Chromatography Terminology when a Russian scientist Tswett in 1906 used a glass columns packed with finely divided CaCO3 to separate plant pigments extracted by hexan The pigments appeared as colour bands that come out of the column one by one.
Tswett was the first to use the term “chromatography” derived from two Greek words “Chroma” meaning color and “graphein” meaning to write.
Definition of Chromatography
Tswett (1906) stated that ” chromatography is a method in which the components of a mixture are separated on adsorbent column in a flowing system”. In case of separation by adsorption the stationary phase is solid so it is called: Liquid-Solid Chromatography (LSC). If separation occurs through partition the stationary phase is liquid so it is called: Liquid -Liquid Chromatography
IUPAC definition (International Union of pure and applied Chemistry) (1993):
Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction.
The stationary phase may be a solid, or a liquid supported on a solid or gel, the mobile phase may be either a gas or a liquid.
Principles of Chromatography
Chromatography is a physical process. Any Chromatography system is composed of three Components :
Mixture to be separated
We can only control stationary and mobile phase as mixtures are the problem we have to deal with. Chromatography is a dynamic process in which the mobile phase moves in definite direction.
Classification of Chromatography Methods
Different methods were attempted for classification of chromatography:
A – According to mechanism of separation:
The mechanism of separation depends mainly on the nature of the stationary phase. Based on separation mechanisms chromatography can be classified into:
Adsorption Chromatography: It is the oldest and most common type of chromatography. The stationary phase is a solid with adsorption power. Mixture components will be adsorbed on the surface of the stationary phase with different powers and that account for separation. Silica gel is the most common stationary phase in adsorption chromatography
Partition Chromatography : The stationary phase is a liquid forming a thin film on an inert solid acts as support. The stationary liquid is usually more polar than the mobile liquid. The two liquids must be immiscible with each other. Cellulose powder and wet silica gel are examples of supports in partition chromatography that carry film of water act as stationary phase. Partition chromatography is preferable over adsorption when dealing with polar compounds
Ion Exchange Chromatography: It is used for separation of charged molecules. The stationary phase is an ion exchange resin to which a cationic or anionic groups are covalently bonded. Ions of opposite charges (counter ions) in the mobile phase will be attracted to the resin and compete with the components of the mixture for the charged group on the resin. Both the mixture components and the mobile phase must be changed. Mixture of Alkaloids (compounds with positive charges) can be separated on anionic exchanger, while mixture of organic acids (negative charges) can be separated using cationic exchanger. Both types are used for desalination of water
Molecular Exclusion (Size Exclusion) Chromatography: Stationary phase has pores of defined diameter. Very large molecules cant enter into the pores and so elute first, Large molecule enter with difficulties and so elute second. Small molecules enter all pores and elute as last ones
Affinity Chromatography: It uses the affinity of proteins to specific ligands such as enzymes. The ligand is attached to suitable polysaccharide polymer such as cellulose – agarose – dextran.
Chiral Chromatography: In this type we can separate enantiomers – we used chiral stationary phase that react with one enantiomer more then the other so separation takes place.
B – According to the mobile phase
Gas Chromatography (GC): Where the mobile phase is inert gas nitrogen or helium. Again if the stationary phase is solid it is called: Gas–Solid Chromatography (GSC). When stationary phase is liquid it is called: Gas-Liquid Chromatography (GLC).
C- According to the technique applied
(methods of holding the stationary phase)
1- Planar or Plane Chromatography: In this type of chromatography the stationary phase is used in the form of layer. Plane chromatography is further classified into:
a- Thin Layer Chromatography (TLC): The stationary phase in the form of fine powder is spread on glass or plastic or aluminum sheets.
b- Paper Chromatography (PC): A specific type of papers is used as stationary phase in the form of sheets.
2- Columnar or Column Chromatography (CC): The stationary phase is held in to a tube made of glass or metal.
D- According to purpose of use
Chromatography can be used for analytical work and also to obtain pure materials from mixtures.
1- Analytical Chromatography:
a- Qualitative Chromatography: In this case Chromatography can be used to:
1- Confirm the absence or probable presence of certain constituent in the sample under investigation:
2- Give an idea about the complexity of the mixture and the least number of compounds present.
3- Check purity and identity of any compound.
4- Establish a (finger print ) pattern for extracts , volatile oils or pharmaceutical preparations. These finger prints can be then used to check the identity and purity in the future.
5- Monitor both column chromatography and organic chemical reactions.
b- Quantitative Chromatography: The development of modern instruments enable the use of chromatography to determine the amount of any component in a mixture as absolute amount or relative to another component HPLC/ GC/ HPTLC can be used for there applications.
2- Preparative and Industrial scale Chromatography : This was the first and is the main application of chromatography. The technique was developed primarily for this purpose. Chromatography is used to obtain reasonable quantities of pure compounds from mixtures.
Glossary of Terms
Activity. In adsorption chromatography, the relative strength of the surface of the packing. For silica gel, the more exposed the silanol groups, the more active the surface. Activity can be controlled by adding water or another polar modifier, which is hydrogen-bonded to the active sites, thereby reducing the surface activity.
Affinity Chromatography. A technique in which, for the macromolecule of interest, a biospecific adsorbent is prepared by coupling a specific ligand (such as an enzyme, antigen or hormone) to a solid support (or carrier). This immobilized ligand will interact only with molecules that can selectively bind to it. Molecules that will not bind elute unretained. The retained compound can later be released in a purified state. Affinity chromatography is a selective filtration rather than a chromatographic technique.
Asymmetry (As). Asymmetry (skew) is a factor describing the shape of a chromatographic peak. Theoretically it is assumed peaks are symmetrical and of Gaussian shape. Practically, the peak asymmetry factor is measured as shown below. A value of >1 indicates a tailing peak and <1 a fronting peak.
Bar. A unit of pressure equal to one atmosphere. It is equivalent to 14.5 pounds per square inch (psi) or 0.1 Megapascal.
BET Method. A method developed by Bruner, Emmett and Teller for measuring surface area. The level of liquid nitrogen adsorption within the pores of the phase is measured at very low temperatures. Pore volume and pore size distribution methods can also be obtained by this method.
Biocompatible. It refers to a tubing or fitting material that will not change the biological activity of material coming into contact with it during the HPLC analysis time.
Bonded. Term which implies that the stationary phase is chemically bonded to the surface of the supporting material.
Bonded Phase Coverage. It refers to the amount of bonded phase on a silica support. Coverage is usually described in mmol/m2 or in terms of percentage carbon.
Capacity Factor (k’). A factor which measures sample retention (tR) independently of eluent flow rate or column
length. Retention times are measured relative to the column dead-time (t0). Its calculation is shown on the end of this page
Channelling. Occurs when voids created in the packing material of a column may cause eluent and accompanying solutes to move more rapidly than the average flow velocity, resulting in band broadening. The voids are created by poor packing or by erosion of the packed bed.
Check Valve. A device built into an HPLC pump which allows the flow of eluent in one direction only.
Column Dead-time (t0). The time taken for solvent molecules or other non-retained peaks to move through the column.
Column Efficiency (N). A term used to express the width of a peak produced by a column. Efficiency is measured in terms of the number of plates, a parameter which is inversely related to the square of the peak width.(See above under Assymetry) for the full calculation.
Counterion. In an ion-exchange process, the ion in solution used to displace the ion of interest from the ionic site. In ion-pairing, it is the ion of opposite charge added to the eluent to form a neutral ion pair in solution.
Dead Volume. A measure of solvent accessible volume between injector and detector after the space occupied by the column packing material has been subtracted. Both interstitial (intraparticle and interparticle) column volumes and system (injector, detector, connecting tubing and end fittings) volumes contribute. The dead volume can be determined by injecting an inert compound (ie. a compound that does not interact with the column packing) and measuring its retention volume.
Degassing. The practice of removing dissolved gases in the eluent. It can be achieved by helium sparging, applying vacuum to the eluent, ultrasonification or heating.
Denaturing. The process of destroying the tertiary and quaternary structure of a protein.
Desalting. A technique in which low molecular weight salts and other compounds are removed from non-ionic and high molecular weight compounds. An example is the use of size exclusion columns to exclude large molecules and retain lower molecular weight salts.
Eluotropic Series. A series of solvents with an increasing degree of polarity, generally used to explain solvent strength.
Endcapping. The reaction of a silylating reagent with unreacted accessible silanols remaining on the silica surface after the initial bonding reaction. The process may reduce undesirable adsorption of basic or polar molecules which otherwise may cause peak tailing.
Exclusion Limit. In SEC, the upper limit of molecular weight (or size), beyond which molecules will elute at the same retention volume (exclusion volume). Many SEC packings are referred to by their exclusion limit. For example, a 105 column of porous silica gel will exclude any compounds with a molecular weight higher than 100,000 based on a polystyrene calibration standard.
Flash Chromatography. A very fast form of classic LC used by synthetic organic chemists for rapid purification. Performed primarily in the normal-phase mode, sometimes with reversed-phase chromatography.
Fractionation Range. In SEC, refers to the range in which the packing can separate molecules based on their size. Molecules that are too large to diffuse into the pores are excluded. Molecules that can diffuse into all of the pores totally permeate the packing, eluting unseparated at the permeation volume.
Fronting. A term describing a peak shape whose front has a leading edge. Gel Filtration Chromatography (GFC). SEC carried out with aqueous eluents. It is sometimes referred to as aqueous GPC. Most gel filtration separations involve biopolymers.
Gel Permeation Chromatography (GPC). SEC carried out with organic eluents. Used for the separation and characterisation of polymers.
Gradient Elution. The process by which the strength and composition of the eluent is increased during the chromatographic run thereby reducing analysis time.
Guard Column. A short column placed between sample injector and the inlet of the main column. It protects the analytical column against contamination from sample particulates and strongly retained solutes. The guard column is usually of cartridge format and packed with the same material as in the main column.
Helium Sparging. The process of bubbling helium through the eluent to remove dissolved gas.
High Pressure Mixing. A procedure in which two or more solvents are mixed on the high pressure side of the pumping system to form a final eluent. One pump is required per solvent.
Hydrophilic. A description of compounds, solvents or bonded phases that either readily dissolve in water or prefer water to non-polar organic solvents, ie. ‘water-loving’.
Hydrophilic Interaction Chromatography (HILIC). The use of polar stationary phases and partially aqueous eluents to separate compounds in order of increasing hydrophilicity (polarity).
Hydrophobic. A term describing compounds, solvents or bonded phases that dissolve easily in non-polar organic solvents such as hexane or prefer such solvents to water, ie. ‘water-hating’.
Hydrophobic Interaction Chromatography (HIC). A protein separation technique in which reversed-phase materials are used with eluents containing high salt concentrations. Gradients are run by decreasing salt concentrations with time.
Injection Solvent. The solvent the sample is dissolved in prior to chromatographic analysis.
Interaction in Chromatography
Hydrophobic interaction: Hydrophobicity is the physical property of a molecule (known as a hydrophobe )
Ion-exchange Capacity. A measure of the number of ionic sites that can take part in the exchange process. Exchange capacity is expressed in mequiv/g. Ion Exclusion. The process in which ionised solutes can be separated from non-ionised or partially ionised solutes using ion-exchange resins. Ionised solutes will move faster down the column.
Ion-pair Chromatography. A form of reversed-phase chromatography in which ions in solution can be paired or
neutralised prior to separation as an ion-pair. Ion-pairing reagents are usually ionic compounds that contain a hydrocarbon chain. The latter imparts a certain hydrophobicity to the resultant ion-pair allowing it to be retained on a reversed-phase column.
Isocratic. Chromatographic conditions in which a constant composition eluent is used.
Isoelectric Point. The pH point at which a molecule no longer has a net charge.
Life expectancy of used and unused HPLC column We don’t have any documentation that gives specific recommendations about how long HPLC columns can be kept. There are no set ‘rules’ and people have widely differing opinions about how long columns should be kept before they are used for the first time. When an older column is found not to perform as might be expected you can never be sure that it has not ‘aged’ in some way and so the performance is not as good as it would have been when it was new. This ‘aging’ might be method dependent and effect some types of sample more than others.
We believe that it is good practice for columns not to be kept any longer than a year after they have been manufactured before they are sold for use. We actively manage our stock of manufactured and distributed columns to try and ensure that this does not happen and to minimise the amount of time that a column spends waiting on the shelf.
Ligand-exchange Chromatography. A technique in which chelating ligands are added to the eluent. On adsorption onto the stationary phase they act as chelating agents. An example is the use of copper amine chelates for the separation of amino acids.
Low Pressure Mixing. A pumping procedure in which two or more solvents are mixed on the low pressure side of the pump. Only one pump is required.
Mean Pore Diameter. A term that refers to the average diameter of the pores within a phase.
MegaPascal (Mpa). A unit of pressure. One Mpa equals about 10 bar (atmospheres) or 145 pounds per square inch (psi).
Modifier. A chemical added to reversed-phase solvent systems designed to optimise the chromatographic separation.
Monomeric Phase. A bonded phase in which individual molecules are bonded to a support. For silica, monomeric phases are typically prepared by the reaction of an alkyl monochlorosilane.
Overload. A saturation of the stationary phase by the solute which is evidenced by band broadening, tailing and
flat edged chromatographic peaks.
Particle Size Distribution. A measure of the distribution of the particles used to manufacture a column. In HPLC a narrow particle size distribution is desirable. For a 10μm size particle, a particle size distribution of dp±10% means that 90% of the particles have a 9-11μm size.
Particle Size (dp). This term refers to the average particle size of the material packed into a column.
Peak Broadening. The tendency of a chromatographic peak to broaden as it passes through the column. It is also known as peak spreading or peak dispersion. The peak width or the number of theoretical plates in the column (N) is a measure of peak broadening.
Polarity. A measure of the separation of charges within a molecule. Polar molecules interact more strongly with and are best separated on polar stationary phases.
Polymeric Packing. Packings based on polymeric materials, usually in the form of spherical beads. Common polymers include polystyrene-divinylbenzene, polymethacrylate and polyvinylalcohol.
Polymeric Phase. A bonded phase in which typically a di- or trichlorosilane is reacted with more than one reactive silanol group on the surface of silica.
Pore Size (Mean Pore Diameter). The average pore diameter of the pore in a porous packing. The pore diameter is important in that it must allow free diffusion of solute molecules into and out of the pore so that the solute can interact with the stationary phase. In SEC, the packings have different pore diameters, and therefore molecules of different sizes can be separated. For a typical adsorbent such as silica gel, 60Å and 100Å pore diameters are most popular. For packings used for the separation of biomolecules, pore diameters >300Å are used.
Pore Volume. The total volume of the pores in a porous packing, usually expressed in ml/g. It is measured by the BET method of nitrogen adsorption or by mercury intrusion, in which mercury is pumped into the pores under high pressure.
Precolumn. A column packed with silica placed between the pump and the injector. It presaturates the eluent with stationary phase minimising loss of the latter from the main column. It will also remove particulate material
Pressure Drop. The difference in pressure between the inlet and outlet of a column during flow caused by the hydrodynamic resistance of the packed bed.
Residual Silanols. These are the silanol (-SiOH) groups that remain on the surface of a silica after a bulky phase is chemically bonded to its surface. Their numbers can be reduced by further reacting (endcapping) the silica surface with a small organosilane.
Resolution. A measure of the separation of two adjacent peaks. The higher the resolution value the greater the separation (see HPLC Calulation below)
Retention Time. The elapsed time between sample injection and the appearance of the chromatographic peak apex.
Sample Capacity. The term refers to the amount of sample that can be injected onto a column without overloading it. In preparative applications it is typically expressed as grams of solute per gram of stationary phase.
Selectivity (α). The ratio of capacity factors of adjacent peaks (see p.342). Also referred to as separation factor or relative retention ratio.
Siloxane Bond. The main -Si-O-Si- bond found in silica.
Size Exclusion Chromatography (SEC). A mode of HPLC used mainly to separate high molecular weight samples and to determine their molecular weight distribution by virtue of their size in solution. Also known as gel permeation, gel filtration or steric exclusion chromatography.
Surface Area. The total area of the phase’s solid surface. For silica it is typically 100-600 m2/g. Tailing. The phenomenon in which a peak has an asymmetry factor >1. The downside of the peak will be skew.
Theoretical Plate. Measure of column efficiency. Length of column relating to this concept is called height equivalent to a theoretical plate (HETP).
Void. The formation of a space, usually at the head of the column, caused by a settling or dissolution of the packing. A void in the column leads to decreased efficiency and loss of resolution.
Void Volume (V0). The total volume of eluent in the column, the remainder being taken up by packing material. Can be determined by injecting an unretained substance.
Zero Dead Volume (ZDV). It refers to a fitting which adds no extra volume to the system. In practice ZDV fittingshave a finite but insignificant volume.
Zwitterions. Compounds that carry both positive and negative charges in solution.
AA Amino acid
ADP Adenosine 5′-diphosphate
AGP α1-acid glycoprotein
amu Atomic mass unit
ATP Adenosine 5′-triphosphate
AU Absorbance units
AUFS Absorbance units full scale
BDS Base deactivated silica
BSA Bovine serum albumin
CCC Countercurrent chromatography
CE Capillary electrophoresis
CEC Capillary electrochromatography
CI Chemical ionisation
CSFC Capillary supercritical fluid chromatography
CSP Chiral stationary phase
CT Charge transfer
CTAC Cetyltrimethylammonium chloride
CZE Capillary zone electrophoresis
DAC Dynamic axial compression
DACC Donor-acceptor complex column
DAD Diode array detection
Dabsyl 4-Dimethylaminoazobenzene-4-sulphonyl chloride
Dansyl 5-Dimethylaminonaphthalene-1-sulphonyl chloride
DEG Diethylene glycol
DHPLC Denaturing HPLC
DMSO Dimethyl sulphoxide
DNA Deoxyribonucleic acid
DRI Differential refractive index
ECD Electrochemical detection
EDTA Ethylenediaminetetraacetic acid
EI Electron impact
ELISA Enzyme-linked immunosorbent assay
ELSD Evaporative light-scattering detector
FAB Fast atom bombardment
FAME Fatty acid methyl ester
FFAP Free fatty acid phase
FIA Flow-injection analysis
FID Flame ionisation detection
FLEC 1-(9-fluorenyl)ethyl chloroformate
FMOC 9-Fluorenylmethoxycarbonyl chloride
FPD Flame photometric detection
FPLC Fast protein liquid chromatography
FTIR Fourier transform infrared
GC-MS Gas chromatography-mass spectrometry
GFC Gel filtration chromatography
GLC Gas-liquid chromatography
GPC Gel permeation chromatography
HAC Hydroxyapatite chromatography
HEPES Hydroxyethylpiperazineethanesulphonic acid
HETP Height equivalent to a theoretical plate
HFBA Heptafluorobutyric acid
HIC Hydrophobic interaction chromatography
HILIC Hydrophilic interaction chromatography
HPLC High performance liquid chromatography
HPTLC High performance thin layer chromatography
HRGC High resolution gas chromatography
HSA Human serum albumin
IAM Immobilised artificial membrane
IC Ion chromatography
ICP Inductively coupled plasma
ICR Ion cyclotron resonance
IEC Ion-exchange chromatography
IMAC Immobilised metal-ion affinity chromatography
IPA Isopropanol (propan-2-ol)
IPC Ion pair chromatography
ISRP Internal surface reversed-phase
IUPAC International Union of Pure and Applied Chemistry
LDR Linear dynamic range
LDV Low dead volume
LEC Ligand exchange chromatography
LIF Laser-induced fluorescence
MECC Micellar electrokinetic capillary chromatography
MEKC Micellar electrokinetic chromatography
MEPS Microextraction in packed syringe
MS Mass spectrometer
MSD Mass-selective detection
NIR Near infrared
NPC Normal-phase chromatography
NPR Non-porous resin
OTLC Open-tubular liquid chromatography
PAD Pulsed amperometric detection
PAGE Polyacrylamide gel electrophoresis
PAH Polycyclic aromatic hydrocarbon
PAT PEEK alloyed with Teflon
PCR Polymerase chain reaction
PCR Post column reaction
PEO Polyethylene oxide
PHRED Photochemical reactor enhancement detection system
PID Photoionisation detection
PIPES Piperazine-1,4-diethanesulphonic acid
PLOT Porous-layer open-tubular
PRP Polymeric reversed-phase
RAM Restricted access media
RI Refractive index
RPLC Reversed-phase liquid chromatography
SAX Strong anion-exchanger
SCOT Support-coated open-tubular
SCX Strong cation-exchanger
SDS Sodium decyl (or dodecyl) sulphate
SEC Size (or steric) exclusion chromatography
SFC Supercritical fluid chromatography
SFE Supercritical fluid extraction
SIM Selected (single) ion monitoring
SMB Simulated moving bed
SPE Solid phase extraction
SPME Solid phase microextraction
SPS Semipermeable surface
SRM Standard reference material
TBAHS Tetrabutylammonium hydrogensulphate
TEAA Tetraethylammonium acetate
TFA Trifluoroacetic acid
WAX Weak anion-exchanger
WCX Weak cation-exchanger
WCOT Wall-coated open-tubular
ZDV Zero dead volume