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  1. High Performance Liquid Chromatography M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar

  2. Introduction • 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 .

  3. contd • 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 • Reside only on stationary phase ( no migration ) , • Reside only in the mobile phase ( migration with mobile phase ) , • Distribute between two phases ( differential migration ) .

  4. contd • 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

  5. Column Chromatography • 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 .

  6. principle • 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 .

  7. contd • 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

  8. Contd • 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 .

  9. contd • 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 .

  10. contd • 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 .

  11. contd • 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 .

  12. contd • Peak asymmetry has many causes , • Application of too much analyte to the column , • Poor packing of the column , • Poor application of the sample to the column or solute support interactions .

  13. contd • 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

  14. contd • 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 .

  15. contd • Good resolution is determined by the following 3 functions : • Selectivity , • Efficiency , • 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 .

  16. contd • 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 .

  17. Principle of HPLC • 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 .

  18. contd • 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 .

  19. Instrumentation • 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 .

  20. (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.

  21. Solvent reservoir • 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 .

  22. Pump • 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 .

  23. Injection port • 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 .

  24. contd • 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 .

  25. contd • 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 .

  26. Contd • 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 .

  27. Sample preparation • 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 .

  28. Sample 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 .

  29. contd • 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

  30. Column • 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.

  31. Matrices & Stationary phases • Three form of column packing matrices are available they are : • Microporous supports : ( micropores ramify through the particles which are generally 5 – 10 µm in diameter ), • 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 , • Bonded phases : in which stationary phase is chemically bonded to an inert support such as silica .

  32. contd • 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 .

  33. contd • 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.

  34. contd • 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 .

  35. contd • 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 .

  36. Column packing • 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 .

  37. Mobile phase • 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 .

  38. contd • 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 .

  39. contd • 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 .

  40. contd • 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 .

  41. Pumping system • The purpose of the pump is to provide a constant , reproducible flow of solvent through the column . • Two types of pumps are available : • Constant pressure pump , • Constant volume pump .

  42. contd • 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 .

  43. contd • Two types of constant displacement pumps are available : • Motor driven syringe type pump , • 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 .

  44. Detector • 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 ) .

  45. contd • 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 .