32 Chromatography

Dr. Swagat K. Mohapatra

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Contents

  1. Introduction
  2. Chromatography Theory
  3. Branches of Chromatography
  4. Classification of Chromatography methods
  5. Adsorption Chromatography
  6. Partition Chromatograph:
  7. Ion-exchange Chromatograph:
  8. Molecular exclusion / gel permeation Chromatograph
  9. Planar chromatography
  10. Column Chromatography
  11. Gas chromatography
  12. Liquid chromatography
  13. High Performance Liquid Chromatography (HPLC)
  14. References

 

Introduction:

Chromatography is a technique mainly used for separating and/or recognizing the components in a chemical mixture. The origin behind this technique is that the chemical components of the mixture have different affinities to adsorb onto a surface and/or dissolve in an organic solvent. This technique is widely accepted in industry on a large scale to distinguish and purify the components and the respective products in various organic syntheses.

Mikhail S. Tswett, a Russian scientist, first identified this techniques an efficient method for separation, during the beginning of twentieth century. He used a simple form of liquid-solid chromatography to separate a number of plant pigments, such as chlorophyll, carotenes, and xanthophylls. Since these components are of different colors such as green, orange and yellow respectively, they gave this technique its name as “color writing”. However, since then this technique was not popular among scientists for a long time. In 1941, A. J. P. Martin and R. L. M. Synge at Cambridge University, UK discovered partition chromatography for which there were awarded the Noble Prize in 1952.

Today chromatography is one of the most powerful and useful techniques available to the modern chemist. In a single step process it can separate a mixture into its individual components and simultaneously provide a quantitative estimate of each constituent. Samples may be gaseous, liquid or solid in nature and can range in complexity from a simple blend of two isomers of the same chemical species to a multi component mixture containing widely differing chemical species. Furthermore, the analysis can be carried out, at one extreme, on a simple, inexpensive thin layer plate, &/or on the other hand on a very costly and complex instrument.

Thus chromatography has been defined as “a separation technique that is achieved by distributing the components of a mixture between two phases, a stationary phase and a mobile phase. Those components held preferentially in the stationary phase are retained longer in the system than those that are distributed selectively in the mobile phase. As a consequence, the solutes are eluted from the system as local concentrations in the mobile phase in the order of their increasing distribution coefficients with respect to the stationary phase; thus a separation can be achieved”.[1]

 

Chromatography Theory:

Chromatography comprises a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase. A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase.

Techniques such as H.P.L.C. (High Performance Liquid Chromatography) and G.C. (Gas Chromatography) use columns – narrow tubes packed with stationary phase, through which the mobile phase is forced. The sample is transported through the column by continuous addition of mobile phase. This process is called elution. The average rate at which an analyte moves through the column is determined by the time it spends in the mobile phase.

Distribution of analytes between phases: The distribution of analytes between phases can often be described quite simply. The analyte A is in equilibrium between the two phases; ⇌

The equilibrium constant, K, is termed the partition coefficient; defined as the molar concentration of analyte in the stationary phase (Cs) divided by the molar concentration of the analyte A in the mobile phase (Cm).

 

A chromatogram is a graph showing the detector response as a function of elution time (figure 2).

The time between sample injection and an analyte peak reaching a detector at the end of the column is termed the retention time (tR). Each analyte in a sample will have a different retention time. The time taken for the mobile phase to pass through the column is called dead time (tM). tR and tM are easily obtained from chromatogram.

The term Retention factor or Capacity factor (Rf) is often used to describe the migration rate of an analyte on a column. The retention factor for analyte A is defined as;

When an analytes retention factor is less than one, elution is so fast that the accurate determination of the retention time is very difficult. High retention factors (greater than 20) mean that elution takes a very long time. Ideally, the retention factor for an analyte is between one and five.

 

Selectivity factor (α) is a quantity, which describes the separation of two species (A and B) on the column:

When calculating the selectivity factor ‘α’, species A elutes faster than species B. The selectivity factor is always greater than one.

 

Classification of Chromatography methods:

Chromatography methods can be mainly categorized on the basis of followings: 1) the stationary phase (adsorption, partition, ion-exchange, and molecular exclusion), 2) chromatographic bed shape (planar, column), 3) the mobile phase (gas chromatography, and liquid chromatography).

 

1. On the basis of the stationary phase:

i) Adsorption Chromatography: It includes a mobile liquid or gas phase which is adsorbed onto the surface of a stationary solid phase. The equilibration between the two phases (mobile and stationary) differentiates the solutes, and thus help to separate.

 ii) Partition Chromatograph: It involves the formation of a thin film on the surface of a solid supported by the liquid stationary phase.

iii) Ion-exchange Chromatograph: In this case a resin is used as the solid stationary phase, which is required to covalently attract ions (cations/anions) towards it. The oppositely charged ions of the solute present in the mobile phase liquid undergo an electrostatic force of attraction and attach to the resin.

 iv) Molecular exclusion / gel permeation Chromatograph:

This method does not hold the attraction between the stationary phase, and the solute. The mobile phase, either a liquid or gaseous phase, when pass through a porous gel, are separated based on their size. As the pore size of the gel are kept usually small, this allows the smaller molecules to go into the gel while excluding the larger solute particles. This retards the flow rate of the smaller molecules while passing through the column, where as the large molecule pass through the column at faster rate.

 

2.On the basis of chromatographic bed shape:

i) Planar chromatography: Here the stationary phase is presented as or on a plane. This can be a paper; can be used as such or some substance can be impregnated on the surface as the stationary bed (called paper chromatography). It can also be a layer or solid particles uniformly spread over a glass plate, called thin layer chromatography (TLC). The different components in the organic mixture, while passing through the planar surface vertically, can be distinguished over time based on the distance they travel. This mainly depends on the interaction between the solute and the stationary phase with respect to the mobile phase. The specific retention factor of each component will also be used to characterize the unknown substance.

 ii) Column Chromatography: In this case the stationary bed is kept inside a glass tube or column. There are to possibilities in such arrangements. In the first case the solid stationary phase or support coated with a liquid stationary may be tightly packed the whole inside volume of the column, called as Packed column chromatography. While in the second the solid stationary phase concentrate on or along the inside wall of the column, leaving an open, unrestricted path for the mobile phase at the middle portion of the column, called Open tubular column.

 

3. On the basis of the mobile phase:

i) Gas chromatography (GC): [2,3]

Gas Chromatography deals with separation and purification of compounds between two phases. One if fixed, called to be stationary phase, which may be either a solid or a liquid held by a solid. The second one is mobile, called to be mobile phase, which may be a gas, liquid or vapor. There are two method of separation based on the state of stationary phase: 1) gas-solid chromatography (GSC), this involves the mobile phase is a gas, and the stationary phase as an active solid like silica gel, alumina; 2) gas-liquid chromatography (GLC), where the moving phase is a gas, and the stationary phase is a liquid distributed on an inert solid support. GLC is mainly used for the preparation and identification of a variety of organic compounds. GSC is used mainly for the separation of gases.

 

Principle: The principle of operating gas chromatograph involves the incorporation of small amount of sample either in gaseous or liquid state, containing nano gram amounts of analytically pure gaseous or vaporizable component, which are inserted into a column holding the stationary phase by an inert carrier gas (N2 or He) under controlled temperature. Phase equilibria happen between the components in the sample mixture, separate the components on the basis of their difference in adsorption, bonding nature, and solubility. Each fraction then pass through the column at a varied flow rate, and thus begin to separate. Further the carrier gas coming out of the column moves through a detector, thus develops a distinct signal with respect to the quantity of each of the component present in the sample mixture. Then the detector response is amplified on the recorder as a peak, called chromatogram which is a plot of time versus the intensity of peaks, and represent the eluted components in the carrier stream. The retention time, which is time needed for each of the peaks to reach the detector, and characteristics to each of the component present under a set chromatographic conditions. Thus it characterizes each of the component of the mixture, and further area of the peaks give a quantitative measurement.

An appropriate choice of the injection port, column, and the detector temperature, column materials, the detector measures the efficiency of the chromatographic separations of each component present in the sample.

 

Basic Design: There are many commercial models of gas chromatograph available till date, with considerable variations in design and arrangement of components. The basic design of the apparatus includes: (a) carrier gas system, (b) sample injector, (c) column, (d) thermostat, (d) detector, and (e) recorder (figure 3).[3]

THE ROYAL SOCIETY OF CHEMISTRY

b) In Toxicology: Gas chromatograph is also being useful in the field of toxicology, particularly for the substances like steroids, lipids, barbiturates, drugs, and blood alcohols. Usually the samples available in the field of toxicology are very limited, and in low concentrations, thus Gas chromatograph is a worth full method for their complete analysis, and identification of the sample mixture. For example, to monitor the trace constituents of atmospheric components, a small gas chromatograph-mass spectrometer was sent to space in a simulated manned spacecraft. Gas chromatograph is also used in the aerospace and nuclear submarine field to analyze the air quality of working and sleeping environments. This has also been employed in the remote sampling and analysis of tunnel atmosphere.

 

 c) In Industry: Chromatographic method has also been seen to be useful in the field of industrial This is used to identify and quantitatively measure the organic solvents present in breath samples. There are toxic organic substances who has neither any identifiable biological metabolite nor whose presence can be compared with atmospheric concentration of the existing substances. It is thus making difficult to evaluate their effect of exposure. Thus breath analysis by Gas chromatography is found to be useful to analyze quantitatively such organic substances.

Furthermore, this method is running by several industrial hygiene departments for product identification programs for complete analysis of proprietary products. In industrial laboratory, chromatographic method also plays a major role. For example: analysis of organic solvents used for various chemical syntheses, analysis of products prepared from different batches, analysis of organic compounds of unknown compositions and for full identification.

Many commercial products upon addition can increase to some extent the toxicity of less hazardous materials. For example, mixing carbon tetrachloride with typewriter cleaner, which contains only ethylene, mixing benzene to gasoline for increase the efficiency of the engine.

 

d) In Air analysis:

Air quality analysis can also be determined using Gas chromatograph. For examples, in various industrial plants, the air samples containing organic solvents can be separated and purified by chromatography avoiding any tedious chemical method. Substances such as chlorinated hydrocarbons can be easily monitored in a mixture and individual components can also be identified in chromatography, whereas chemical method can only determine the total chlorinated hydrocarbon. Presence of nitrogen oxides in air can be monitored by this method. Scientists Bethea, [4] and Lawson [5] were first proposed Unilever such analysis.

Using a two section column, Bethea [4] was able to determine the nitrogen dioxide with a lower detection limit of 200 ppm. Gas chromatography of gases emanating from the soli has been successful in separating the components mixture using a three-column system equipped with a thermal detector. A short silica column has been used to determine nitrogen dioxide at 50-60 °C with hydrogen as a carrier gas. Nitrogen dioxide has been collected and concentrated on Molecular Sieves and determined by chromatography.

FIGURE 4: SCHEMATIC DIAGRAM OF A LIQUID

CHROMATOGRAPHY COLUMN

 

ii) Liquid chromatography

Liquid chromatography method is similar to gas chromatography but uses a liquid instead a gas as the mobile phase. The stationary phase is usually an inert solid such as silica gel (SiO2.xH2O), or alumina (Al2O3.xH2O) packed in a glass column (figure 4)

The adsorbing properties of silica and alumina are reduced if they absorb water, but the reduction is reversed by heating to 200 –400 °C. Silica is slightly acidic, and  Liquid chromatography method is similar to gas chromatography but uses a liquid instead a gas as the readily adsorbs basic solutes. On the other hand, alumina is slightly basic and strongly adsorbs acidic solutes. Other stationary phases that can be used include

mobile phase. The stationary phase is usually an inert solid such as silica gel (SiO2.xH2O), or alumina magnesia, MgO.xH O (good for separating unsaturated organic compounds); and dextran (a polymer of glucose) cross-linked with propan-1,2,3-triol (glycerol, (Al2O3.xH2O) packed in a glass column (figure 4). CH OHCHOHCH OH), which is sold as Sephadex and can separate compounds such as purines. Sephadex has the structure shown in Fig. 16.

molecular weights having a very low volatility. This is where Gas chromatography lacks in separating. Furthermore, this technique can run even with samples in trace amounts, such as in drugs and drug metabolites in blood.

In principle, the sample mixture needs to pass through a short length of tube packed column with an adsorbent, which selectively removes the material of interest. The adsorbent is then washed and the adsorbed material extract with a small amount of suitable solvent and the solvent then inject onto the column.

A more elaborate variation on liquid chromatography is high performance liquid chromatography (HPLC). This technique generally THEROYAL uses very small packing particles with a relatively high pressure.

 

High perforance liquid chromatography(HPLC)

The effectiveness in separating the various components of an organic mixture increase with the decrease in the particle size in the stationary phase, due to the rapid equilibrate of solute between the two phases. Further, when particles sizes are smaller, the capillary action increases, as a result the draining of the solvent through the column become difficult under gravity. Therefore, a high pressure has to be needed to apply on the solvent to run the column. This is where a new chromatographic method called High pressure Liquid Chromatography aroused. This is widely known as High

long the pressure needed would be excessive.) Columns are typically 10–30 cm long, with an internal diameter of 4 mm.    Reproducibility is essential, and this is only possible if a constant flow rate is maintained. This means that the pump used must be capable of generating a uniform pressure; twin cylinder reciprocating pumps are typical.  This type of pump has two chambers with pistons 180o out of phase, and can generate pressures up to 10MNm -2 (10 MPa/100 atmospheres). The high pressures involved mean that the instrumentation has to be very strong, and the ‘plumbing’ is usually constructed from Chromatography stainless steel. The pump and the piping must be inert to the solvent and solutesC being passed through them. The flow rates of HPLC columns are slow – often in the range 0.5–5 cm3 min-1.

The volumes of the columns are very small, and this means that the injection of the sample must be very precise and it must be quick without disturbing the solvent flow.

Performance Liquid Chromatography (HPLC).

A schematic diagram of the HPLC system is given in Figure 5. [6]

Here the stationary phase is made up of uniform porous silica particles of 10-6 surface pores are having diameter range between 10-6 to 10-9, which results a area.

The particles bonded to the non-volatile liquid permits to interact the solutes with different polarities. The particles of the stationary phase are tightly packed into the HPLC column, and kept in place using glass fibers coated with inert alkyl silane molecules.

Generally the length of columns are not required to be longer because of their effectiveness in the separating the components of the organic mixture. In the case of long columns, the pressure applied needs to be increased.

A constant flow rate is always necessary to have the reproducibility, which is indispensable for such measurements. This demands a suitable pump, which should be able to produce uniform pressure. Twin cylinder reciprocating pumps are idea for this purpose. These kind of pumps generally contain two chambers with piston of 180 ° out of phase, and is able to generate pressure around 10 MNM-2. Further all the tubing in the pump, and in the instrument as well should be inert to the organic solvents, and solutes being go through them.

HPLC columns normally have slow flow rates, between the range 0.5 – 5 cm3.min-1. Further these columns are very narrow. Thus injection of the sample should be accurate, and this should not disturb the solvent flow in the column. In case of Gas Chromatograph, the flow rate of solutes passing through the column can be increased by raising the temperature of the column. While in case of HPLC, this is achieved by modifying the composition of the mobile phase. For example, in the concentration gradient of methanol and water, the ratio of methanol can be increased linearly from 10 – 60 % of the total volume, during the separation.

 

As the concentration of the solute in their respective solution passing through the column are very low. Instead of extracting them from the solution, they are analyzed directly for identification once they come through the pump. In often cases the different components of the organic mixture are separated by HPLC absorb ultraviolet light. The eluate is generally made to pass through a small cell so that can come across the ultraviolet radiation.

 

Application:

Gas Chromatograph analyzes compounds which are stable at high temperatures, while High Performance Liquid Chromatography (HPLC) technique is ideal for those sample which are likely to decompose at high temperature or low volatile organic solids. It is one of the widely adopted analytical procedure; its application involve in a range of areas such quality control, process control, forensic analysis, environmental monitoring and clinical testing. Furthermore HPLC technique remains as one of the most sensitive analytical method and is an exclusive method which is suitable for multi-component mixtures.

An example of the use of this technique is in the identification of tetrahydrocannabinol carboxylic acid in urine, which is generally present in the urine of those persons who have smoked marihuana. The tetrahydrocannabinol carboxylic acid can be extracted from the urine by means of a solid state extraction cartridge packed with a C18 reverse phase (a strongly dispersive packing containing bonded octyldecyldimethyl chains). The urine sample can be used direct, without pretreatment and the materials of interest are irreversibly adsorbed on the reverse phase solely by dispersive interactions. [2]The chromatograms obtained from a urine sample and a reference standard by this procedure is shown in figure 6. This is a characteristic application for liquid chromatography using solid phase extraction cartridges. It is seen that the tetra hydrocannabinol carboxylic acid is clearly and unambiguously separated from the contaminating materials with an extraction efficiency of over 90%. [2]

A similar example of using this technique is analysis of tricyclic antidepressant drugs in blood serum.

 

Pharmaceuticals: HPLC provides a reliable quantitative analysis of pharmaceutical products with high accuracy and precision in a single run. An appropriate method for preparing sample for solid dosage is dispersion in water or a mixture of water and organic solvents such as acetonitrile/methanol. HPLC provides various possibilities for separating chiral organic molecules into their respective enantiomers, which involves precolumn derivatization to form diastereomers. Furthermore, typical columns may be prepared with cyclodextrins for special chiral moieties as stationary phases. The common use in analyzing the pharmaceutical products are: Assay, Related Substances, Analytical Method Validation, Stability Studies, Compound Identification, Working Standards.

 

Foods: HPLC technique has been proved to be suitable in analyzing food components. Food components are usually in complex forms and extraction of these components is not easy. Again both the desirable and undesirable substances are often seen to be present in trace amounts, and thus their extraction, and identification become further complicated and thus do not provide the necessary amount of accuracy and precision. HPLC provides a doable solutions because of a variety of choices available in selecting the stationary and mobiles phases. The Common components in food those can be analyzed in food are: fat and water soluble vitamins, Residual pesticides, Antioxidants, Sugars, Cholesterol and sterols, Dyes and synthetic colors, Mycotoxins, Amino acids, Residual antibiotics, Steroids and flavonoids, Aspartame and other artificial sweeteners, and some active ingredients of farm products.

 

Air analysis: HPLC technique is also being introduced in detecting the level of several pollutants present in air. For example, recently the extent of particulate matter with aerodynamic diameter < 2.5 (PM2.5) bound polycyclic aromatic hydrocarbons (PAHs) present in air is measured by HPLC method with fluorescence-ultraviolet detector (HPLC-FLD). Incomplete combustion and pyrolysis of fossil fuels and other organic materials are generally the source for PAHs in the atmosphere. Their presence in the atmosphere are serious threats to the human life due to their toxic, carcinogenic, and mutagenic effects. In particular, epidemiological evidence confirms the incidences of respiratory diseases, including lung cancer in persons from the exposure of PM2.5 bound PAHs.

you can view video on Chromatography

References:

  • Scott Raymond P. W., Principles and Practice of Chromatography, Chrom-Ed Book Series, 01-02, www.library4science.com.
  • http://www.cdc.gov/niosh/pdfs/74-177-k.pdf
  • http://media.rsc.org/Modern%20chemical%20techniques/MCT5%20Chromatogra
  • R. M. Bethea, M. C. Meador Journal of Chromatographic Science, 7, 655 (1969)
  • A. Lawson, H. G. McAdie, Journal of Chromatographic Science, 8, 723 (1970)
  • http://faculty.ksu.edu.sa/Dr.almajed/Books/practical%20HPLC.pdf

Textbooks:

i)Chromatography, By B. K. Sharma, Goel Publishing House, Krishna Prakashan Media (P) Ltd, fift edition 2007.

ii) Analytical Chromatography, By Gurdeep R. Chatwal, Himalaya Publishing House 2006.

iii)Chromatography, By David Robert Browning, McGraw-Hill, 2008.

 

Websites:

i)http://media.rsc.org/Modern%20chemical%20techniques/MCT5%20Chromatogra

ii) http://faculty.ksu.edu.sa/Dr.almajed/Books/practical%20HPLC.pdf

iii)http://shodhganga.inflibnet.ac.in/bitstream/10603/3866/7/07_chapter%201.pdf

iv)http://lincolnparkhs.org/ourpages/auto/2010/9/26/37121222/Chapter%2018%20-%20Chromatography.pdf