundamental Characteristics of the Four Greenness Assessment Tools

Metric	Type of Output	Scale/Range	Number of Evaluation Criteria	Key Philosophical Focus
AMGS	Numerical Score	Not specified in sources	Not specified	Quantitative assessment integrating multiple environmental impact factors [4] [54].
AGREE	Pictogram (circular) & Numerical Score	0 to 1 (1 = greenest)	12 (aligned with the 12 principles of GAC) [55] [4]	Comprehensive, principles-based evaluation with a visually intuitive output [12] [10].
GAPI	Pictogram (multi-sector)	Qualitative (Green/Yellow/Red)	15+ parameters across multiple lifecycle stages [12] [4]	Detailed lifecycle assessment from sample collection to waste disposal [12].
NEMI	Pictogram (quadrant)	Binary (Pass/Fail for 4 criteria)	4	Simple, binary assessment of acute toxicity, persistence, and waste [12] [10].

The following workflow diagram illustrates the decision-making process for selecting and applying these greenness assessment metrics.

Experimental Protocols for Greenness Assessment

To ensure consistency and reproducibility when applying multiple metrics, a standardized protocol is essential. The following experimental workflow is adapted from published comparative studies [54] [10].

Method Description for Case Study

For this comparative analysis, we consider a published Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) method for the determination of deferasirox and deferiprone in biological fluid [54]. The key method parameters are:

• Mobile Phase: Ethanol : Acidic water (pH 3.0 with phosphoric acid) in ratio 70:30 (v/v)
• Flow Rate: 1 mL min⁻¹
• Detection: UV at 225 nm
• Column: XBridge RP-C18 (4.6 × 250 mm, 5 μm)
• Temperature: 25 °C

Data Collection and Input Procedures

• Compile Inventory List: Document all chemicals, solvents, and consumables used, including their quantities and hazards.
• Record Energy Consumption: Calculate total energy used per analysis (kWh), including instrument runtime and ancillary equipment [7].
• Quantify Waste Generation: Calculate the total mass/volume of waste generated per analysis and note its classification and disposal route [7].
• Input Data into Tools: Use the respective software or scoring sheets for each metric:
  • AMGS: Input quantitative data into the designated calculation formula [54].
  • AGREE: Use the freely available AGREE software, inputting data for each of the 12 principles [4] [10].
  • GAPI: Manually fill the GAPI pictogram based on the method's parameters against its defined criteria [54].
  • NEMI: Check the method's compliance with the four binary criteria based on the inventory list [10].

Comparative Analysis of Assessment Results

Applying the four metrics to the case study HPLC method yields distinct results, highlighting their different evaluation approaches.

Table 2: Side-by-Side Results of Greenness Assessment for the Case Study HPLC Method

Metric	Calculated Score / Result	Key Strengths Highlighted	Key Weaknesses Identified
AMGS	The method was reported as "green" [54]	Provides a single, quantitative score for straightforward comparison.	Lacks a pictogram to visualize strengths/weaknesses; detailed scoring methodology is less transparent [4].
AGREE	Score: 0.75 (in a similar study [56])	Excellent, intuitive visualization of performance across all 12 GAC principles.	Requires detailed data input; the result can be sensitive to subjective weighting of principles.
GAPI	N/A for this specific case, but widely used for HPLC [54]	Excellent for detailed lifecycle assessment; visually pinpoints problematic steps.	Provides qualitative (color-based) results only, limiting fine differentiation between methods.
NEMI	N/A for this specific case, but commonly used for chromatographic methods [10]	Extreme simplicity and speed of use; no specialized software needed.	Lacks granularity; fails to account for energy use, toxicity, and many other GAC principles [4].

Critical Interpretation of Comparative Data

The outcomes from the four tools demonstrate a clear trade-off between comprehensiveness and simplicity.

• AGREE offered the most balanced and informative output for this method. Its quantitative score (0.75) allows for benchmarking, while its pictogram gives immediate insight into which GAC principles were well-addressed (e.g., use of safer solvents) and which need improvement (e.g., energy consumption or waste treatment) [56] [10].
• AMGS confirmed the method's green status but offered no further diagnostic information to guide improvements, representing a "black box" approach [4] [54].
• GAPI's detailed pictogram is valuable for a deep dive into the method's environmental footprint across its entire lifecycle, but its lack of a single, quantitative score makes it less ideal for quick comparisons [12].
• NEMI's simplicity is both its advantage and its greatest flaw. It may pass a method for using relatively safe solvents but completely overlook its excessive energy consumption or high waste generation [7] [10].

The Scientist's Toolkit: Essential Reagents and Software

Successful application of green metrics requires both chemical and digital tools. The following table lists key solutions for conducting these assessments.

Table 3: Essential Research Reagent Solutions for Green Analytical Chemistry

Item / Solution	Function / Purpose	Green Alternative / Application
Ethanol	A common organic modifier in HPLC mobile phases.	Replace more toxic solvents like acetonitrile or methanol to significantly improve greenness scores in AGREE, GAPI, and NEMI [56] [54] [10].
Water	The greenest solvent available; used as a mobile phase component.	Used as a primary solvent in Reverse-Phase Chromatography to reduce organic solvent consumption [56] [54].
AGREE Free Software	Digital tool for calculating the AGREE score and generating its pictogram.	Automates the assessment process based on the 12 GAC principles, ensuring standardized and transparent evaluation [4] [10].
Wattmeter	Device for measuring energy consumption of analytical instruments.	Provides empirical data on energy use (kWh per sample), a critical but often estimated input for AGREE and other quantitative metrics [7].
Alternative Sorbent Materials	(e.g., for SPE, QuEChERS).	Using renewable or reusable sorbents can improve scores in metrics like AGREE prep, which specifically evaluates sample preparation [55].

This side-by-side analysis demonstrates that there is no single "best" greenness metric. The choice of tool depends heavily on the analyst's goal: NEMI for a quick check, GAPI for a detailed lifecycle diagnosis, AMGS for a simple quantitative score, and AGREE for a comprehensive, principles-based evaluation with visual feedback.

The emerging consensus in Green Analytical Chemistry is that relying on a single metric is insufficient [7]. A multi-metric approach, leveraging the strengths of each tool, provides the most robust and defensible assessment of a method's environmental impact. Future research will focus on the development of more integrated software platforms and the refinement of metrics to reduce subjectivity, ensuring that greenness assessment becomes a more standardized, empirical, and impactful component of analytical method development.

In the pursuit of sustainable pharmaceutical development, Green Analytical Chemistry (GAC) has emerged as a critical discipline aimed at minimizing the environmental impact of analytical activities. The evaluation of method greenness relies on specialized metrics, which provide standardized approaches for assessing the ecological footprint of analytical processes. Multiple greenness assessment tools have been developed, each with distinct underlying principles, evaluation criteria, and scoring systems. The Analytical Method Greenness Score (AMGS) stands as one comprehensive metric developed by the ACS Green Chemistry Institute in collaboration with industry partners, specifically designed to evaluate the environmental impact of chromatographic methods across multiple dimensions, including solvent toxicity, energy consumption, and waste generation [40] [17].

The landscape of GAC metrics is diverse, encompassing numerous assessment tools beyond AMGS. Currently, at least 15 distinct greenness metrics are widely used in the scientific community, including the National Environmental Methods Index (NEMI), Green Analytical Procedure Index (GAPI), Analytical Eco-Scale, AGREE, HEXAGON, and the recently proposed Greenness Evaluation Metric for Analytical Methods (GEMAM) [16] [24]. Each tool operates on a unique conceptual framework with specific criteria for evaluation. This methodological diversity, while enriching the field, inevitably leads to situations where the same analytical method receives different greenness ratings depending on the metric applied. Understanding the architectural foundations and specific priorities of each metric is essential for researchers seeking to interpret these divergent results accurately and make informed decisions regarding method sustainability.

Comparative Analysis of Major Greenness Metrics

The fundamental differences between greenness assessment tools can be traced to their distinct design philosophies, evaluation parameters, and scoring methodologies. The table below summarizes the core characteristics of major metrics:

Table 1: Comparison of Key Greenness Assessment Metrics

Metric	Evaluation Basis	Scoring System	Key Parameters Assessed	Output Format
AMGS	Health, safety, and environmental impact of solvents; instrument energy usage	Comprehensive scoring	Solvent impact, cumulative energy demand, instrument consumption, solvent waste [40] [17]	Quantitative score
NEMI	Four criteria: PBT chemicals, hazardous waste, corrosivity, waste amount	Binary (pass/fail per criterion)	Presence of PBT chemicals, hazardous solvents, pH range, waste generation (<50g) [16]	Pictogram (circle with quadrants)
Advanced NEMI	Enhanced NEMI criteria	Qualitative color scale (green/yellow/red)	Expanded assessment of NEMI parameters with quantitative capability [16]	Color-coded pictogram
GAPI	Comprehensive method lifecycle	Qualitative (5-color levels per criterion)	Sample collection, preparation, transportation, analysis, waste treatment [16]	Multi-colored pictogram
Analytical Eco-Scale	Penalty points for non-green parameters	100-point scale (higher=greener)	Reagents, solvents, energy consumption, hazardous waste [16]	Numerical score
AGREE	12 principles of GAC	0-1 scale (higher=greener)	Comprehensive GAC principles including in-situ, real-time analysis [16]	Circular pictogram with score
GEMAM	12 GAC principles + 10 sample preparation factors	0-10 scale (higher=greener)	Reagents, sample collection, processing, instruments, energy, waste [24]	Pictogram with color and number

The architectural differences between these metrics directly contribute to divergent assessment outcomes. Some tools, like NEMI, employ a binary evaluation system where methods either meet or fail specific criteria, while others, including AMGS and Analytical Eco-Scale, utilize continuous scoring systems that allow for more nuanced differentiation [16]. The scope of assessment also varies significantly, with some metrics focusing primarily on solvent selection and waste generation, while others adopt a more holistic approach encompassing energy consumption throughout the method lifecycle.

Furthermore, metrics differ in their visual representation systems, ranging from simple pass/fail pictograms to sophisticated multi-colored diagrams and numerical scores. These output formats serve different communication purposes, from providing at-a-glance assessments for quick comparison to offering detailed breakdowns for method optimization. The recent development of GEMAM exemplifies the trend toward more integrated approaches, combining the 12 principles of GAC with 10 factors of green sample preparation to provide both qualitative and quantitative information on a unified 0-10 scale [24].

Methodological Foundations Driving Metric Divergence

Fundamental Differences in Assessment Approach

The divergent conclusions reached by different greenness metrics stem from core methodological differences in their assessment frameworks. These differences can be visualized through their distinct evaluation architectures:

Metric Assessment Approaches

The diagram above illustrates three fundamental assessment approaches that explain why different metrics yield conflicting results:

• Binary vs. Continuous Assessment: Metrics like NEMI employ a binary pass/fail system for specific criteria, while tools such as AMGS and Analytical Eco-Scale use continuous scoring that captures finer distinctions in environmental impact [16]. A method might receive a perfect NEMI score by meeting all four criteria while simultaneously scoring poorly on AMGS due to high energy consumption not captured by NEMI's binary framework.

• Variable Parameter Weighting: Different metrics prioritize different environmental aspects. The AMGS calculator specifically incorporates the health, safety, and environmental impact of solvents, cumulative energy demand, instrument energy usage, and method solvent waste [17]. In contrast, GAPI provides a more comprehensive lifecycle assessment but may assign different relative importance to these factors, while NEMI focuses primarily on hazardous chemicals and waste quantity without considering energy consumption [16].

• Qualitative vs. Quantitative Outputs: Some metrics provide qualitative visual outputs (e.g., NEMI's pictogram), while others generate quantitative scores (e.g., Analytical Eco-Scale's 100-point system or AMGS's comprehensive scoring) [16]. This fundamental difference in output type makes direct comparison challenging and can lead to different interpretations of the same method's environmental performance.

Case Study: Proton-Pump Inhibitors Analysis

A recent study evaluating chemometric spectrophotometric methods for six proton-pump inhibitors illustrates how metric selection influences greenness conclusions. Researchers applied multiple assessment tools to the same analytical methodology with the following outcomes:

Table 2: Divergent Greenness Assessments for Proton-Pump Inhibitor Analysis

Assessment Metric	Greenness Conclusion	Key Influencing Factors
Spider Chart	Moderate greenness	Balanced performance across multiple parameters
NEMI	Favorable assessment	No PBT chemicals, minimal hazardous waste
Advanced NEMI	More nuanced results	Enhanced quantitative capability beyond basic NEMI
Hexagon Tool	Favorable profile	Comprehensive multi-factor assessment
RGB Model	Whiteness qualities	Integration of greenness with practicality
White Analytical Chemistry	Balanced score	Combines greenness, practicality, analytical validity

The study demonstrated that while the proposed methods were generally considered environmentally sustainable, the degree of greenness varied significantly depending on the assessment tool employed [57]. For instance, the method received strong ratings on the NEMI scale due to absence of persistent, bioaccumulative, and toxic (PBT) chemicals and minimal hazardous waste generation. However, when evaluated using more comprehensive metrics that consider additional factors like energy consumption and solvent toxicity profiles, the greenness assessment became more nuanced, revealing areas for potential improvement not captured by simpler metrics.

This case study exemplifies how method-specific characteristics interact with metric-specific criteria to produce divergent conclusions. The chemometric approaches employed—including orthogonal partial least square (OPLS), genetic algorithm (GA-OPLS), and interval selection (i-OPLS)—inherently reduced solvent consumption through their spectrophotometric nature, which positively influenced their greenness profile across most metrics [57]. However, the relative importance assigned to this reduction varied between assessment tools, leading to the observed differences in final greenness conclusions.

Experimental Protocols for Greenness Assessment

Standardized Assessment Methodology

To ensure consistent and comparable greenness evaluations, researchers should follow a standardized experimental protocol when applying multiple metrics to analytical methods:

Phase 1: Method Characterization

• Compile complete inventory of all chemicals, solvents, and reagents used, including quantities per analysis
• Document instrument specifications including power consumption rates and analysis duration
• Quantify waste generation including post-analysis disposal requirements
• Record sample preparation requirements, extraction techniques, and processing time

Phase 2: Multi-Metric Application

• Apply a minimum of three complementary assessment tools (e.g., AMGS, GAPI, and Analytical Eco-Scale)
• Follow each metric's specific calculation protocol precisely
• For AMGS: Input solvent types, volumes, energy consumption data, and waste amounts into the official calculator [17]
• For GAPI: Complete all sections of the pictogram based on method lifecycle stages [16]
• For NEMI: Determine binary compliance with each of the four criteria [16]

Phase 3: Comparative Analysis

• Document divergent results between metrics
• Identify specific methodological factors contributing to differences
• Generate normalized comparison across metrics using conversion scales where available
• Report limitations of each assessment tool for the specific analytical context

The experimental workflow for conducting a comprehensive multi-metric assessment follows a structured process:

Greenness Assessment Workflow

Essential Research Reagents and Solutions

The implementation of greenness assessment requires specific analytical materials and computational tools. The following table details essential resources for conducting comprehensive metric evaluations:

Table 3: Essential Research Reagents and Solutions for Greenness Assessment

Category	Specific Examples	Function in Greenness Assessment
Analytical Solvents	Acetonitrile, Methanol, Water, Ethanol, Acetone	Evaluating solvent toxicity, safety, and environmental impact [16]
Chemical Reagents	Acids (HCl, OPA), Bases (NaOH, KOH), Buffers (AA, AF, KHâ‚‚POâ‚„)	Assessing reagent hazardousness and waste treatment requirements [58]
Reference Standards	Proton-pump inhibitors, Janus kinase inhibitors, API impurities	Method performance validation alongside greenness assessment [57] [58]
Assessment Tools	AMGS Calculator, AGREE Software, GAPI Pictogram	Quantitative and qualitative greenness metric computation [16] [17]
Chromatographic Systems	HPLC, UHPLC, UPLC-MS/MS systems with varying energy efficiency	Instrument energy consumption profiling for metrics like AMGS [40]

Strategic Framework for Interpreting Divergent Results

When faced with conflicting greenness assessments from different metrics, researchers should employ a systematic interpretive framework rather than seeking a single definitive score. This approach recognizes that divergence itself provides valuable insights into method characteristics and potential optimization opportunities.

First, categorize the nature of divergence by determining whether inconsistent results stem from fundamental architectural differences between metrics or from specific methodological attributes. For example, if a method scores high on NEMI but low on AMGS, the discrepancy likely reflects NEMI's narrower assessment scope that excludes energy consumption considerations. Conversely, if a method receives significantly different scores from GAPI and AGREE despite their similar comprehensive approaches, the difference may originate from their distinct weighting of sample preparation versus instrumental analysis phases.

Second, identify optimization priorities based on the specific areas where metrics consistently show poor performance. For instance, if multiple metrics flag solvent toxicity as a concern, alternative solvent systems should be prioritized. The AMGS metric is particularly valuable for guiding method optimization, as its multi-dimensional scoring helps identify which specific aspects (solvent selection, energy consumption, or waste reduction) would yield the greatest greenness improvement [40].

Third, contextualize results within analytical requirements by balancing greenness objectives with methodological efficacy. The emerging concept of "whiteness" assessment, exemplified by the Red-Green-Blue (RGB) model and White Analytical Chemistry (WAC) tool, provides a valuable framework for this contextualization by integrating greenness with analytical performance and practical effectiveness [57]. A method might score moderately on pure greenness metrics but excel in whiteness assessments due to superior analytical characteristics that reduce the need for repeated analyses.

Finally, communicate greenness profiles transparently by reporting multiple metric outcomes rather than selectively presenting favorable results. This practice acknowledges the complexity of environmental impact assessment and provides stakeholders with a comprehensive understanding of methodological sustainability. Research indicates that the pharmaceutical industry is increasingly adopting multi-metric reporting approaches, with organizations like AstraZeneca utilizing tools like AMGS to trend data as a mode of continuous process verification and internal benchmarking [40].

The interpretation of divergent results from different greenness metrics requires a sophisticated understanding of each tool's underlying principles, assessment criteria, and inherent limitations. Rather than representing methodological inconsistencies, these divergences reflect the multi-faceted nature of environmental impact assessment and the legitimate differences in prioritization between various metric frameworks.

The Analytical Method Greenness Score (AMGS) contributes significantly to this landscape through its specific incorporation of energy consumption metrics alongside solvent and waste assessments, providing a particularly relevant tool for chromatography-intensive pharmaceutical analysis [40] [17]. However, no single metric currently captures all dimensions of methodological environmental impact, necessitating a complementary multi-metric approach.

Researchers should select assessment tools based on their specific analytical context, with simpler metrics like NEMI providing rapid screening for obvious environmental concerns, while comprehensive tools like AMGS, GAPI, and AGREE offer deeper insights for method optimization. The strategic interpretation of divergent results enables more nuanced sustainability assessments, ultimately supporting the pharmaceutical industry's progress toward greener analytical practices that maintain methodological efficacy while reducing environmental footprints. As green chemistry continues to evolve, the ongoing refinement of assessment metrics and the development of standardized interpretive frameworks will further enhance our ability to make meaningful comparisons between analytical methods and drive continuous improvement in environmental sustainability.

The drive toward sustainable practices in the pharmaceutical industry has extended into the realm of analytical chemistry, necessitating robust tools to quantify the environmental impact of analytical methods. Greenness metrics provide a standardized approach to evaluate and improve the sustainability of techniques like chromatography, which, despite operating on a small scale, generate significant cumulative waste [1]. Several metrics have been developed, each with a unique approach to assessment.

The Analytical Method Greenness Score (AMGS) is a comprehensive metric developed by the American Chemical Society's Green Chemistry Institute in collaboration with industry partners. It is designed to provide a holistic evaluation of the environmental impact of chromatographic methods [1]. This guide provides a detailed comparison of AMGS with other established greenness metrics, supporting informed selection for researchers and drug development professionals.

Comparative Analysis of Greenness Metrics

The following table summarizes the core characteristics, strengths, and limitations of key greenness assessment tools.

Table 1: Comparison of Green Analytical Chemistry Assessment Metrics

Metric	Core Approach & Output	Key Advantages	Inherent Limitations & Shortcomings
Analytical Method Greenness Score (AMGS)	A multi-dimensional metric evaluating solvent energy, solvent EHS (Environment, Health, and Safety), and instrument energy consumption [1].	Provides a holistic, chromatographically-focused evaluation [1].Uniquely incorporates instrument energy consumption into the score [1].Enables direct, quantitative comparison and trending of method improvements over time.	Has constraints, such as not accounting for the impact of mobile phase and sample diluent additives [1].Primarily designed for chromatography; applicability to other techniques is less defined.
AGREE (Analytical GREEnness)	A comprehensive assessment based on 12 principles of green chemistry, outputting a score from 0-1 and a visual radar chart [1].	Offers a broad, multi-principle foundation [1].Visual, easily interpretable output facilitates communication [1].An online calculator is available to support users [1].	The assessment model remains relatively coarse for chromatography-specific parameters [1].Does not distinctly weight factors like instrument energy consumption.
GAPI (Green Analytical Procedure Index)	A pictogram with five pentagrams color-coding the environmental impact of each stage of an analytical procedure [1].	Provides a detailed, visual breakdown of each analytical step [1].Useful for identifying specific areas within a procedure that require improvement.	The output is primarily qualitative and semi-quantitative, making it less suited for tracking incremental improvements.
Analytical Eco-Scale	A semi-quantitative tool assigning penalty points for reagents, energy, and waste; a higher final score (from 100) indicates a greener method [1].	Simple and straightforward to calculate and interpret [1].Effectively identifies specific areas for improvement to enhance method greenness.	Its semi-quantitative nature can limit precision and granularity for comparative analysis.

Experimental Protocols for Metric Application

Case Study: Applying AMGS in Pharmaceutical Development

A documented protocol from AstraZeneca illustrates the practical application of the AMGS metric for assessing and improving the sustainability of chromatographic methods within a drug development portfolio [1].

1. Objective: To evaluate the current environmental impact of the chromatographic methods in use and identify opportunities for developing more sustainable alternatives.

2. Methodology:

• Portfolio Assessment: The AMGS tool is applied to all chromatographic methods across the drug project portfolio. The score is calculated based on the instrument's energy consumption and the solvents used, considering their energy of production, disposal, and EHS profiles [1].
• Data Analysis and Trending: The calculated AMGS scores are analyzed at departmental, drug project, and individual method levels. Internal software tools are used to trend this data, creating a baseline and tracking progress as a form of continuous process verification [1].
• Identification and Implementation of Improvements: Methods with poor sustainability credentials are targeted for redevelopment. General strategies to reduce the AMGS score include:
  • Switching to solvents with greener EHS and energy profiles.
  • Reducing overall solvent consumption by optimizing runtime or flow rates.
  • Minimizing instrument energy usage [1].

3. Outcome: This systematic protocol allows an organization to move from a theoretical understanding of green chemistry to a strategic, data-driven implementation, ultimately reducing the environmental footprint of its analytical activities.

Workflow for a Comparative Metric Evaluation

Researchers comparing different metrics can follow this general experimental workflow:

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 2: Key Reagents and Materials for Green Chromatographic Analysis

Item	Function & Role in Green Chemistry
LC-MS Grade Solvents	High-purity solvents (e.g., Acetonitrile, Methanol) are essential for mobile phase preparation. Selecting solvents with greener EHS and energy profiles is a primary lever for improving a method's AMGS [1].
Superficially Porous Particle (SPP) Columns	These chromatography columns offer high efficiency and allow for faster separations or lower backpressures, potentially reducing runtime and solvent consumption.
Column Heater/Oven	A precise thermostat is critical for method robustness and transfer. Optimizing temperature can help reduce runtime, thereby saving energy and solvent.
HPLC/UHPLC System	The instrument platform itself is a direct source of energy consumption and solvent waste. Modern UHPLC systems often operate at higher pressures, enabling faster, more solvent-efficient separations.
Waste Collection Containers	Proper containment and disposal of solvent waste is a fundamental requirement. The volume and toxicity of this waste are direct inputs for greenness calculations [1].

The choice of a greenness metric is not one-size-fits-all but should be guided by specific research goals. The AMGS metric excels in environments where a detailed, quantitative, and chromatography-specific understanding of environmental impact—particularly regarding solvent lifecycle and instrument energy—is required for strategic decision-making and continuous improvement. Its primary strength lies in its holistic and granular nature.

In contrast, AGREE is highly valuable for a broad, principle-based overview and communication, while GAPI and the Analytical Eco-Scale are useful for rapid visual and semi-quantitative assessments, respectively. A comprehensive sustainability strategy may leverage the strengths of multiple metrics, using AMGS for deep, quantitative optimization of key methods and other tools for screening or communication purposes.

The pharmaceutical industry, a significant generator of hazardous waste, faces increasing pressure to adopt sustainable practices, driving the rapid emergence of Green Analytical Chemistry (GAC) principles [59]. In analytical chemistry, particularly for pharmaceutical analysis, this has triggered the development of multiple greenness assessment metrics to evaluate and minimize the environmental impact of methods like High-Performance Liquid Chromatography (HPLC) and Thin-Layer Chromatography (TLC) [10] [56]. However, this proliferation of tools creates a fundamental reproducibility challenge: the subjective interpretation of metric criteria can significantly influence a method's perceived greenness, complicating direct comparisons and reliable sustainability claims.

Framed within broader research comparing the Analytical Method Greenness Score (AMGS) with other metrics, this guide examines the core problem of subjectivity. We objectively compare metric performance and provide experimental data demonstrating how inconsistent application affects reliability, offering scientists a clearer path toward reproducible green assessments.

Understanding the Metric Landscape: From Green to White Analytical Chemistry

A comprehensive understanding requires categorizing available tools based on their assessment focus.

Table 1: Categories of Greenness and Complementary Assessment Metrics

Category	Metric/Acronym	Full Name	Primary Focus
Greenness-Focused	NEMI	National Environmental Method Index [10]	Environmental impact (hazard, waste)
	AES	Analytical Eco-Scale [10]	Penalty points for un-green aspects
	GAPI	Green Analytical Procedure Index [10] [11]	Holistic pictogram for entire method
	AGREE	Analytical GREEnness Metric [10] [11]	Scores all 12 GAC principles; pictogram
	AGREEprep	AGREE for Sample Preparation [60] [11]	Scores 10 sample prep principles
	AMGS	Analytical Method Greenness Score [58] [56]	Quantitative greenness score
Performance & Practicality	RAPI	Red Analytical Performance Index [6]	Analytical performance (precision, sensitivity, etc.)
	BAGI	Blue Applicability Grade Index [6] [61]	Practicality & economic aspects
Holistic Models	WAC	White Analytical Chemistry [6]	Overall balance of Green, Red, and Blue criteria
	RGB	Red-Green-Blue Model [60] [61]	Visual assessment combining all three aspects

The movement towards White Analytical Chemistry (WAC) acknowledges that a method's sustainability is not just about its environmental footprint (Green) but also its analytical performance (Red) and practicality and cost-effectiveness (Blue) [6]. A "whiter" method achieves a better balance among these three attributes. The recent introduction of the Red Analytical Performance Index (RAPI) specifically addresses the "red" criteria by providing a standardized tool to score key analytical performance parameters, helping to objectify this aspect of assessment [6].

Diagram 1: The WAC framework balances Green, Red, and Blue attributes, each measured by specific tools.

The Core Challenge: Subjectivity and Its Impact on Reproducibility

Despite standardized criteria, significant subjectivity arises during metric application, affecting scores and compromising comparability.

Case Study: HPTLC Analysis of Ertugliflozin

A 2024 study directly compared Normal-Phase (NP) and Reversed-Phase (RP) HPTLC methods, assessing both with four greenness tools [10]. While all tools consistently agreed the RP method (using ethanol-water) was greener than the NP method (using chloroform-methanol), the degree of difference and the absolute score for each method varied between tools. This demonstrates that while trends can be reliable, the final quantitative output is metric-dependent. The study's authors made interpretive decisions, such as how to account for waste volume and solvent toxicity, which directly influenced the final scores.

Variable Outputs for an FT-IR Method

A green FT-IR method for quantifying antihypertensive drugs was assessed with three tools, yielding different but complementary scores: a MoGAPI score of 89, an AGREEprep score of 0.8, and an RGB model score of 87.2 [60]. Each score reflects the tool's specific focus and weighting. AGREEprep's 0.8 score (on a 0-1 scale) highlights the minimal, solventless sample preparation, while the MoGAPI score of 89 reflects the greenness of the entire analytical procedure. This variability is not necessarily a flaw but underscores the need to understand what each metric is measuring.

Comparative Experimental Data: A Quantitative Look at Metric Scores

The following tables synthesize experimental data from published studies, providing a direct comparison of how different metrics evaluate the same analytical methods.

Table 2: Greenness Metric Scores from Experimental Case Studies

Analytical Method & Target	Key Green Feature	AGREE	AGREEprep	AES	GAPI/ MoGAPI	NEMI	Ref.
RP-HPTLC (Ertugliflozin)	Ethanol-water mobile phase	Reported	N/A	Reported	N/A	Reported	[10]
FT-IR (Amlodipine/Telmisartan)	Solventless, minimal waste	N/A	0.8	N/A	89 (MoGAPI)	N/A	[60]
1H-qNMR (Aspirin/Omeprazole)	Non-destructive, no derivatization	Used in study	N/A	Used in study	Used in study	Used in study	[61]
AQbD-HPLC (Irbesartan)	Ethanol-based mobile phase	0.75	N/A	N/A	N/A	N/A	[56]

Table 3: Illustrative WAC Assessment of a Green 1H-qNMR Method Data derived from a 2025 study on aspirin and omeprazole analysis [61].

Assessment Dimension	Tool Used	Score/Result	Rationale Based on Experimental Protocol
Greenness	AGREE, GAPI, AES	High Scores	No organic solvent waste; minimal sample preparation; low energy consumption per sample (1.0 kWh).
Analytical Performance (Red)	RAPI (Principles)	High	Validation showed high linearity (r=0.9999), precision (%RSD < 1.08), accuracy, and low LOD/LOQ (~0.01/0.03 mg mL⁻¹).
Practicality (Blue)	BAGI (Principles)	Moderate to High	No labeling/pretreatment needed; fast analysis; but cost of deuterated solvent (DMSO-d₆) and NMR instrumentation is high.
Overall Whiteness	RGB/WAC Concept	High	The method achieves an excellent balance of being eco-friendly, analytically robust, and practically feasible for its intended use.

The Scientist's Toolkit: Essential Reagents and Materials for Green Assessment

Beyond the analytical methods themselves, conducting a robust greenness assessment requires a "toolkit" of conceptual resources and software.

Table 4: Essential Research Reagent Solutions for Green Metric Evaluation

Tool/Reagent	Function in Green Assessment	Example Use Case
AGREE & AGREEprep Software	Open-source calculators providing a pictogram and overall score for the entire method (AGREE) or sample prep (AGREEprep).	Evaluating a microextraction-HPLC method for UV filters in cosmetics [11].
BAGI Software	Open-source tool for assessing the practicality and economic feasibility ("blueness") of an analytical method.	Complementing greenness scores to give a full WAC picture of a 1H-qNMR method [6] [61].
RAPI Methodology	Framework for scoring the "redness" or analytical performance (e.g., precision, sensitivity, linearity) of a method.	Objectively comparing the validation parameters of two HPLC methods claiming to be green [6].
Solvent Selection Guide	A guide to substitute hazardous solvents (e.g., acetonitrile, chloroform) with safer alternatives (e.g., ethanol, water).	Justifying the higher greenness score of RP-HPTLC (ethanol-water) over NP-HPTLC (chloroform-methanol) [10] [56].
White Analytical Chemistry (WAC) Framework	A conceptual model ensuring a balanced evaluation of greenness, analytical performance, and practicality.	Explaining why a very green but impractical or inaccurate method is not ideal for routine use [6].

The reproducibility challenge in green metric assessment is non-trivial. Subjective interpretation of criteria can lead to inconsistent scores, potentially undermining the credibility of sustainability claims in pharmaceutical analysis. This comparison demonstrates that no single metric provides a perfect, holistic assessment.

The most robust strategy for evaluating an analytical method's sustainability is the triangulation of results from multiple tools, such as using AGREE for general greenness, AGREEprep for sample preparation, RAPI for analytical performance, and BAGI for practicality [6] [11]. This multi-faceted approach, guided by the White Analytical Chemistry framework, provides a more reproducible and reliable foundation for comparing methods and advancing truly sustainable scientific practices. Future work should focus on developing more standardized weighting for metric criteria and integrated software that minimizes subjective user input, further enhancing the reliability and comparability of greenness assessments.

Conclusion

The journey towards sustainable analytical practices is underpinned by a robust and evolving toolkit of greenness metrics. While AMGS stands out for its chromatographic specificity, integration of cumulative energy demand, and strong industry adoption, no single metric is a panacea. The future lies in a nuanced, multi-metric approach that leverages the unique strengths of tools like AMGS, AGREE, and GAPI based on the analytical context. For biomedical and clinical research, this translates to developing greener, more efficient quality control methods, reducing the environmental footprint of drug development, and aligning with broader corporate and regulatory sustainability goals. Future directions will likely involve the incorporation of machine learning for predictive greenness, the expansion of metrics to techniques like Gas Chromatography, and a stronger focus on standardizing weighting practices to enhance objectivity and comparability across the scientific community.

References

• 1. Utilisation of Analytical Method Greenness Score to drive ... [https://pubs.rsc.org/pt-br/content/articlehtml/2025/gc/d5gc01574j]
• 2. About the AMGS Calculator [https://acsgcipr.org/tools/about-the-amgs-calculator/]
• 3. Analytical greenness metrics for metabolomics [https://pubmed.ncbi.nlm.nih.gov/40830591/]
• 4. Greenness evaluation metric for analytical methods and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12318332/]
• 5. Green and white analytical approach for parallel ... [https://www.nature.com/articles/s41598-025-07056-9]
• 6. Red analytical performance index (RAPI) and software [https://pubs.rsc.org/en/content/articlehtml/2025/gc/d4gc05298f]
• 7. How to correctly evaluate greenness, whiteness and other ... [https://pubs.rsc.org/en/content/articlehtml/2025/gc/d5gc00615e]
• 8. HPLC 2025 Preview: The Road To Sustainable Analytical ... [https://www.chromatographyonline.com/view/hplc-2025-preview-the-road-to-sustainable-analytical-chemistry]
• 9. Green analytical chemistry metrics for evaluating the ... [https://www.sciencedirect.com/science/article/pii/S2095177924001102]
• 10. Comparing the Greenness and Validation Metrics of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11137692/]
• 11. Comparison of the Greenness Assessment ... [https://www.mdpi.com/2673-4532/4/4/32]
• 12. Green metrics and green analytical applications [https://www.sciencedirect.com/science/article/pii/S2772577424000685]
• 13. About – ACSGCIPR [https://acsgcipr.org/about/]
• 14. ACS Green Chemistry Institute Pharmaceutical Roundtable ... [https://www.nesacs.org/acs-green-chemistry-grants/]
• 15. A Perspective on Current and Future Metric Tools - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC12590457/]
• 16. Green analytical chemistry metrics for evaluating the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11697060/]
• 17. Analytical Method Greenness Score (AMGS) Calculator [https://chemistryforsustainability.org/tools-metrics/analytical-method-greenness-score-amgs-calculator]
• 18. Greenness metrics for analytical methodologies linked to ... [https://www.sciencedirect.com/science/article/pii/S2772577424000491]
• 19. Overview of sixteen green analytical chemistry metrics for ... [https://www.sciencedirect.com/science/article/abs/pii/S0165993623002984]
• 20. Tools & Metrics | Chemistry For Sustainability [https://chemistryforsustainability.org/tools]
• 21. Making the move towards modernized greener separations... | CoLab [https://colab.ws/articles/10.1039%2Fc8gc03875a]
• 22. Expanding the Green Chemistry Toolkit: ACS GCIPR I... - ACS... [https://communities.acs.org/t5/GCI-Nexus-Blog/Expanding-the-Green-Chemistry-Toolkit-ACS-GCIPR-Invests-in-Three/ba-p/98584]
• 23. An Inside Look at the Analytical Method Greenness Score in High... [https://www.chromatographyonline.com/view/an-inside-look-at-the-analytical-method-greenness-score-in-high-performance-liquid-chromatography-enantioseparations-an-earth-day-interview-with-daniel-armstrong]
• 24. Greenness evaluation metric for analytical methods and ... [https://www.sciencedirect.com/science/article/pii/S209517792500019X]
• 25. Tools – ACSGCIPR [https://acsgcipr.org/tools/]
• 26. Evaluating the greenness of wine analytical chemistry [https://ives-openscience.eu/53211/]
• 27. Modified GAPI (MoGAPI) Tool and Software for the Assessment of... [https://www.mdpi.com/2673-4532/5/3/30]
• 28. a selective Janus kinase (JAK) inhibitor | Discover Chemistry [https://link.springer.com/article/10.1007/s44371-025-00310-z]
• 29. Separation of 15 impurities in semi-synthetic paclitaxel in a ... [https://pubmed.ncbi.nlm.nih.gov/40752188/]
• 30. Green Extraction Tree: A Novel Tool to Greenness Metric ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12593995/]
• 31. Improving Throughput and Analytical Greenness in ... [https://www.waters.com/nextgen/us/en/library/application-notes/2025/improving-throughput-and-analytical-greenness-in-pharmaceutical-discovery-using-ultrashort-hplc-columns.html?srsltid=AfmBOoq8P3El6TzbJvSnfct1QGIbF-PVOIaGE2clylY7kGEjTO0EX2sZ]
• 32. AGREE—Analytical GREEnness Metric Approach and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7588019/]
• 33. Ultra-short columns for the chromatographic analysis of ... [https://www.sciencedirect.com/science/article/pii/S0021967323005101]
• 34. Comparison Techniques for HPLC Column Performance [https://www.chromatographyonline.com/view/comparison-techniques-hplc-column-performance]
• 35. Evaluation of ultrahigh-performance liquid chromatography ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4206774/]
• 36. Green Approaches in High-Performance Liquid ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12430196/]
• 37. Assessing the Environmental Impact of Analytical Methods [https://www.chromatographyonline.com/view/the-green-component-assessing-the-environmental-impact-of-analytical-methods]
• 38. A Sustainable Approach to Green Sample Preparation and ... [https://www.chromatographyonline.com/view/a-sustainable-approach-to-green-sample-preparation-and-analytical-performance-using-hs-spme-gc-qtof-ms]
• 39. HPLC and GC: 6 Simple Differences to Enhance Your ... [https://bitesizebio.com/29109/run-fly-comparison-hplc-gc/]
• 40. Utilisation of Analytical Method Greenness Score to drive ... [https://pubs.rsc.org/en/content/articlelanding/2025/gc/d5gc01574j]
• 41. Comparison of greenness and whiteness of selected ... [https://pubs.rsc.org/en/content/articlehtml/2025/gc/d4gc05097e]
• 42. Applying Analytical Method Greenness Scoring to the USP ... [https://www.waters.com/nextgen/us/en/library/application-notes/2024/applying-analytical-method-greenness-scoring-to-the-usp-monograph-of-naproxen-improving-the-sustainability-of-validated-methods-by-modernizing-to-newer-technology.html?srsltid=AfmBOoqTOBPoeBgVpBuQp4yKhPhJ7pB5_keJARTSmT2D-5lj9Jsh5ih6]
• 43. Green solvents in membrane separation [https://pubs.rsc.org/en/content/articlelanding/2025/gc/d5gc03161c]
• 44. The development of a novel, green, efficient, and eco ... [https://www.sciencedirect.com/science/article/abs/pii/S0039914024015595]
• 45. Recent application of green analytical chemistry: eco-friendly ... [https://fjps.springeropen.com/articles/10.1186/s43094-024-00658-6]
• 46. Analytical Green Star Area (AGSA) as a new tool to assess ... [https://www.sciencedirect.com/science/article/pii/S2352554125001494]
• 47. Green metric tools for analytical methods assessment ... [https://www.sciencedirect.com/science/article/abs/pii/S0165993623002832]
• 48. Guidelines on the proper selection of greenness and ... [https://www.sciencedirect.com/science/article/abs/pii/S0003267025004465]
• 49. Evaluation of analytical greenness metric for an eco-friendly ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11365310/]
• 50. Emerging Tools for Holistic Evaluation of Analytical Methods [https://www.chromatographyonline.com/view/emerging-tools-for-holistic-evaluation-of-analytical-methods]
• 51. MA tool—Multi-color assessment platform for white ... [https://www.sciencedirect.com/science/article/abs/pii/S0026265X25021290]
• 52. On the greenness of separation modes containing compressed fluids [https://pubmed.ncbi.nlm.nih.gov/39489969/]
• 53. Analytical chemistry in the era of sustainability: evaluating ... [https://www.sciencedirect.com/science/article/pii/S2214158825000182]
• 54. Quality by design approach for green HPLC method ... - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC9084420/]
• 55. Sustainability assessment in food analysis [https://www.explorationpub.com/Journals/eff/Article/101056]
• 56. Eco-Friendly AQbD-Driven HPLC: A Sustainable Approach ... [https://jyoungpharm.org/10.5530/jyp.20250087]
• 57. Greenness-sustainability metrics for assessment smart- ... [https://pubmed.ncbi.nlm.nih.gov/38577411/]
• 58. A case study of a multipurpose novel janus kinase inhibitor [https://www.sciencedirect.com/science/article/pii/S2772577425000072]
• 59. Embracing Sustainable Processes in the Pharmaceutical ... [https://scifiniti.com/3079-1421/1/2025.0013]
• 60. Green vibrational spectroscopic approach for simultaneous ... [https://www.nature.com/articles/s41598-025-97485-3]
• 61. Greenness, blueness, and whiteness evaluation of a ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12053856/]