Chromatography-the bed rock of separation science. A junior quality control analyst in a pharmaceutical laboratory is given a mixture consisting of different chemical components. How can he separate and identify all these components? Is there any specific analytical technique useful for this purpose? Well, there is one analytical separation and purification technique quite useful for this task i.e., chromatography. What is chromatography, how it is used, its working principle and its possible applications in a diversity of fields. This article will address all these preliminary questions that forms the basis of this very interesting topic called ‘’chromatography’’.
The word chromatography is derived from a combination of two words: chroma which means colour and graphy derived from graphein meant for writing or recording. Thus, chromatography literally means colour writing. This word was used for the first time in 1903 by a Russian botanist ‘’Mikhail Tsvet’’. He invented and used the technique for the first time to separate plant pigments such as chlorophyll, xanthophyll and carotenoids from a complex mixture.
Chromatography was initially used by artists and colour theorists only for the sake of perfecting industrial dyes in the textile manufacturing industry. Further research and advancement in the technique however led chromatography to become a widely popular analytical separation and purification tool in chemistry. The colour aspect has become a bit irrelevant today but the basic working principles governing chromatographic separation stays the same.
Chromatography is a technique which is used to separate a mixture into its constituent chemical components followed by the identification and purification of each component. The separation is based on the relative ability of each component to interact with the stationary phase and/or the mobile phase. This interaction can be developed via different means such as on the basis of shape, size, chemical bonding, polarity and/or other sources of affinity.
Before understanding the principle of chromatographic separation in more detail, one should be aware of the chromatographic terms that are frequently used.
- Stationary phase: Stationary phase is defined as a substance that stays fixed or unmoved in a chromatographic process. A chromatographic stationary phase is either a solid or a liquid coated on a solid surface.
- Mobile phase: Mobile phase is the solvent that moves past the stationary phase in chromatographic analysis. The word mobile refers to the ability to flow thus a mobile phase could either be a liquid or a gas in chromatographic analysis.
- Solute: A solution consists of a solute and a solvent. In the context of chromatography, solute refers to the analytical components undergoing separation. The mixture to be analyzed is usually dissolved in a particular solvent, each component to be separated is then referred to as a solute molecule.
- Solvent front: Solvent front is an important term particularly with reference to planar (2-dimensional) chromatography. It refers to the maximum distance travelled by the mobile phase.
- Retention time: In simpler terms, retention time is the time that an analyte component spends onto the stationary phase. In other words, it can be defined as the time required for the mobile phase to sweep past a component from the stationary phase. In more complex chromatographic processes, retention time refers to the time that analyte components spend inside a column. You will understand this concept better as we proceed further in this article.
- Retention/Retardation factor: Retention factor is the ratio of the distance travelled by a solute to the maximum distance travelled by the mobile phase i.e., the solvent front. The retention factor is also known as retardation factor and/or denoted as an Rf value.
- Retention volume: Retention volume is defined as the volume of mobile phase required to recover the analyte component from the stationary phase. This term again holds greater significance in complex column chromatographic processes which will be discussed more thoroughly later in the article and in subsequent articles in the series.
Working Principle of Chromatography
A basic chromatographic principle is based on the separation of analytical components depending on their ‘’relative affinities’’ or their extent of interaction with the mobile phase and a stationary phase. The analyte components distribute themselves between the two phases relatively. Based on this concept, all the components do not travel at the same rate. The ones with greater affinity towards the mobile phase travel faster while the components strongly adhered to the stationary phase travels at a retarded rate.
Example: A mixture M is given. The quality control analyst John (we discussed about him at the beginning) has to separate M into its constituent components A, B and C. John is provided with water, some organic solvents (ethanol and methanol), a chromatographic plate and other stationery essentials. He is also given a chart stating the Rf values for pure A, B and C. John will follow the step-by-step guide given below to carry out his analysis.
Step I: Take a lead pencil and draw a solid line just above the base of the chromatographic plate.
Step II: Prepare the mobile phase by mixing water, ethanol and methanol in the ratio 2:1:1.
Step III: Take a thin capillary tube. Use it to apply a small spot of the reaction mixture (M) at the center of the solid line drawn in step I.
Step IV: Vertically situate the chromatographic plate containing the mixture spot M into the container containing the mobile phase. Just dip the lower edge of the plate into the mobile phase.
Step V: Allow the solvent to travel up the chromatographic plate under capillary action. You will observe the mixture separated into distinct spots of different colours as it travels up the chromatographic plate with the mobile phase solvent.
Step VI: Mark the maximum distance travelled by the mobile phase at the top of the plate as solvent front.
Step VII: Note the distance travelled by each solute component and the position of the solvent front to calculate Rf value for each component using the formula given below.
where dc= distance travelled by solute component and ds= distance travelled by mobile phase solvent.
Step VIII: Compare the Rf value obtained for each component with the Rf values given for pure compounds to identify A, B and C.
Whatsoever, this example is the simplest version of a chromatographic separation. Further detail on such type of chromatographic separations can be found in our separate articles on paper chromatography and thin layer chromatography.
Types of Chromatography
Chromatographic techniques can be classified on numerous different bases as discussed in this section.
1. Chromatography classification on the basis of stationary phase
|Adsorption Chromatography||Partition Chromatography|
|Solid stationary phase||Liquid stationary phase coated on a solid support|
|Analyte components are separated based on differential adsorption on the stationary phase versus their movement with the mobile phase molecules||Analyte components are separated based on relative partitioning between the molecules of the stationary phase and the mobile phase|
2. Chromatography classification on the basis of mobile phase
|Liquid Chromatography||Gas Chromatography|
|Liquid mobile phase||Gas mobile phase|
|The stationary phase in this case could either be a solid or liquid||The stationary phase in this case as well could either be a solid or a liquid|
|In case of a solid stationary phase, it is solid-liquid chromatography||A solid stationary phase and a gas mobile phase gives the chromatographic separation its name: gas-solid chromatography|
|In case of a liquid stationary phase, it is liquid-liquid chromatography||In case of a liquid stationary phase, it is called gas-liquid chromatography|
3. Chromatography classification on the basis of geometry
|2-Dimensional/Planar Chromatography||3-Dimensional Chromatography|
|Stationary phase held on a plane||Stationary phase packed in a column|
|Examples include paper chromatography and thin layer chromatography||Examples include all types of column chromatography such as liquid chromatography and gas chromatography|
4. Chromatography classification on the basis of difference in polarities of the two phases
|Normal phase Chromatography||Reverse phase Chromatography|
|Stationary phase is more polar than mobile phase||Stationary phase is less polar than mobile phase|
|Mobile phase is less polar than stationary phase or is entirely non-polar||Mobile phase is more polar than stationary phase|
|A higher percentage of organic solvents such as hexane present in the mobile phase||A higher percentage of water or polar organic solvents such as ethanol, methanol and acetonitrile present in the mobile phase|
|Silica or alumina based stationary phases are commonly employed||Silica coated with long hydrocarbon chains are commonly employed as stationary phases|
5. Chromatography classification on the basis of sample
|Analytical Purposes||Preparative Purposes|
|Separation and identification of components of a mixture on a small scale||Purification of a significant quantity of any compound from a mixture on a larger scale|
|Minute quantities less than one nanogram are separated and analyzed||Hundreds of kilograms of materials are processed in a single batch|
|Thin capillary columns required||Bigger columns with higher loading capacity required|
6. Chromatography classification on the basis of mechanism of separation
|Chromatography Type||Separation Mechanism|
|Paper chromatography||Partitioning of solute components between mobile phase and stationary phase|
|Thin layer chromatography||Separation of solute components on the basis of adsorption on silica gel|
|Ion exchange chromatography||Separation on the basis of charge present on an analyte|
|Molecular or Size exclusion chromatography||Separation on the basis of size of molecules present in the analyte mixture|
|Affinity chromatography||Separation based on selective interaction and chemical bonding of the solute molecules with the functional groups present on the stationary phase|
|Liquid column chromatography|
Gas column chromatography
|Both liquid and gas chromatography can be performed on any of the separation mechanisms discussed above|
Why is Chromatography important
Chromatographic analyses are fruitful for a plethora of applications, some of the very common applications of chromatography are as discussed below.
Chromatography is frequently performed for the qualitative and quantitative analysis of chemical compounds and drugs. Development of a new drug requires a precise pharmaceutical dosage with high purity. Chromatography can be used to ensure all these quality control parameters. Active pharmaceutical ingredient of a drug can be separated from trace impurities using chromatography.
Chromatography can be used as biomarkers in diagnosis and evaluation of infectious diseases. Gas chromatography can be performed to analyze clinical samples such as bodily fluids (blood and urine) and tissue homogenates (enzymes and bilirubin). It helps in detecting chemical compounds responsible for causing infections such as hepatitis and metabolic disorders.
Quality control analysis by chromatography is important in the food industry as well, as means of isolating proteins, vitamins and preservatives etc. The nutritional value of processed foods can be determined by analyzing any chemical additives present in it.
Biological fluid samples collected as evidence from crime sites can be analyzed by chromatography to catch criminals. Chromatography on blood samples can also help in determining alcohol consumption of a person.
Research and Development
Chromatography holds a special importance in the research and development sector. Extraction of valuable phytochemicals from a plant requires further identification. Gas chromatography can be performed for analyzing volatile organic compounds such as essential oils. High performance liquid chromatography (HPLC) can be used for the separation and purification of fatty acids, flavonoids and terpenoids etc. Learn more about Gas Chromatography and HPLC here.
Hence concluding that chromatography is a very useful technique for performing a diversity of applications in the modern scientific world.
For more interesting information on extensive applications of chromatography, you might like the following sources:
1. Calderon, L. d. A. (2016). Chromatography: The Most Versatile Method of Chemical Analysis.
2. M.Younas (2017). Organic Spectroscopy and Chromatography.
3. Poole, C. F. (2003). The essence of chromatography, Elsevier.
4. Smith, I. (2013). Chromatography, Elsevier.