This article is specifically dedicated to discussing the uses of chromatography. Chromatography is an extremely versatile analytical separation and purification technique that finds multiple uses in scientific research and exploration. Chromatography is defined as a technique that is used to separate a complex chemical mixture into its individual components. It is based on a stationary phase and a mobile phase. The stationary phase is held fixed while the mobile phase is usually a solvent or a gas that moves past the stationary phase carrying analyte molecules with it.
Analytical separation occurs based on the relative affinity of analyte interaction with the stationary phase versus the mobile phase. There are several different types of chromatography. A chromatographic separation could be as simple as paper chromatography. Conversely, it may require sophisticated technology such as that needed for high-performance liquid chromatography (HPLC), gas chromatography (GC),supercritical fluid chromatography (SFC),etc. In either case, there are several different uses of chromatography.
A specific chromatographic technique may be required for a special application. What are these uses and applications and what is their scope in industrial research, similarly how do these impact our everyday lives? Let’s find out all of it through this article.
Qualitative and Quantitative analysis using chromatography
Qualitative analysis refers to separating and identifying the different chemical constituents of a sample mixture. Conversely, quantitative analysis refers to finding the quantity or amount of a chemical constituent in a sample. Chromatography can help accomplish both. Simple chromatographic techniques such as paper chromatography and thin-layer chromatography (TLC) are largely used for qualitative analysis. On the other hand, column chromatographic techniques fascinated with detectors allow both qualitative as well as quantitative analysis.
Many different types of detectors can be coupled with an HPLC instrument such as an ultraviolet (UV) detector. UV detector helps in qualitative and quantitative analysis of UV light absorbing organic compounds. Quantitative analysis using HPLC-UV follows Beer Lambert’s law.
The following step-by-step guide shows how a quantitative analysis using the UV detector is done:
Step I: A standard stock solution is first prepared followed by the preparation of 5-6 different dilutions from this stock solution in the respective solvent.
Step II: The least concentrated solution is scanned through the 195-370 nm range of the electromagnetic spectrum to determine a lambda max (λmax) value at which it absorbs maximum UV light.
Step III: The detector response is then recorded for all concentrations at this λmax value. A standard calibration curve plotting absorbance versus standard concentration is obtained.
Step IV: This straight-line graph is finally used to determine the unknown concentration of an analyte sample whose absorbance is known.
Similarly, GC coupled with a mass spectrometric (MS) detector helps separate and quantify volatile chemical components from a complex sample mixture. In this way, chromatography can be used for quality control and evaluation of foodstuff, pharmaceutical drugs, industrial mixtures, cosmetic products, biological macromolecules, etc.
Uses of chromatography in the food industry
Food testing for quality control is an essential requirement of the food industry.
- Food quality control can be performed by isolating chemicals such as artificial sweeteners, food colors, pesticides, and preservatives from a food sample.
- The shelf-life and expiration date of foodstuff can be determined using chromatography.
- The nutritional value of processed foods can be determined by chromatography which helps detect any chemical additive present in it and its amount.
- Food adulteration by substitution or addition of certain chemical ingredients in dairy products (cheese, milk) and honey can be readily detected using HPLC or GC-MS.
- Trace levels of contaminants present in beverages (cold drinks and juices) and dietary supplements can be detected and purified using highly sensitive chromatographic techniques like UHPLC.
- Allergens and mycotoxins present in animal-based foods (meat) can also be detected through UHPLC thus chromatography is important for food safety.
- The level of sugars, proteins, and vitamins present in a foodstuff can be analyzed to validate the authenticity of the nutritional data given on its packaging.
Uses of chromatography in the fragrance industry
Gas chromatography (GC) holds a particular significance for chromatographic analysis in the fragrance industry.
- Essential oils, ethers, alcohols, and esters make up the basic components of any fragrance such as perfumes, deodorants, and air fresheners. All these components are extremely volatile. Therefore, GC-MS is the most used technique for their analysis.
- Essential oils extracted from fruit peels (such as orange) and plants (marigold, chamomile, geranium, etc.) used in aromatherapy, therapeutic teas, and scented candles are also analyzed via gas chromatography.
Uses of chromatography in the pharmaceutical sector
A major application of chromatography lies in the evaluation of pharmaceutical drugs and medicinal materials.
- Chromatography plays an important role in drug development. Any impurities or unknown compounds present in a drug sample can be detected through HPLC.
- Enantiomers are isomers identical in all aspects except their chirality. But one enantiomer can be used as a medicinal drug while the other can be toxic. Chiral stationary phases can be developed in HPLC or in higher techniques such as convergence chromatography for isolating enantiomeric compounds.
- Precise chromatographic techniques such as hydrophobic interaction chromatography can be used in vaccine development and testing, identifying which antibodies function best against a certain disease. The SARS-COVID 19 spike proteins were also isolated and analyzed via liquid chromatography.
Uses of chromatography in the environmental evaluation
Environmental pollution results in harmful effects on human health. Chromatography facilitates environmental protection by analysis of a wide range of chemical compounds.
- Low concentration of analytes and pollutants present in complex environmental matrices such as air, soil, and water can be detected through high-pressure chromatographic techniques like HPLC and UHPLC.
- The concentration of pollutants such as perfluoroalkyl substances (PFAS) as well as industrial contaminants leaching out in drinking water can be detected through chromatography.
- Trace quantities of dangerous pesticides such as DDT (dichloro-diphenyl-trichloroethane) that become a part of groundwater near agricultural lands can be detected and quantified via chromatography.
- Water purification can be done through ion-exchange chromatography.
- GCMS can help monitor ambient air quality index (AQI) by identifying gaseous air pollutants.
Uses of chromatography in research, development, and chemical industry
- Gas chromatography is used in the petrochemical industry to detect and quantify volatile organic compounds during oil refining and also to remove crude products.
- During chemical compound synthesis, any unwanted intermediate compounds can be detected through chromatography and separated.
- The progress of a chemical reaction can be monitored at different time intervals by spotting the reactants on a TLC plate.
- High-efficiency chromatographic techniques such as fast protein liquid chromatography (FPLC) and perfusion chromatography are often applied at the preparative stage in the research and development of new molecules.
- FPLC can be used in the preparative analysis of bulky proteins and polymers. Special peptide sequences called protein tags can be introduced onto valuable proteins so that they can be retained on an affinity column.
Uses of chromatography in biological and clinical analysis
- Blood and urine samples can be used to determine the alcohol consumption of an individual via chromatography.
- Metabolites are by-products produced as result of a chemical reaction in the animal/human body. These metabolites can be used as biomarkers during disease diagnosis. The study of these metabolites is called metabolomics. Chromatography plays an integral role in metabolomics.
- Bile acids are chemicals produced by cholesterol metabolism in the liver. An excess of bile acids can act as a biomarker for liver diseases such as liver cirrhosis. TLC, HPLC, and GC can be used for the analysis of bile salts in a blood sample.
- Liquid chromatography is also used in studying protein levels such as Hb2 to diagnose thalassemia and enzymes in liver function tests (LFTs) to diagnose hepatitis.
- Size exclusion chromatography can be used to assess different levels of protein folding under the influence of pH, temperature changes or denaturants, etc.
- Size exclusion chromatography can also be used for determining the molecular weight of a polymer, studying protein-complex association, and for protein desalting.
Uses of chromatography in forensic analysis
Chromatography is important in the crime scene and forensic analysis.
- Bodily fluids collected from a crime scene such as the blood spilled on the floor, blood stains on weapons, fabric, hair, and nails can be used as samples for chromatographic analysis.
- Gel electrophoresis can be used in genome sequencing and DNA fingerprinting to reveal the identity of the alleged criminal and also for parental recognition.
- Chromatography is also very important for identifying fluids and different chemical compounds in a body postmortem as a prospective sign of poison ingestion before death.
Having discussed in-depth, the many different uses of chromatography, we would like to conclude that chromatography is inevitably essential in all fields of life. Wherever you would hear the words analysis, separation, purification, etc., in chemistry, the discussion of chromatography will become indispensable.
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