CH ROM ATO GRA PHY. Jigarkumar B. Patel K.B.I.P.E.R. INTRODUCTION. History of chromatography. Chromatography was first developed and defined by the russian botanist Mikhail S. Tswett in 1903.
Jigarkumar B. Patel
Chromatography was first developed and defined by the russian botanist Mikhail S. Tswett in 1903.
He produced a colorful separation of plant pigments using a column of calcium carbonate (chalk).
The word chromatography comes from the Greek words for color "chroma" and write "graphein". So chromatography means \'to write with color\'
1938 Paper and TLC
1952 Gas-liquid chromatography GLC
1968 High pressure liquid chromatography HPLC
1980s Super critical fluid chromatography SFC
In all chromatography there is a mobile phase and a stationary phase.
The stationary phase is the phase that doesn\'t move and the mobile phase is the phase that does move.
The mobile phase moves through the stationary phase picking up the compounds to be tested.
As the mobile phase continues to travel through the stationary phase it takes the compounds with it.
At different points in the stationary phase the different components of the compound are going to be absorbed and are going move with the mobile phase.
This is how the separate from the other components is achieved.
USP 2000: “Chromatography is procedure by which solutes are separated by dynamic differential migration process in a system consisting of two or more phases, one of which moves continuously in given direction and in which the individual substance exhibits different mobilities by reasons of differences in adsorption, partition, solubility, vapor pressure, molecular size or ionic charge density. The individual substance thus separated can be identified or quantitatively measured by suitable analytical method.”
Chromatography is used by scientists to:
Analyze – examine a mixture, its components, and their relations to one another
Identify – determine the identity of a mixture or components based on known components
Purify – separate components in order to isolate one of interest for further study
Quantify – determine the amount of the a mixture and/or the components present in the sample
Real-life examples of uses for chromatography:
Pharmaceutical Company – determine amount of each chemical found in new product
Hospital – detect blood or alcohol levels in a patient’s blood stream
Law Enforcement – to compare a sample found at a crime scene to samples from suspects
Environmental Agency – determine the level of pollutants in the water supply
Manufacturing Plant – to purify a chemical needed to make a product
Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase)
Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)
Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase)
Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)
Basically, molecules can be separated from one another if there are differences in one or more of the following:
Separation of closely related components of a complex mixture. Mobile phase forced through a stationary Phase (column or a planar surface).Components of a mixture distribute between mobile and stationery phase to varying degree. More solubility in Mobile Phase – fast progress- low retention Time (Rt). More solubility in stationery phase – slow progress – long retention time(Rt)
In the animation below the red molecules have more affinity towards the stationary phase than the green molecules.
For example, suppose we have a mixture of two solutes, A and B, dissolved in a suitable buffer solution.
Detector signal vs. retention time or volume
Let us assume, for the purpose of this illustration, that solute B binds more strongly to the matrix than solute A.
Nature of mobile phase :
On the basis of types of Mobile Phase and Stationary Phase and the kinds of equilibrium involved in the transfer of solutes between the two phases. Liquid Chromatography Gas Chromatography Supercritical Chromatography
Based on instrument/ apparatus
Based on working principle/
Mode of chromatography
Nature of Stationary Phase
1-3: Gives separation basically
4-7: give separation and separated compound is analyzed quantitatively and qualitatively.
Ion exchange Chromatography
Size exclusion Chromatography
3) Thin Layer Chromatography:
Separation and identification of very small quantity of drug (µg), limit test, assay.
Advantage: fast as compared to PC.
a) Two Dimensional Thin Layer Chromatography
b) Continuous development TLC
c) High Performance Thin Layer Chromatography (HPTLC)
4) Gas Chromatography:
For gaseous and volatile substance.
a) Gas-Solid Chromatography (GSC)
b) Gas-liquid Chromatography (GLC)
5) High Performance/Pressure Liquid Chromatography:
a) Normal Phase HPLC
b) Reverse Phase HPLC
6) Super Critical Fluid Chromatography
7) Electro Chromatography/ Capillary Electrophoretic Chromatography
Adsorption chromatography is probably one of the oldest types of chromatography around.
It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase.
The equilibriation between the mobile and stationary phase accounts for the separation of different solutes.
2) Partition (liquid-liquid) Chromatography:
Substance having similar chemical type.
Both the phases must be liquid.
one having greater partition co-efficient in m.p. than s.p., it will move fast and vice versa.
Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography)
This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase.
Solute equilibriates between the mobile phase and the stationary liquid
3) Ion-exchange Chromatography:
Ionic and inorganic Substance.
Separation is based upon ionic charge density.
S.P.: ion exchangers which has capacity to exchange ions.
Support chemically bonded to anionic or cationic group
Separation on the basis of charge on the solute on the principle of opposite charges attracting each other.
Resolution influenced by pH & ionic strength of buffer.
Cationic Sulfonate exchanger strongly acidic
Carboxylate exchanger weak
Anionic Quaternary ammonium strongly basic
Tertiary amine weak
In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it.
Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.
4) Size exclusion(MolecularExclusion/gel permeation) chromatography:
Size exclusion chromatography is a type of chromatography in which the stationary phase is a molecular sieve (Different gel material).
Therefore, the molecules are separated according to differences in their molecular size.
Non interactive mode of separation.
The particles of column packing have various size pores and pore networks
Solute molecules are retained or excluded on the basis of their hydrodynamic volume.
- semi rigid cross linked - polymer gels
- rigid controlled pore size glasses or silicas
Also known as gel permeation or gel filtration,
This type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size.
The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.
This is the most selective type of chromatography employed.
It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase.
For example, the immobilized molecule may be an antibody to some specific protein.
When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase.
This protein is later extracted by changing the ionic strength or pH.
Two types of partition chromatography:1. Liquid-liquid --- liquid stationary phase is held on support particles through physical adsorption, easy to lose during the mobile phase running.2. Bonded-phase --- the stationary phase is chemical bonded to support surfaces.In early partition chromatography, liquid-liquid type dominated, but nowadays bonded phase has become predominant.Partition chromatography, now normally referred as to Bonded-phase chromatography, has two types:
Partition Chromatography: a Typical LC
Normal-phase LC:The stationary phase is polar (silica or chemically modified surface) and the mobile phase is non-polar (hexane, ether or other non-polar organic solvents). Good for separation of non-polar solutes.Reversed-phase LC:column packing is made of chemically modified silica (normally with non-polar surface) and the mobile phase is polar (water, or polar organic solvents like alcohols, acetonitrile). Excellent for solubilizing and separating polar solutes. This type of LC is very useful for biological or medical research which normally deal with water solutions.LC plays with balancing average concentration in mobile phase versusconcentration in the stationary phase.Keq = Cstat/Cmobile
Nature of stationary phase
i) Normal phase
Stationary phase - silica, alumina
Mobile phase - CH2Cl2, IPA, MeOH, Hexane
ii) Reverse phase
Stationary phase - non polar
Mobile phase - MeOH, ACN, Aqueous buffers
1.Displacementofsolvent molecules which are adsorbed on the stationary phase.
2. Sorption of solute molecule into a layer of solvent molecule without their displacement from stationary phase.
Retention of solutes decreases with decreasing polarity
1 – Uracil
2 – Phenol
3 – Acetophenone
4 – Nitrobenzene
5 – Methyl Benzoate
6 – Toluene
Stationary phase selection criterion is highly dependent on the particular application and separation aims.
1. C18 (Octyldecylsilane – ODS) Bonded Ligand
The most popular and functional of the available reversed-phase stationary phases.
Particularly useful for the separation of non-polar analyte species.
2. C8 Bonded Ligand
While less non-polar than the C18, it provides similar selectivity while giving appreciably shorter run times.
3. C4 / C3 Bonded Ligand
These shorter-chain hydrocarbons are not as stable or as retentive as their longer chain C18 and C8 counterparts, but provide good separations for a variety of protein and polypeptide analytes.
4. CN (Cyano) Bonded Ligand
Interacts with polar functional groups, allowing its use in both reversed-phase and normal phase chromatography.
5. NH2 (Amino) Bonded Ligand
Interacts with polar functional groups. Commonly used for the separation of sugars and polysaccharides.
6. Phenyl Bonded Ligand
Exhibits a more polar nature than either the C18 or C8, the π electron clouds providing sites of interaction for a variety of aromatic (ring) analytes.
Analyte retention increases with increasing stationary phase carbon chain length.
Some typical bonded phases used in reversed-phase HPLC are described below:
In normal-phase HPLC, the stationary phase is polar, often the silica itself, and the mobile phase is relatively non-polar (such as hexane, iso-propylether, toluene,etc.).
The least polar component elutes first.
Increasing the polarity of the mobile phase decreases the elution time.
In reversed-phase HPLC, the stationary phase is non-polar, often a hydrocarbon, and the mobile phase is relatively polar (such as water, methanol or acetonitrile).
The most polar component elutes first.
Increasing the polarity of the mobile phase increases the elution time.
1 Normal phase
CN rugged, fairly polar, general utility
OH more polar than cyano
NH2 highly polar, less stable
Silica rugged, cheap, less convenient to operate,used in prep LC
2 Reversed phase
C18 rugged, highly retentive
C8 slightly less retentive than C18
C3, C4 less retentive, mostly for peptides & proteins
C1 least retentive, least stable
Phenyl moderately retentive, some selectivity difference
CN moderately retentive, both normal &reverse phase
NH2 weak retention, less stable, carbohydrates
Polystyrene pH 1 - 13, better shape & longer life for some separations
Today there is a large number of bonded phases available with an extensive range of polarities but, of these, the reverse phases are by far the most commonly used in modern LC.
In liquid chromatography, liquid-liquid systems are unstable as, however small the solubility of the stationary phase may be in the mobile phase, the stationary liquid phase will be eventually stripped from the column.
A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
A stationary phase chemically bonded to a support that is used for the separation.
It is the most commonly used LC mode.
The most popular support used is microparticulate silica gel.
An organosilane, such as octadecyl (for reversed-phase chromatography), is the most accepted type of bonded phase.
Approximately 70 percent of all HPLC is carried out on chemically bonded phases.
Some Commonly Used Silane Reagents Used in Bonded Phase Synthesis
Alternately treating the bonded phase with water and a dichlorsilane,a series of methyloctylsilyl residues can be attached to each other that builds up the oligomeric reverse phase.
When the last oligomeric synthesis has been completed and the final oligomer attached, the remaining silanol groups are capped with trimethyl chlorsilane or hexamethyltrisilazane.
In the second stage it is presumed that the \'octyldichloro\' chain reacts with some of the excess water to from a \'octyldihydroxy\' chain.
When the silica surface is saturated with water and is treated with octyltrichlorsilanea reaction occurs with both the hydroxyls of the silica surface and the adsorbed water, causing a cross-linking reaction and the octylsilanyl polymer is built up on the surface.
As a result of the polymerization process, the stationary phase assumes a multi-layer character and that is why it is termed a \'bulk\' phase.
The \'octyldihydroxy chain can, in turn react, with two more octyltrichlorsilane molecules and start building the polymer on the surface.
The Distribution of Solvents A and B as a Mono-layer on a Silica Gel Surface
The Distribution of Solvents A and B as a Bi-layer on a Silica Gel Surface