HPLC instrument

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HPLC instrument

  1. 1. M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar
  2. 2.  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. 3.  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 ) .
  4. 4.  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. 5.  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. 6.  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. 7.  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. 8.  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. 9.  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. 10.  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. 11.  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. 12.  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 .
  13. 13.  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. 14.  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. 15.  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 .
  16. 16.  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. 17.  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. 18.  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. 19.  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. 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. 21.  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. 22.  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. 23.  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. 24.  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. 25.  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. 26.  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. 27.  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. 28.  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. 29.  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. 30.  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. 31.  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 .
  32. 32.  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. 33.  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. 34.  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. 35.  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. 36.  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. 37.  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. 38.  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. 39.  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. 40.  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. 41.  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 .
  42. 42.  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. 43.  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 .
  44. 44.  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. 45.  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 .
  46. 46.  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 .
  47. 47.  UV detectors have many positive characteristics : highly sensitive , small sample volumes , linearity over wide range concentrations , non destuctiveness to sample & suitability for gradient elution.
  48. 48.  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 .
  49. 49.  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 .
  50. 50.  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 .
  51. 51.  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.
  52. 52.  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 .
  53. 53.  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
  54. 54.  For diagnosis of different porphyrias .  Thyroxine , uric acid .  Nucleic acid analysis, oliginucleotides , steroids , amino acids , serotonin , measurement of isoenzymes .
  55. 55.  (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.

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