Implementation of green analytical chemistry in the quality control laboratory of the company UPSA

The Ministry for the Economy, Finance and the Recovery describes Corporate Social Responsibility (CSR) as the “the contribution of companies to the challenges of sustainable development” targeting in particular working conditions and the environment.

Well before this recent concept of CSR, the UPSA Quality Control laboratory introduced the concept of green analytical chemistry in 2004, in order to prevent or reduce pollution at its source.

The Quality Control Laboratory drew inspiration from the principles of green chemistry (1) in order to develop new analytical methods that minimize risk and maximize efficiency.

The emphasis was thus placed on eliminating toxic organic solvents and phosphate, and on reducing the quantity of organic solvents used, water consumption and energy use.

1. Design of green analytical chemistry for the UPSA laboratories

A study was conducted of 3 alcoholic solutions that are representative of products used in the field: a sterile 70% ethanol solution, a sterile 70% isopropyl alcohol solution and a pure 99.6% isopropyl alcohol solution. The analyses were conducted with a Sievers M9 Portable TOC Analyzer equipped with a sample feeder (Suez). The vials used for sample preparation were vials treated specifically for TOC Analyzer use, certified as having a low TOC content.


2. Why change the analysis methods?

Up until then, the analytical methods applied in the laboratory stipulated the use of numerous organic solvents such as acetonitrile, methanol, toluene, hexane and chloroform, etc. both for sample preparation and for HPLC (High Performance Liquid Chromatography) or TLC (Thin Layer Chromatography) mobile phases, for example, which led to high levels of consumption.

However, all these organic solvents present hazards. Is their use actually unavoidable or are they just a habit among chemists who develop analysis methods?

Consequently, the use of such toxic reagents implies the need for laboratory fume hoods that consume energy. This collective protection equipment is nevertheless to be considered a palliative measure and therefore insufficient in the spirit of green chemistry, where sources of contamination are to be considered.

The indiscriminate use of this type of reagent raises a question in terms of the protection of persons and of the environment, of the preservation of resources, and of traces left by waste for future generations. So, in 2004, the challenge of making analysis methods compliant with Environmental, Health and Safety principles was taken up.

An analysis method transformation phase was initiated, revolving around three distinct steps:

  1. Formulation of 10 golden rules of development.
  2. Development and validation of methods following these rules, in agreement with the applicable ICH frames of reference.
  3. Filing of analytical method variations in Marketing Authorisation dossiers with the Health Authorities.

2.1. The 10 golden rules for developing a green analytical method

The development of a green analytical chemistry method requires principles to be defined to apply to the method.

On the basis of the principles of green chemistry,(1) well before the appearance of publications on green analytical chemistry,(2, 3, 4) 10 golden rules were defined in order to guide this transition.

  1. Reduction of waste

It is better to avoid producing waste than to then have to process it or get rid of it

  1. Atom savings

Implementation of analysis methods which reduce or eliminate consumption of atoms (reagents, solvent, materials, consumables, etc.).

  1. No or fewer hazardous chemical products

Everything must be done so that an analysis method uses and produces substances that present little or no toxicity to humans and the environment.

  1. Safer solvents and auxiliary substances

Renouncing the use of synthetic auxiliary substances (solvents, separation agents, etc.) or choosing harmless auxiliary substances when they are necessary.

  1. Preserved functional efficacy

The analytical method preserves functional efficacy while reducing toxicity.

  1. Design of energy efficiency

The energy expenditure necessary for the implementation of an analytical method must be examined from the standpoint of its impact on the environment and efficiency, and be reduced to the minimum. As far as possible, operations must be carried out under ambient temperature and pressure conditions.

  1. The use of existing apparatus and renewable resources

The energy expenditure necessary for the replacement of an apparatus or an existing technology should be examined from the standpoint of its impact on the environment and efficiency.

Energy expenditure should include: destruction of existing equipment and the manufacture of the replacement apparatus, the robustness of the new equipment in comparison with the existing equipment (wear of spare parts, nature of composite materials used, consumables, electricity consumption, information technology, etc.).

Using a natural resource or a renewable raw material rather than fossil or synthetic products, as far as technique and efficiency allow.

  1. Reduction of derivatives or extractions

Making every effort to eliminate or reduce the use of extraction processes using organic solvents in sample preparation to a minimum.

Separation and purification operations should be avoided in order to minimize energy consumption and the production of waste.

  1. Design for degradation

The chemical products used in analytical chemistry should be able to decompose into harmless biodegradable waste. They must not linger in the environment.

  1. Safer analytical chemistry for the prevention of accidents at work / occupational diseases

The chemical substances and materials used in a chemical analysis process should be chosen so as to prevent accidents such as hazardous emissions, explosions and fires, chemical risk (non-use of toxic, carcinogenic, mutagenic or reprotoxic compounds), musculoskeletal disorders.

  • Development of a green analytical method for HPLC

The development of a “green” HPLC method revolves around the following:

Establishment of the criteria of the “green” HPLC method

*The criteria for a green HPLC method were the elimination of toxic organic solvents and phosphate, the reduction of energy consumption, the reduction of the quantity of organic solvents used, the replacement of organic solvents by aqueous solvents (if possible) and the reduction of water consumption.

Choice of technology

Research and development

  • Bibliography

– Chemical structure and physicochemical properties of the compounds to be analysed

– Bibliography of existing analytical methods for a given compound (e.g.: paracetamol)

  • Modelling of a potential method

– Integration of the sampling plan

– Preparation of samples for analysis

– Choice of the analytical method (HPLC, Gas phase chromatography, UV spectrometry)

– Choice of the mobile phase (HPLC)

– Choice of column (HPLC)

– Choice of detector

  • Development

– Application of the model

– Optimization

  • Validation and report

2.3. Results

Two approaches were implemented in the UPSA Quality Control laboratory to transition to green analytical chemistry.

The first approach was to reduce the number of HPLC systems and the number of methods used in the laboratory (for a constant analysis volume) by developing methods with column couplings and/or UV dual detection with different optical paths. Column coupling allowed the development of methods that reduce two analysis methods into just one, thus decreasing occupancy time and the number of HPLC systems.

The second approach was to work with solutions with a high concentration of analytes in order to reduce the volumes of test solutions and to be able to analyse samples in accordance with sampling plans. In order to avoid saturating the detectors, volumes in the order of 1/10 μL were injected for standard and test solutions using internal standards to correct variations caused by the very small injection volumes. In addition, cells with a 3 mm optical path length were used. The development of the green HPLC method required the elimination of phosphates and toxic organic solvents (methanol and acetonitrile) from the mobile phases. The phosphates were replaced principally by succinate, formiate or acetate.

In addition, there was a significant reduction in the proportion of organic solvents in the mobile phases. These contain percentages of organic solvents of between 0.8 % and 30 % (more often close to 3 %) versus 30 % to 85 % in the previous mobile phases.

The preparation of test solutions also incorporated the principles of green analytical chemistry. Organic solvents (methanol, acetonitrile, hexane, toluene, chloroform) were replaced by aqueous solutions for the preparation of test solutions. Solutions chemistry principles were implemented to solubilize the samples by exploiting the conditional solubilities of analytes (example: pH-dependent solubility). In some cases, a small quantity of ethanol was added to aid solubilization of analytes.

In order to comply with point 7 of the 10 principles of green analytical chemistry, the option chosen by the laboratory was to conduct development with the existing HPLC devices. UHPLC (Ultra High Performance Liquid Chromatography) was not chosen.

Methods were developed with short columns to reduce analysis time and because of the robustness of HPLC columns compared to those of UHPLC.


I. Method using column couplings and/or dual detection

Column coupling is the connection of columns in series. The principle is to couple columns with different stationary phases allowing separation of analytes by different chromatographic techniques.

In the following examples, couplings of reversed phase chromatography columns (C1, C8, C18 …) with an ion exchange chromatography column (NH2) allow reversed phase chromatography and ion exchange chromatography to be performed in series.

Chimie Analytique : Figure 1
Figure 1 : couplage colonne

I.1. Assay of acetylsalicylic, salicylic and ascorbic acids in effervescent tablets

Two short columns (50 mm x 3 mm) have been coupled in series. The first column is a C1 column (reversed phase) and the second is an NH2 column (ion chromatography).

Chimie Analytique : Figure 2
Figure 2 : interactions polaires et apolaires couplage colonne/phase mobile/ analytes

The mobile phase is a mixture of ethanol and 0.05 M sodium succinate buffer pH=5.5 (6 % / 94 % v/v). Succinic acid (natural compound) allows a more rapid elution of acids retained by the NH2 column. The HPLC system is fitted with two detectors (DAD: Diode-Array Detection) in series with different optical paths. The first detector with a 3 mm optical path allows quantification of ascorbic acid and acetylsalicylic acid at λ=266 nm and the second with a 60 mm optical path searches for degradation products such as salicylic acid at λ=296 nm.

These three analytes are quantified in the same 7 minute run. The sample is prepared in an ethanol / 0.1 M HCl mixture (5% / 95 %: v/v).

Chimie Analytique : Figure 3


This method replaced two HPLC methods one assaying ascorbic acid the other assaying acetylsalicylic acid and salicylic acid. The characteristics of the analysis are detailed below:

Chimie Analytique : Tableau 1



The benefits of the development of a “green” analytical method are multiple: a number of methods with a direct effect on the pool of HPLC systems required, analysis time and above all, elimination of chemical reagents harmful to humans and the environment.

I. 2. Assay of paracetamol, pheniramine and ascorbic acid in powders in sachets.

In this context, two columns have been coupled in series. The first column is a C18 column (150 mm x 4.6 mm) and the second is a NH2 column (50 mm x 4.6 mm). The mobile phase is a mixture of ethanol and a 0.05M sodium succinate buffer pH=5.5 (19 % /81 % v/v).

The HPLC system is fitted with two detectors (DAD) in series with different optical paths (3 mm for quantification of paracetamol and ascorbic acid – 60 mm for quantification of pheniramine). The three analytes are quantified in the same run (13 minutes). The sample is prepared in purified water.

Chimie Analytique : Figure 4
Figure 3: Chromatogramme – paracétamol, acide ascorbique et phéniramine.

The benefits so obtained are identical to those obtained in the previous example.

I.3. Analysis method using an internal standard

– Assay of ascorbic acid in effervescent tablets.

This method was developed to maximally reduce consumption of the solvent used to prepare the solution for analysis. The sample is prepared in purified water containing acetylcysteine as an antioxidant.

The final ascorbic acid concentration of sample solutions is 20 mg/mL. Such a concentration of ascorbic acid saturates classic DAD detectors which have an optical path of 10 mm for injection volumes of a few μL. The use of an internal standard (potassium acesulfame) makes it possible to be free of variations associated with injection volumes in the order of 1/10 μL. The column used in this case is an NH2 column (50 mm x 3 mm). The mobile phase is a mixture of ethanol and a 0.05M sodium acetate /0.05M acetic acid buffer (10 % / 90 % v/v). The analysis time by HPLC is 14 minutes. The injection volume is 0.1 μL.

Chimie Analytique : Figure 4
Figure 4: Chromatogramme – dosage acide ascorbique

The benefit in this context is represented by the use of smaller volume laboratory glassware, with reduced purified water consumption as a direct consequence; the long-term handling of smaller containers reduces the risk of the appearance of musculoskeletal disorders.

I.4. How do these new methods developed in the Control Laboratory comply with the 10 principles of green analytical chemistry?

The methods developed met the initially established rules of green analytical chemistry:

  • Reduction of waste: less mobile phase, consumption of smaller volumes of test solutions, etc.). The new HPLC methods made reduced analysis time possible by using short columns or couplings of short columns. Reduction of solvent volumes (water or organic solvents) for preparation of the sample: use of internal standards allowing the injection of small volumes (0.1 μL to 0.4 μL). Detector coupling allowing analysis methods to be paired within one method.
  • Atom consumption reductions: significant drop in solvent consumption, less consumption by HPLC columns). Implementation of analysis methods with reduction of organic solvents used in mobile phases and replacement of organic solvents with aqueous solvents in sample preparations.
Chimie Analytique : Tableau 1
Evolution consommation solvants pour analyses HPLC – Laboratoire Contrôle Qualité
  • No or fewer hazardous chemical products

Elimination of toxic organic solvents (methanol, acetonitrile, hexane, toluene, chloroform, etc.)

  • Safer solvents and auxiliary substances

Water, ethanol and isopropyl alcohol have replaced methanol, acetonitrile, hexane, toluene, chloroform, etc.

  • Designing to preserve functional efficacy

The alternative methods are more robust that those replaced while increasing precision, repeatability and accuracy.

  • Design of energy efficiency

.By using one HPLC system with two detectors to replace two HPLC systems.

.By reducing the analysis time by HPLC.

.By having robust methods in terms of HPLC column consumption.

.By developing analysis methods that do not require laboratory hoods for the preparation of samples for analyses.

.By developing HPLC methods at ambient temperature (HPLC column and preparation of samples).

  • Use of existing apparatus and renewable resources

The development was carried out with existing equipment (use of existing HPLC systems rather than a UHPLC system).

Durability of materials through the choice of small HPLC columns that are more robust than UHPLC columns (comparative tests carried out in the control laboratory: durability of HPLC columns vs UHPLC columns: 5 to 50 times more injections for HPLC columns). Water and ethanol (natural resource or renewable raw material) replaced the acetonitrile and methanol that are very widely used in HPLC, as the latter are derived from chemical synthesis. Acetonitrile, a by-product of the production of acrylonitrile, itself derived from petrochemistry, is not a renewable resource. Methanol is produced industrially as it is not present in significant quantities in nature.

  • Reducing derivatives and extractions

Separation and purification operations were avoided in order to minimize energy consumption and production of waste.

  • Design for degradation

Aqueous solvents have replaced methanol, acetonitrile, toluene, chloroform and ether for the preparation of samples for analyses. Sample solutions are for the most part prepared in water. Solubilization of analytes was mainly by acting on the pH. When this was necessary, ethanol was added to the aqueous phases to increase the solubilization of certain compounds. The phosphate buffers for mobile phases have been replaced by acetate, formiate or succinate (natural, non-toxic organic compounds).

  • Safer analytical chemistry for the prevention of accidents

Preparation using a weight-based method has replaced the volumetric method. Preparations are thus carried out in erlenmeyers replacing flasks, at the same time eliminating manual agitation. The non-use of toxic, carcinogenic, mutagenic, reprotoxic compounds through the replacement of the solvents methanol*, acetonitrile*, toluene* and chloroform* by aqueous solvents, to which ethanol can be added if needed.

*Methanol classified as toxic with proven risk of serious effects on organs, acetonitrile classified as Category D – Poisonous and infectious materials, 1. Materials causing immediate and serious toxic effects by the Canadian Health Authority, toluene classified as mutagenic, carcinogenic, reprotoxic (CMR), chloroform classified as Toxic and CMR.

Taking account of musculoskeletal disorders in the preparation of samples for analysis (elimination of manual agitation, elimination of grinding tablets in the mortar).


Since 2004, the UPSA quality control laboratory has developed several analysis methods making possible the elimination of toxic organic solvents and phosphate, the use of compounds that do not persist or bioaccumulate in organisms or the environment, the reduction of energy consumption, the reduction of the quantity of organic solvents used, the replacement of organic solvents by an aqueous solvent if possible, the reduction of water consumption, and the use of existing devices.

In total, a dozen methods have been developed for assays of active principles, degradation products and preservatives for more than fifty finished product formulas. All these methods have been filed in the corresponding MA dossiers (marketing authorisation) and applied on approval from the Health Authorities. All these developments were carried out without investment. The only purchases made concern four detectors with different optical lengths. These detectors allow the grouping of analytical methods within a single HPLC system.

This work has allowed the reduction of overall organic solvent consumption (from 1,721 L of organic solvent in 2005 to 468 L in 2016) and has enabled a very significant drop in toxic solvents (example: 1,068 L of methanol in 2005 versus 109 L in 2016).

The number of HPLC systems fell from 22 systems in 2005 to 16 in 2016 then to 12 in 2021 for the analyses of finished products, for an analysis volume that is at least constant and even increasing.

The reduction in the number of HPLC systems has enabled decreases in the consumption of energy and consumables and simplification in terms of maintenance and metrology.

The choice to develop HPLC analytical methods was made because this type of equipment was already present in the laboratory. UHPLC was not selected for several reasons: a problem with the robustness of UHPLC columns, a larger quantity of consumables for the maintenance of systems and saturation of detectors (because of the finer peak) involving additional dilution steps during the preparation of test solutions.

To the question: “Can we develop analytical methods that are mindful of working conditions and mindful of the environment? “, the answer is certainly yes.

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Myriam DESCAZAL – François-Xavier MARTIN – – Mylène RABINEAU – Amandine VISENTIN


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