HPLC instrument

  • View

  • Download

Embed Size (px)



Text of HPLC instrument

  • 1.M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar

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. 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. 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. 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. 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. 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. 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. 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. K = tR tM = VR VM tM VM Capacity ratios characterize the column performance . The success of any chromatographic procedure is measured by its ability to separate completely ( resolve ) one analyte from a mixture of similar compounds . Peak resolution ( Rs )is related the properties of the peaks . 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. 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. 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. 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. 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 , . 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. 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. 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. The increased resolution achieved in HPLC compared to classical chromatography is primarily the result of adsorbents of very small particle size ( less then 20m )& large surface areas . The smallest gel beads used in gel exclusion chromatography are superfine grade with diameters of 20-50m . 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 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. 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. 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. 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. 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. 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 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. 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. 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 is made up of stainless steel . Column has to withs