M.Prasad Naidu

MSc Medical Biochemistry,

Ph.D.Research Scholar

Chromatography is a physical process whereby components ( solutes ) of a sample mixture are separated by their differential distribution between stationary & mobile phases .

Planar & column are two basic forms of chromatography .

High performance liquid chromatography is a form of column chromatography .

During column chromatography process mobile phase carries the sample through the column containing stationary phase .

As the mobile phase flows through the stationary phase the solutes may

1) Reside only on stationary phase ( no migration ) ,

2) Reside only in the mobile phase ( migration with mobile phase ) ,

3) Distribute between two phases ( differential migration ) .

The basis of all forms of chromatography is partition or distribution coefficient ( Kd ) .

Kd describes the way the solute distribute it self between two immiscible phases .

Distribution coefficient is a constant at a given temperature for two immiscible phases A & B .

concentration in phase A Kd = concentration in phase B

In column chromatography , the stationary phase may be pure silica or polymer , or it may be coated onto , or chemically bonded to, support particles .

The stationary phase may be coated into a tube , or it is coated on inner surface of the tube .

When the mobile phase is liquid it is called liquid chromatography ( LC ) .

When the stationary phase in LC consists of smaller diameter particles the technique is high performance liquid chromatography .

In analytical liquid chromatography the mobile phase or eluent , exits from the column & passes through a detector or a series of detectors that produce a series of electronic signals that are plotted as a function of time distance or volume , the resulting graph is a chromatogram .

The retention time ( tR ) is the time taken for each analyte peak to emerge from the column .

Under defined chromatographic conditions tR is a charcteristic of the analyte .

The volume of the mobile phase required to elute the analyte under defined chromatographic conditions is referred to as retention ( or ) elution volume ( VR ) .

VR = tR Fc

Eluting solutes are displayed graphically as a series of peaks , they are frequently referred to as chromatographic peaks .

These peaks are described in terms of peak width , peak height & peak area .

The data represented by the chromatogram are used to help identify & quantify the solutes .

Most important parameter in column chromatography is the partition ratio ( or ) capacity ratio K’ .

Capacity ratio has no units & it is a measure of the additional time the analyte takes to elute from the column relative to an unretained or excluded analyte that does not partition into stationary phase .

K’ = tR – tM = VR – VM tM VM Capacity ratios characterize the column

performance . The success of any chromatographic procedure

is measured by it’s ability to separate completely ( resolve ) one analyte from a mixture of similar compounds .

Peak resolution ( Rs )is related the properties of the peaks .

Rs = 2 ( tRB – tRA ) WA + WB tRA & tRB are the retention times of compounds

A & B respectively , & WA & WB are base widths of peaks for A & B , respectively .

When Rs = 1.5 the separation of the two peaks is 99.7 % complete .

In most practical cases Rs value of 1.0 corresponds to 98 % of separation , are adequate for quantitative analysis .

Peak asymmetry has many causes ,

1) Application of too much analyte to the column ,

2) Poor packing of the column , 3) Poor application of the sample to the column

or solute support interactions .

Chromatography columns consists of number of adjacent zones each zone is called theoretical plate & its length in the column is called plate height .

The more efficient the column the greater the number of theoretical plates are involved .

N = 16 ( tR/W )2

The plate number can be increased by increasing the column length, but there is a limit to this because the retention time & peak width increases proportionally L , where as the peak height decreases as the square root of N .

Good resolution is determined by the following 3 functions :

1) Selectivity , 2) Efficiency ,3) Capacity . Selectivity is a measure of inherent ability

of the system to discriminate between structurally related compounds .

Two structurally related compounds differ in Kd or K’ .

Ratio of partition coefficient of two compounds gives relative retention ratio ,α .

Efficiency is the measure of diffusion effects that occur in the column to cause peak broadening & over lap .

Capacity is a measure of the amount of material that can be resolved without causing peaks to overlap irrespective of actions like gradient elution .

The limit to the length of the column is due the problem of peak broadening .

The number of theoretical plates is related to the surface area of the stationary phase therefore smaller the particle size of the stationary phase , the better is the resolution.

The Smaller the paritcle size , the greater is the resistance to flow of the mobile phase .

The resistance in flow causes back pressure in the column that is sufficient to damage the matrix structure of the stationary phase .

The new smaller particle size stationary phases that can withstand high pressures caused dramatic development in the column chromatography .

The increased resolution achieved in HPLC compared to classical chromatography is primarily the result of adsorbents of very small particle size ( less then 20µm )& large surface areas .

The smallest gel beads used in gel exclusion chromatography are superfine grade with diameters of 20-50µm .

A combination of high pressure & adsorbents of smaller size leads to high resolution power & short analysis time in HPLC .

(1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position", (6') Switching valve in "load position", (7) Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.

Solvent reservoir should have a capacity of at least 500 ml for analytical applications , but larger reservoirs are required for preparative work .

In order to avoid the bubbles in the column & detector the solvent must be degassed .

Several methods are there for degassing : 1) By warming the solvent ,2) By vigorous stirring with magnetic stirrer ,3) By ultrasonication ,4) By subjecting solvent to vacuum or by

bubbling helium gas through the solvent reservoir .

Typical requirements for a pump are :1 ) it must be capable of pressure outputs of at

least 500 psi & preferably up to 5000 psi . The main feature of good pumping system is

that it can capable of outputs of at least 5x107 pascals ( 7200 psi ) .

2) Pump should have a controled , reproducible flow delivery of about 1ml/min for anlytical applications & up to 100ml/min for preparative applications .

3 ) it should yield pulse free solvent flow 4) It should have a small hold up volume .

The correct application of the sample on to the HPLC column is particularly important factor in achieving successful separations .

Two injection methods are existing First method makes use of a microsyringe to

inject the sample either directly on to the column packing or onto a small plug of inert material immediately above the column packing .

The second method of sample injection retains the column pressure by use of a loop injector .

Metal loop has as fixed small volume that can be filled with sample .

By means of an appropriate valve switching system , the eluent from the pump is channelled through the loop , the outlet of the loop leads directly onto the column .

Therefore sample is flushed on to the column by eluent without interruption of flow to the column .

Repeated application of highly impure samples such as sera , urine , plasma or whole blood are preferably deproteinated because they decrease the resolving power of the column .

To prevent the above problem a guard column is frequently installed between the injector & the analytical column .

Guard column is a short column of the same internal diameter & packed with material similar to analytical column .

The packing in the guard column retains contaminating material & can be replaced at regular intervals .

Sample preparation is essential preliminary action in HPLC , particularly for the test compounds in a complex matrix such as plasma , urine , cell homogenate .

For analysis of drugs in biological fluids sample preparation is relatively much simpler.

Sample preparation is done by clean up techniques they are :Solvent extraction ,Solid phase extraction ,Column switching & newer supercritical fluid extraction ( under research )Derivatization .

For HPLC analysis many analytes are chemically derivatized before or after chromatographic separation to increase their ability to be detected .

Eluted amino acids are reacted with ninhydrin in post column reactor , the resulting chromogenic species are detected by photometer .

Aliphatic amino acids , carbohydrates , lipids & other substances do not absorb UV can be detected by chemical derivatization with UV absorbing functional groups .

Precolumn derivitization for amino acids & peptides is by phenyl isothiocyanate , dansyl chloride for UV column detection .

Precolumn derivatization for fatty acids , phospholipids is by phenacyl bromide for UV column detection .

Post column derivatization for carbohydrates is by orsinol & sulphuric acid for UV column detection

Column is made up of stainless steel . Column has to withstand pressures of up to 5.5

X 107 pascal . Straight columns of 15 – 50 cm length & 1 –

4mm diameter & has flow rate of 2 cm3/ min.

Preparative columns have an internal diameter of 25 mm & has flow rate of 100 cm3 / min.

Three form of column packing matrices are available they are :

1) Microporous supports : ( micropores ramify through the particles which are generally 5 – 10 µm in diameter ),

2) Pellicular ( superficially porous ) supports : in which porous particles are coated on to an inert solid core such as a glass bead of 40 µm in diameter ,

3) Bonded phases : in which stationary phase is chemically bonded to an inert support such as silica .

For adsorption chromatography , adsorbents such as silica & alumina are available as microporous or pellicular forms which are suitable for HPLC .

Pellicular forms have high efficiency but low sample capacity therefore microporous supports are preferred .

For partition chromatography bonded phases are used .

In normal phase liquid chromatography the stationary phase is a polar compound such as alkylnitrile or alkylamine & the mobile phase is a nonpolar solvent such as hexane .

For reversed phase liquid chromatography stationary phase is a nonpolar compond such as octasilane (OS) or octadecylsilane (ODS), & the mobile phase is a polar solvent such a water / acetonitrile or water / methanol.

Cross linked microporous polystyrene resins are widely used suitable ion exchange resins for HPLC .

Stationary phase for exclusion separations are porous silica , glass , polystyrene or polyvinylacetate beads & are available in a range of pore size .

The support for affinity separation are similar to those for exclusion separations .

The spacer arm & ligand are attached to the supports by chemical bonding .

Chiral stationary phases contain proteins that are composed of amino acids each of which has a stereocenter ( except glycine ) commonly used are alfa 1 acid glycoproteins ( AGP ) ,human serum albumin ( HAS ) .

Semirigid as well as nonrigid gels have limitted role in HPLC stationary phase .

The major priority in packing of a column is to obtain a uniform bed of material with no cracks or channels .

Rigid solids as well as hard gels should be packed as densely as possible but without fracturing the packing process .

Most widely used technique for column packing is the high pressure slurrying technique .

The choice of mobile phase to be used in any separation will depend on the type of separation to be achieved .

Eluting power of the solvent is related to its polarity.

The components of the applied sample are separated by the continuous passage of the mobile phase through the column , this is known as elution development .

Column development is of 2 types :1)Isocratic elution ,2)Gradient elution . Column development using a single liquid as the

mobile phase is known as an isocratic elution . In order to increase the resolving power of the

mobile phase , it is necessary continuously to change it’s pH , ionic concentration or polarity this is known as gradient elution .

In order to produce a suitable gradient , two eluents have to be mixed in the correct proportions prior to their entering the column.

Gradient elution uses separate pumps to deliver two solvents in proportions predetermined by a gradient programmer .

All solvents for use in HPLC systems must be specially purified because traces of impurities can affect the column & interfere the detection system especially when measuring absorbance below 200nm .

Purified solvents are available commercially , but even with these solvents 1 – 5 µm microfilter is generally introduced into the system prior to the pump .

All solvents are degassed before use .

Gassing can alter column resolution & interfere with continuous monitoring of the effluent .

The purpose of the pump is to provide a constant , reproducible flow of solvent through the column .

Two types of pumps are available :

1) Constant pressure pump ,2) Constant volume pump .

Constant pressure pumps produce a pulseless flow through the column , but any decrease in the permeability of the column will result in lower flow rates for which the pumps will not compensate .

Constant pressure pumps are seldom used in contemporary liquid chromatography .

Constant displacement pumps maintain a constant flow rate through the column irrespective of changes within the column .

Two types of constant displacement pumps are available :

1) Motor driven syringe type pump ,2) Reciprocating pump ( most commonly used

form of constant displacement pump ) . All constant displacement pumps have in built

safety cut off mechanisms , so that if the pressure within the chromatographic systems changes from preset limits the pump is inactivated automatically .

The sensitivity of the detector system must be high & stable to respond to the low concentrations of each analyte in the effluent.

Most commonly the detector is a variable wave length detector based upon UV – visible spectrophotometry since few compounds are colored visible detectors are of limited value .

Detector is capable of measuring absorbance units down to 190 nm wave length & has sensitivities as low as 0.001 absorbance units for full – scale deflection ( AUFS ) .

Variable wave length detector operates at a wave length selected from a given wave length range .

Thus the detector is tuned to operate at the absorbance maximum for a given analyte or set of analytes which enhances greatly the applicability & selectivity of the detector.

Acetonitrile & methanol two widely used solvents in reversed phase chromatography have minimum UV absorption at 200nm .

Most biomolecules like proteins , nucleic acids, vitamins , steroids , pigments & aromatic amino acids absorb strongly in 220 – 365 nm range .

Aliphatic amino acids , carbohydrates , lipids & other substances do not absorb UV can be detected by chemical derivatization with UV absorbing functional groups .

UV detectors have many positive characteristics : highly sensitive ,

small sample volumes , linearity over wide range concentrations , non destuctiveness to

sample & suitability for gradient elution.

Fluorescence detectors are extremely valuable for HPLC because of their sensitivity but the technique is limited by the fact that relatively few compounds fluoresce .

Electrochemical detectors are extremely sensitive for electro active species .

The sensitivity of UV absorption , fluorescence & electrochemical detection can be increased significantly by the process of derivatisation , where by the analyte is converted pre or post column to a chemical derivative .

Diode arrays are used as HPLC detectors because they rapidly yeild spectral data over the entire wave length range of 190 – 600 nm in about 10 milliseconds .

Incorporation of computer technology into HPLC has resulted in cost effective , easy to operate automated systems with improved analytical performance .

The area or height of each chromatographic peak is determined from the stored data in computer & used to compute the analyte concentration represented by each peak .

Fast protein liquid chromatography :this provides a link between classical column chromatography ,& HPLC .

FPLC uses experimental conditions intermediate those of column chromatography & HPLC .

Narrow-bore columns (1-2 mm) are used for in this application .

Liquid chromatography-mass spectrometry (LC-MS, or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry.

HPLC has had big impact on separation of oligopeptides & proteins .

FPLC a modified version useful in separation of proteins .

HPLC coupled with electrochemical detector is useful in assay of catecholamines ,vitamins (AD&E ,niacin , thiamine) & antioxidants .

HPLC has role in quantification of various hemoglobins in hemoglobinopathies .

HPLC coupled with MS is useful in measuring cortisol in blood & saliva .

HPLC is useful in cytokine measurement . Useful in assay of HbA1c . Useful in assay of fructosamine . 5 – hydroxy idole acetic acid & serotonin can be

assayed. The pharmaceutical industry regularly employs

Reverse Phase HPLC to qualify drugs before their release.

Assay of plasma & urinary catecholamines , plasma & urinary metanephrines

For diagnosis of different porphyrias .

Thyroxine , uric acid .

Nucleic acid analysis, oliginucleotides , steroids , amino acids , serotonin , measurement of isoenzymes .

(1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position", (6') Switching valve in "load position", (7) Sample injection loop, (8) Pre-column (guard column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.