Basic Chromatography Techniques, Chemistry tutorial

Introduction:

Chromatography (from the Greek chroma 'color' and graphein 'to write') is the collective word for the set of laboratory methods for the separation of mixtures. The mixture is dissolved in a fluid termed as the 'mobile phase' that carries it via a structure holding the other material known as the 'stationary phase'. The different constituents of the mixture travel at various speeds, causing them to separate. The separation is mainly based on differential partitioning between the mobile and stationary phases. The subtle differences in a compound's partition coefficient yield in differential retention on the stationary phase and therefore changing the separation.

Chromatography might be preparative or analytical. The main purpose of preparative chromatography is to separate the components of a mixture for further use (and is therefore a form of purification). The Analytical chromatography is done usually by smaller amounts of material and is for measuring the relative proportions of analyte in a mixture.

History of Chromatography:

Chromatography, exactly 'color writing', was first used by Russian scientist Michael Tswett in the year 1900. He carries on to work by chromatography in the first decade of the 20th century, mainly for the separation of plant pigments like chlorophyll, carotenes and xanthophylls. As such components encompass different colors (that is, green, orange and yellow correspondingly) they gave the method its name. Between the year 1930 and 1940 new kinds of chromatography emerged and the method became helpful for lots of separation processes. Archer John Martin Porter and Richard Laurence Millington Synge throughout the year 1940 and 1950 worked widely on chromatography method which led to the establishment of the principles and fundamental methods of partition chromatography, and their work encouraged the fast growth of some chromatographic methods: paper chromatography, gas chromatography and what would become termed as high performance liquid chromatography. The technology has since then advanced fast. Researchers found that the major principles of Tswett's chromatography could be applied in numerous different ways, resultant in the different varieties of chromatography illustrated below. The separation of increasingly identical molecules is made possible because of persistence enhancement in the technical performance of the chromatography.

Basic Chromatography terms:

A) The analyte is the substance to be separated during chromatography.

B) Analytical chromatography is employed to find out the existence and the concentration of analyte(s) in the sample.

C) A bonded phase is a stationary phase which is covalently bonded to the support particles or to the inside wall of the column tubing.

D) The visual output of the chromatograph is a chromatogram that comprises of various peaks that correspond to various components of the separated mixture.

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Fig: Basic Chromatography

Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for illustration acquired via a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the response made by the analyte exiting the system. The signal is proportional to the concentration of the analyte separated.

A) A chromatograph is equipment which lets a sophisticated separation example: gas chromatographic or liquid chromatographic separation.

B) Chromatography is the physical process of separation in which the components to be separated are distributed between the two phases, one of which is stationary (that is, stationary phase) whereas the other (that is, the mobile phase) moves in the definite direction.

C) The eluate is the mobile phase leaving the column.

D) The eluent is the solvent which will carry out the analyte.

E) The eluotropic sequence is a list of solvents ranked according to their eluting power.

F) An immobilized phase is a stationary phase that is immobilized on the support particles or on the inner wall of the column tubing.

G) The mobile phase is the phase that moves in a definite direction. This might be a liquid (that is, LC and Capillary Electrochromatography (CEC)), a gas (GC), or a supercritical fluid (that is, supercritical-fluid chromatography, SFC). The mobile phase comprises of the sample being separated or analyzed and the solvent which moves the sample via the column. In case of HPLC the mobile phase comprises of a non-polar solvent(s) like hexane in normal phase or polar solvents in the reverse phase chromatography and the sample being separated. The mobile phase moves via the chromatography column (that is, the stationary phase) where the sample interacts by the stationary phase and is separated.

H) Preparative chromatography is employed to purify adequate quantities of a substance for further use, instead of analysis.

I) The retention time is the characteristic time it takes for a specific analyte to pass via the system (that is, from the column inlet to the detector) under set conditions. The sample is the matter examined in chromatography. It might comprise of a single component or it might be a mixture of components. Whenever the sample is treated in the course of an analysis, the phase or the phases having the analyte of interest is/are termed to as the sample while everything out of interest separated from the sample before or in the course of the analysis is termed to as waste.

J) The solute refers to the sample components in the partition chromatography.

K) The solvent refers to any substance able of solubilizing the other substance, and particularly the liquid mobile phase in LC.

L) The stationary phase is the substance that is fixed in place for the chromatography process. Illustration comprises the silica layer in thin layer chromatography.

Techniques by chromatographic bed shape:

Planar chromatography:

Planar chromatography is a separation method in which the stationary phase is present on the plane. The plane can be a paper, or the paper might be impregnated through a substance as the stationary bed (that is, paper chromatography). It could as well be a layer of solid particles spread on a support like a glass plate (that is, thin layer chromatography). Different components of the sample mixture migrate at various rates according to how strongly they interact by the stationary phase as compared to the mobile phase. The specific Retention factor (Rf) of each and every chemical can be employed to help in the recognition of an unknown substance.

a) Paper Chromatography:

The Paper chromatography is a method employed for separating and recognizing mixtures which are either colored or can be colored. The secondary or primary colors in ink can simply be separated by this method. This process is a powerful teaching tool however has been greatly substituted by thin layer chromatography. Complex mixtures of identical compounds like amino acids can be separated by employing a two-way paper chromatograph or else termed as two-dimensional (2-D) chromatography. In this process, two solvents are employed and the paper is rotated at around 90oC in between.

The retention factor (Rƒ) is stated as the ratio of the distance traveled via the substance to the distance traveled via the solvent. Rƒ values are generally deduced as a fraction of two decimal places. Whenever Rƒ value of a solution is zero, the solute remains in the stationary phase and therefore it is immobile. If Rƒ value = 1, this means that the solute consists of no affinity for the stationary phase and travels by the solvent front. To compute the Rƒ value, take the distance traveled via the substance divided by the distance traveled by the solvent. For illustration, if a compound travels 1.5 cm and the solvent front travels 2.2 cm, (1.5/2.2) the Rƒ value equals to 0.68.

Paper chromatography is one technique for testing the purity of compounds and recognizing substances. Paper chromatography is a helpful method since it is relatively quick and needs small quantities of material. 

In paper chromatography, such as thin layer chromatography, substances are distributed between the stationary phase and a mobile phase. The stationary phase is generally a piece of high quality filter paper. The mobile phase is a developing solution which travels up the stationary phase, carrying the samples with it. The components of sample will separate readily according to how strongly they adsorb on the stationary phase versus how readily they dissolve in the mobile phase.

Whenever a colored chemical sample is put on a filter paper, the colors separate from the sample by putting one end of the paper in a solvent. The solvent diffuses up the paper, dissolving the different molecules in the sample according to the polarities of the molecules and the solvent. If the sample includes more than one color, that signifies it should encompass more than one type of molecule. Due to various chemical structures of each and every type of molecule, the chances are extremely high that each and every molecule will have at least a slightly dissimilar polarity, giving each molecule a different solubility in the solvent. The unequal solubility causes the different color molecules to leave solution at various places as the solvent carries on moving up the paper. The more soluble a molecule is, the higher it will migrate up the paper. Whenever a chemical is very non-polar it will not dissolve at all in a very polar solvent. This is similar for a very polar chemical and a very non-polar solvent.

This is significant to note that whenever using water (that is, a very polar substance) as a solvent, the more polar the colour, the higher it will increase on the paper.

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Fig: Chromatography Tank (Paper Chromatography)

Thin layer chromatography (or TLC) is a chromatography method employed to separate mixtures. Thin layer chromatography is completed on a sheet of glass, plastic and aluminum foil that is coated by a thin layer of adsorbent material, generally silica gel, aluminium oxide or cellulose (that is, blotter paper). This layer of adsorbent is termed as the stationary phase.

Subsequent to the sample has been applied on the plate, a solvent or solvent mixture (termed as the mobile phase) is drawn up the plate through capillary action. As various analyte ascend the TLC plate at different rates, separation is accomplished.

Thin layer chromatography can be employed to monitor the development of a reaction, recognize compounds present in a particular mixture, and find out the purity of a substance. Particular illustrations of such applications comprise: analyzing ceramides and fatty acids, detection of pesticides or insecticides in water and food, examining the dye composition of fibers in forensics, assaying the radiochemical purity of radiopharmaceuticals or recognition of medicinal plants and their constituents. 

A number of improvements can be made to the original process to automate the various steps, to raise the resolution accomplished with TLC and to let more accurate quantization. This process is termed to as HPTLC, or 'high performance TLC'.

=> Plate preparation:

TLC plates are generally commercially available, with standard particle size ranges to enhance reproducibility. They are made by mixing the adsorbent, like silica gel, having a small amount of inert binder such as calcium sulphate (gypsum) and water. This mixture is spread as thick slurry on an unreactive carrier sheet, generally glass, thick aluminum foil and plastic. The resulting plate is dried and activated via heating in an oven for thirty minutes at around 110 °C. The thickness of the adsorbent layer is usually around 0.1 to 0.25 mm for analytical purposes and approx 0.5 to 2.0 mm for preparative TLC.

The technique is identical to paper chromatography with the benefit of faster runs, better separations and various stationary phases are available for use. Due to its simplicity and speed TLC is frequently employed for monitoring the chemical reactions and for the qualitative analysis of reaction products.

Thin layer chromatography can be run simply if the given process is carried out:  A small spot of solution having the sample is applied to a plate, around 1.5 centimeters from the bottom edge. The solvent is allowed to fully evaporate off, or else a very poor or no separation will be accomplished. The plate must be dried in a vacuum chamber particularly if a non-volatile solvent was employed to apply the sample.

A small amount of a suitable solvent is poured to an appropriate transparent container to a depth of less than 1 centimeter. The strip of filter paper is placed to the chamber, in such a way that its bottom touches the solvent, and the paper lies on the chamber wall and reaches approximately to the top of the container. The container is closed by a cover glass or any other lid and is left for some minutes to let the solvent vapors ascend the filter paper and saturate the air in the chamber. The TLC plate is then put in the chamber in such a way that the spots of the sample don't touch the surface of the solvent in the chamber, and the lid is closed. The solvent moves up the plate through capillary action, meets the sample mixture and carries it up the plate (that is, elutes the sample). Whenever the solvent front reaches no higher than the top of the filter paper in the chamber, the plate must be eliminated and dried.

=> Separation Process:

Various compounds in the sample mixture travel at different rates because of the differences in their attraction to the stationary phase, and due to differences in solubility in the solvent. By changing the solvent, or possibly by using a mixture, the separation of components (that is, measured by the Rf value) can be adjusted. As well, the separation accomplished by a TLC plate can be employed to approximate the separation of a flash chromatography column.

The separation of compounds is mainly based on the competition of the solute and the mobile phase for binding places on the stationary phase. For illustration, if normal phase silica gel is employed as the stationary phase it can be considered polar. Given two compounds that vary in polarity, the more polar compound consists of a stronger interaction by the silica and is thus more capable to dispel the mobile phase from the binding places. As a result, the less polar compound moves higher up the plate (resultant in the higher Rf value). Whenever the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. This is generally stated that 'strong' solvents (that is, elutants) push the analyzed compounds up the plate, whereas 'weak' elutants hardly move them. The order of strength or weakness based on the coating (that is, stationary phase) of the TLC plate. For silica gel coated TLC plates, the elutant strength rises in the given order: Perfluoroalkane (weakest), Hexane, Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl ether, Ethylacetate, Acetonitrile, Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest). For C18 coated plates the order is reverse. Practically this signifies that if we make use of a mixture of ethyl acetate and hexane as the mobile phase, adding more ethyl acetate yields in higher Rf values for all compounds on the TLC plate. Changing the polarity of the mobile phase will generally not result in reversed order of running of the compounds on the TLC plate. The eluotropic series can be employed as a guide in choosing a mobile phase. Whenever a reversed order of running of the compounds is desired, an apolar stationary phase must be employed, like C18-functionalized silica.

=> Analysis:

As the chemicals being separated might be colorless, some of the methods exist to visualize spots: Often a small quantity of a fluorescent compound, generally manganese-activated zinc silicate, is added to the adsorbent which lets the visualization of spots under a backlight (UV254). The adsorbent layer will therefore fluoresce light green by itself; however spots of analyte quench this fluorescence.

Iodine vapors are a common unspecific color reagent.

Particular color reagents exist to which the TLC plate is dipped or which are sprayed to the plate: Potassium permanganate - oxidation, iodine and bromine.

In case of lipids, the chromatogram might be transferred to a polyvinylidene difluoride (PVDF) membrane and then subjected to further analysis, for illustration mass spectrometry, a method termed as Far-Eastern blotting.

Once visible, the Rf value, or retardation factor, of each and every spot can be found out by dividing the distance the product traveled via the distance the solvent front traveled by employing the initial spotting site as reference. Such values based on the solvent utilized and the kind of TLC plate and are not physical constants.

=> Applications:

As an illustration, the chromatography of an extract of green leaves (for illustration spinach) in 7 phases of development. Carotene elutes rapidly and is only visible until step 2. Chlorophyll A and B are halfway in the final step and lutein the first compound staining yellow.

In one study TLC has been applied in the screening of organic reactions for illustration in the fine-tuning of BINAP synthesis from 2-naphthol. In this process the alcohol and catalyst solution (for example iron(III) chloride) are put separately on the base line, then reacted and then immediately examined.

Column Chromatography:

The separation of a mixture and isolation of the components in larger amounts is made probable via chromatography on a column than through TLC. The column is made up of glass and is packed by particles that comprise the stationary phase. The mixture under test is put on top of a layer of sand on the column, and a slow stream of solvent, the eluant, washes the mixture via it. The function of sand is to prevent the particles being disturbed through the liquid. The substance which is the least attracted to the stationary phase is washed out at the bottom of the column first, followed through the remaining components over a time-period.

=> Preparation of Column:

The column is made up in the following steps:

A) Put a wad of glass wool in the bottom of the tube and pour a layer of sand over this. The sand keeps fine particles and as well gives a flat horizontal base for the adsorbent column.

B) Fill the tube by the first solvent to be employed and then add the dry adsorbent in a fine stream shaking or tapping the tube to dislodge air bubbles, and draining solvent out at the bottom to make room as required, however keeping the solvent level above the adsorbent.

C) If the adsorbent has settled to a compact column, add the other layer of sand at the top of the adsorbent to prevent disturbance of the surface in which the solvent is added.

D) Allow the solvent to drain down to just above the top sand layer. Note that the adsorbent column should be kept covered by solvent throughout the chromatography, or else, channels and cracks will build up.

E) Dissolve the sample in a minimum volume of solvent and add up the solution to the solution to the column by a pipette and bulb. Let the solution to drain to the column and instantly add more solvent. 

In the year 1978, W.C. still introduced a modified version of column chromatography termed as flash column chromatography (that is, flash). The method is very identical to the traditional column chromatography; apart from for that the solvent is driven via the column by applying positive pressure. This allowed most of the separations to be carried out in less than 20 minutes, with enhanced separations compared to the old technique. Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped via the cartridge. Systems might as well be linked by detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in fast separations and less solvent usage.

In the expanded bed adsorption, a fluidized bed is employed, instead of a solid phase made through a packed bed. This lets omission of initial clearing steps like centrifugation and filtration, for culture broths or slurries of the broken cells.

=> Column Chromatogram resolution computation:

Generally, column chromatography is set up by peristaltic pumps, flowing buffers and the solution sample via the top of the column. The solutions and buffers pass via the column where a fraction collector at the end of the column setup collects the eluted samples.

Before the fraction collection, the samples which are eluted from the column pass via a detector like a spectrophotometer or mass spectrometer in such a way that the concentration of the separated samples in the sample solution mixture can be found out.

For illustration, if you were to separate two different proteins having different binding capacities to the column from a solution sample, a good kind of detector would be a spectrophotometer by employing a wavelength of 280 nm. The higher the concentration of protein which passes via the eluted solution via the column, the higher the absorbance of that wavelength.

As the column chromatography consists of a constant flow of eluted solution passing via the detector at varying concentrations, the detector should plot the concentration of the eluted sample over a course of time. This plot of sample concentration versus time is known as chromatogram.

The final goal of chromatography is to separate various components from a solution mixture. The resolution deduces the extent of separation between the components from the mixture. The higher the resolution of the chromatogram, the better the degree of separation of the samples the column provides. This data is a good manner of finding out the column's separation properties of that specific sample. The resolution can be computed from the chromatogram. The separate curves in the diagram symbolize various sample elution concentration profiles over time based on their affinity to the column resin. To compute resolution, the retention time and curve width are needed.

The time from the beginning of signal detection via the detector to the peak height of the elution concentration profile of each and every different sample is termed as retention time whereas the width of the concentration profile curve of the various samples in the chromatogram in units of time is termed as curve width.

A simplified process of computing chromatogram resolution is to make use of the plate model. The plate model supposes that the column can be categorized to a certain number of sections, or plates and the mass balance can be computed for each and every individual plate. This approach estimates a typical chromatogram curve as the Gaussian distribution curve. By doing this, the curve width is predicted as four times the standard deviation of the curve, '4σ'. The retention time is the time from the beginning of signal detection to the time of the peak height of the Gaussian curve. From the variables, the resolution, plate number, and plate height of the column plate model can be computed by using the equations:

Resolution (Rs):

Rs = 2(tRB - tRA)/(wB + wA)

Here:

tRB is the retention time of solute

tRA is the retention time of solute A

wB is the Gaussian curve width of solute B

wA is the Gaussian curve width of solute A

Plate Number (N) = (tR)2/(w/4)2

Plate Height (H) = L/N

Here, L is the length of the column

=> Column Adsorption equilibrium:

For the adsorption column, the column resin (that is, the stationary phase) is comprised of micro beads. Even smaller particles like carbohydrates, proteins, metal ions or other chemical compounds are conjugated to the micro beads. Each and every binding particle which is linked to the micro bead can be supposed to bind in a 1:1 ratio having the solute sample sent via the column which required to be purified or separated.

Binding between the target molecule to be separated and the binding molecule on the column beads can be modeled by employing a simple equilibrium reaction Keq = [CS]/([C][S]) here Keq is the equilibrium constant, [C] and [S] are the concentrations of the target molecule and the binding molecule on the column resin, correspondingly. [CS] is the concentration of the complex of the target molecule bound to the column resin.

By using this as a basis, three different isotherms can be employed to illustrate the binding dynamics of a column chromatography: linear, Langmuir and Freundlich.

The linear isotherm takes place if the solute concentration required to be purified is extremely small relative to the binding molecule. Therefore, the equilibrium can be stated as:

[CS] = Keq[C]

For industrial scale utilizations, the net binding molecules on the column resin beads should be factored in because unoccupied sites should be taken into account. The Langmuir isotherm and Freundlich isotherm are helpful in illustrating this equilibrium. Langmuir Isotherm:

[CS] = (KeqStot[C])/(1 + Keq[C]), here Stot is the net binding molecules on the beads.

Freundlich Isotherm: [CS] = Keq[C]1/n

The Freundlich isotherm is employed whenever the column can bind to numerous different samples in the solution which requires to be purified. As most of the various samples encompass various binding constants to the beads, there are numerous different Keq's. Thus, the Langmuir isotherm is not an excellent model for binding in this case.

Displacement chromatography:  

A molecule having a high affinity for the chromatography matrix will compete efficiently for binding sites, and therefore displace all molecules having lesser affinities. There are various differences between the displacement and elution chromatography. In elution mode, substances generally emerge from a column in narrow, Gaussian peaks. Broad separation of peaks, preferably to baseline, is desired in order to accomplish the maximum purification. The speed at which any component of a mixture travels down the column in elution mode based on numerous factors. However for two substances to travel at different speeds, and thus be resolved, there should be substantial differences in several interactions between the biomolecules and the chromatography matrix. The operating parameters are adjusted to maximize the effect of this difference. In most of the cases, baseline separation of the peaks can be accomplished only by gradient elution and low column loadings. Therefore, the two drawbacks to elution mode chromatography, particularly at the preparative scale, are operational complexity, because of gradient solvent pumping, and low throughput, because of low column loadings. Displacement chromatography has benefits over elution chromatography in that components are resolved to consecutive zones of pure substances instead of 'peaks'. As the method takes benefit of the nonlinearity of the isotherms, a larger column feed can be separated on a given column by the purified components recovered at significantly higher concentrations. Historically, displacement chromatography was applied to preparative separations of amino acids and rare earth elements and has as well been investigated for the isotope separation.

Techniques by physical state of mobile phase:

Gas chromatography (GC):

Gas chromatography (GC) is a general kind of chromatography employed in analytical chemistry for separating and analyzing compounds which can be vaporized devoid of decomposition. Typical utilization of GC comprises testing the purity of a specific substance, or separating the various components of a mixture (that is, the relative amounts of these components can as well be determined). In some conditions, GC might assists in recognizing a compound. In preparative chromatography, GC can be employed to make pure compounds from a mixture.

In gas chromatography, the mobile phase (or moving phase) is a carrier gas, generally an inert gas like helium or an unreactive gas like nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, within a piece of glass or metal tubing termed as a column (that is, an homage to the fractionating column employed in distillation). The instrument utilized to carry out gas chromatography is known as a gas chromatograph (or aerograph, gas separator).

The gaseous compounds being examined interact by the walls of the column that is coated by various stationary phases. This causes each and every compound to elute at a different time, termed as the retention time of the compound. The comparison of retention times is what provides GC its analytical value.

The Gas chromatography is in principle identical to column chromatography (and also other forms of chromatography, like HPLC, TLC), however consists of some notable differences. Firstly, the procedure of separating the compounds in a mixture is taken out between a liquid stationary phase and a gas mobile phase, while in column chromatography the stationary phase is a solid and the mobile phase is liquid. (Therefore the full name of the process is 'Gas-liquid chromatography', referring to the mobile and stationary phases, correspondingly.) Secondly, the column via which the gas phase passes is positioned in an oven where the temperature of the gas can be controlled, while column chromatography (generally) has no such temperature control. Thirdly, the concentration of a compound in the gas phase is only a function of the vapor pressure of the gas.

Gas chromatography is as well identical to fractional distillation, as both methods separate the components of a mixture mainly based on boiling point (or vapor pressure) differences.

Though, fractional distillation is generally employed to separate components of a mixture on a large scale, while GC can be employed on a much smaller scale (that is, micro scale).

Gas chromatography is as well at times termed as vapor-phase chromatography (VPC), or gas-liquid partition chromatography (GLPC). Such alternative names, and also their corresponding abbreviations, are often found in the scientific literature. Strictly speaking, GLPC is the most accurate terminology, and is therefore preferred by most of the writers.

=> GC analysis:

The gas chromatograph is a chemical analysis instrument for separating the chemicals in a complex sample. A gas chromatograph employs a flow-through narrow tube termed as the column, via which various chemical constituents of a sample pass in a gas stream (that is, carrier gas, mobile phase) at various rates based on their different chemical and physical properties and their interaction having a specific column filling, termed as the stationary phase. As the chemicals exit the end of the column, they are detected and recognized electronically. The function of the stationary phase in the column is to separate various components, causing each one to exit the column at a different time (that is, retention time). Other parameters which can be employed to modify the order or time of retention are the carrier gas flow rate, column length and temperature.

In GC analysis, an identified volume of gaseous or liquid analyte is injected to the 'entrance' (head) of the column, generally employing a micro syringe (or, solid phase micro-extraction fibers, or a gas source switching system). As the carrier gas sweeps the analyte molecules via the column, this motion is inhibited through the adsorption of the analyte molecules either to the column walls or to packing materials in the column. The rate at which the molecules progress all along the column based on the strength of adsorption, which in turn based on the kind of molecule and on the stationary phase materials. Since each kind of molecule consists of a different rate of progression, the different components of the analyte mixture are separated as they progress all along the column and reach the end of the column at different times (that is, retention time). A detector is employed to monitor the outlet stream from the column; therefore, the time at which each and every component reaches the outlet and the amount of that component can be found out. In general, substances are recognized (that is, qualitatively) by the order in which they emerge (elute) from the column and via the retention time of the analyte in the column.

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Fig: Schematic diagram of a gas chromatograph

=> Component of a gas chromatograph:

The choice of carrier gas (that is, mobile phase) is significant, having hydrogen being the most proficient and providing the best separation. Though, helium consists of a larger range of flow rates which are comparable to hydrogen in effectiveness, with the added benefit that helium is non-flammable, and works by a greater number of detectors. Thus, helium is the most general carrier gas utilized.

=> Detectors:

Detectors are the flame ionization detector (or FID) and the thermal conductivity detector (or TCD). Both are sensitive to a broad range of components, and both work over a broad range of concentrations. As TCDs are essentially universal and can be employed to detect any component other than the carrier gas (that is, as long as their thermal conductivities are different from that of the carrier gas, at detector temperature), FIDs are sensitive mainly to hydrocarbons, and are more sensitive to them than TCD. Though, an FID can't detect water. Both detectors are as well quite robust. As TCD is non-destructive, it can be operated in-series before an FID (destructive), therefore giving complementary detection of the similar analyte.

The other detectors are sensitive merely to particular kinds of substances, or work well just in narrower ranges of concentrations. Some of the gas chromatographs are joined to a mass spectrometer that acts as the detector. The combination is termed as GC-MS. Some of the GC-MS are joined to an NMR spectrometer that acts as a backup detector. This combination is termed as GC-MS-NMR. Some of the GC-MS-NMR is joined to an infrared spectrophotometer that acts as a backup detector. This combination is termed as GC-MS-NMR-IR. It should, though, be stressed this is extremely rare as most analyses required can be concluded through purely GC-MS.

Liquid chromatography:

Liquid chromatography (or LC) is a separation method in which the mobile phase is a liquid. Liquid chromatography can be taken out either in a column or a plane. Present day liquid chromatography which in general uses very small packing particles and a relatively high pressure is termed to as the high performance liquid chromatography (HPLC).

In HPLC the sample is forced via a liquid at high pressure (that is, the mobile phase) via a column which is packed by a stationary phase comprised of irregularly or spherically shaped particles, a porous monolithic layer or a porous membrane. HPLC is historically categorized to two different sub-classes based on the polarity of the mobile and stationary phases. Techniques in which the stationary phase is more polar than the mobile phase (example: toluene as the mobile phase, silica as the stationary phase) are known as normal phase liquid chromatography (NPLC) and the opposite (example: water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is known as reversed phase liquid chromatography (RPLC). Ironically the 'normal phase' consists of fewer applications and RPLC is thus considerably used more.

Affinity Chromatography:

The Affinity chromatography is a technique of separating biochemical mixtures and based on a highly specific interaction like that between antigen and antibody, enzyme and substrate or receptor and ligand.

=> Principle:

The immobile phase is generally a gel matrix, frequently of agarose; a linear sugar molecule derived from algae. Generally the beginning point is an undefined heterogeneous group of molecules in solution, like a cell lysate, growth medium or blood serum. The molecule of interest will encompass a well known and defined property that can be exploited throughout the affinity purification technique. The method itself can be thought of as an entrapment, by the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in solution will not become trapped as they don't acquire this property. The solid medium can then be eliminated from the mixture, washed and the target molecule discharged from the entrapment in a process termed as elution. Possibly the most general use of affinity chromatography is for the purification of the recombinant proteins.

=> Batch and column setup:

Binding to the solid phase might be accomplished via column chromatography whereby the solid medium is packed to a column, the initial mixture run via the column to let setting, a wash buffer run via the column and the elution buffer consequently applied to the column and collected. Such steps are generally done at ambient pressure. Alternatively binding might be accomplished by employing a batch treatment, by adding the initial mixture to the solid phase in a vessel, mixing, separating the solid phase (for illustration), eliminating the liquid phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and eliminating the eluate. At times a hybrid process is used, the binding is completed by the batch method, then the solid phase having the target molecule bound is packed to a column and washing and elution are completed on the column.

A third technique, expanded bed adsorption that joins the benefits of the two processes illustrated above, has as well been developed. The solid phase particles are put in a column where liquid phase is pumped in from the bottom and exits at the top. The gravity of the particles makes sure that the solid phase doesn't exit the column by the liquid phase.

Affinity columns can be eluted via changing the ionic strength via a gradient. Salt concentrations, pH, pI, charge and ionic strength can all be employed to separate or form the gradient to separate. 

Affinity chromatography can be utilized to:

  • Purify and concentrate a substance from 'ε'
  • Decrease the amount of a substance in the mixture
  • Discern what biological compounds bind to a specific substance
  • Purify and concentrate the enzyme solution.

Size-exclusion chromatography (SEC):

This is a chromatographic technique in which molecules in solution are separated via their size, and in several cases molecular weight. This is generally applied to large molecules or macromolecular complexes like proteins and industrial polymers. Generally, whenever an aqueous solution is utilized to transport the sample via the column, the method is termed as gel-filtration chromatography, versus the name Gel permeation chromatography, which is employed if an organic solvent is utilized as a mobile phase. SEC is a broadly employed polymer characterization process due to its capability to give good molar mass distribution results for the polymers.

=> Applications:

The major application of gel-filtration chromatography is the fractionation of proteins and other water-soluble polymers, whereas gel permeation chromatography is employed to examine the molecular weight distribution of the organic-soluble polymers. Either method should not be confused by gel electrophoresis, where an electric field is utilized to 'pull' or 'push' molecules via the gel based on their electrical charges.

=> Advantages:

The advantages of this technique comprise good separation of large molecules from the small molecules by a minimal volume of eluate, and that different solutions can be applied without interfering by the filtration procedure, all while preserving the biological activity of the particles to be separated. The method is usually joined by others which further separate molecules by other characteristics, like acidity, basicity, charge and affinity for some compounds. With size exclusion chromatography, there are short and well-defined separation times and narrow bands, that lead to good sensitivity. There is as well no sample loss as solutes don't interact by the stationary phase. Drawbacks are, for illustration, that merely a limited number of bands can be accommodated as the time scale of the chromatogram is short, and, in common, there has to be a 10 percent difference in molecular mass to encompass a good resolution.

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