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  1. 1. High Performance Liquid Chromatography HPLC DONE BY : HADEIA MASHAQBEH
  2. 2. H P igh erformance Liquid C hromatograph y
  3. 3. A technique by which a mixture sample is separated into components. Although originally intended to separate and recover (isolate and purify) the components of a sample. Today, complete chromatography systems are often used to both qualitify and quantify sample components. CHROMATOGRAPHY : 3
  4. 4. Chromato-graphy / -graph / -gram / - grapher 4  Chromatography: Analytical technique  Chromatograph: Instrument  Chromatogram: Obtained “picture”  Chromatographer: Person
  5. 5. ( mobile phase) 5
  6. 6. Mobile Phase / Stationary Phase  A site in which a moving phase (mobile phase) and a non-moving phase (stationary phase) make contact via an interface that is set up.  The affinity with the mobile phase and stationary phase varies with the solute.  Separation occurs due to differences in the speed of motion. 6 Strong Weak Mobile phase Stationary phase
  7. 7. Chromatography is based on the principal that : Under the same conditions, the time between the injection of a component into the column and the elution of that component (Retention time) is constant. This characteristic is used to perform qualitative analysis. 7
  8. 8. Liquid Chromatography 8  Chromatography in which the mobile phase is a liquid.  The liquid used as the mobile phase is called the “eluent”.  The stationary phase is usually a solid or a liquid.  In general, it is possible to analyze any substance that can be stably dissolved in the mobile phase.
  9. 9. Advantages of High Performance Liquid Chromatography 9  High separation capacity, enabling the batch analysis of multiple components  Superior quantitative capability and reproducibility  Moderate analytical conditions  Unlike GC, the sample does not need to be vaporized.  Generally high sensitivity  Low sample consumption  Easy preparative separation and purification of samples
  10. 10. Three States of Matter and Chromatography Types Mobile phase Gas Liquid Solid Stationary phase Gas Liquid Solid 10 Gas chromatography Liquid chromatography
  11. 11. Separation Techniques 11 I have two separation techniques in my lab, High Performance Liquid Chromatography and Gas Chromatography. Which should I use?
  12. 12. Comparison of HPLC and GC 12 Sample Volatility Sample Polarity HPLC • No volatility requirement • Sample must be soluble in mobile phase GC •Sample must be volatile HPLC GC •Separates both polar and non polar compounds •PAH - inorganic ions •Samples are nonpolar and polar
  13. 13. Comparison of HPLC and GC 13 Sample Thermal Lability Sample Molecular Weight HPLC •Analysis can take place at or below room temperature GC •Sample must be able to survive high temperature injection port and column HPLC GC •No theoretical upper limit •In practicality, solubility is limit. •Typically < 500 amu
  14. 14. Comparison of HPLC and GC 14 Sample Preparation Sample Size HPLC •Sample must be filtered •Sample should be in same solvent as mobile phase GC •Solvent must be volatile and generally lower boiling than analytes HPLC GC •Sample size based upon column i.d. •Typically 1 - 5 L
  15. 15. Comparison of HPLC and GC 15 Separation Mechanism HPLC •Both stationary phase and mobile phase take part GC •Mobile phase is a sample carrier only
  16. 16. Principle of HPLC : 16
  17. 17. Chromatogram
  18. 18. The Chromatogram 18 Injection to tR mAU time tR to - elution time of unretained peak tR- retention time - determines sample identity Area or height is proportiona to the quantity of analyte.
  19. 19.  HPLC is used for: 1) Qualitative Analysis The identification of individual compounds in the sample by the standard sample is measured, yielding a peak at specific minutes. This peak should be correspond to the sample itself under the same conditions (the type of column, column sizes, column temperature, composition of the mobile phase, and flow rate)  the most common parameter for compound identification is its Retention time
  20. 20. Retention time: The time at which a specific analyte elutes (emerges from the column) after injection
  21. 21. 2) Quantitative analysis  The measurement of the amount of a compound in a sample (concentration)  Two main ways a) determination of the peak height of a chromatographic peak as measured from the baseline
  22. 22. b) determination of the peak area in order to make a quantitative assessment of the compound
  23. 23.  Peak Area or Peak Height For most HPLC analyses, peak areas are used for quantitative calculations, although, in most cases, equivalent results may be achieved with peak height. Peak area is especially useful because HPLC peaks may be tailed. In this case, because peak heights may vary (although area will remain constant).
  24. 24. Quantitative Analysis 24  Calibration curve created using a standard.  Absolute calibration curve method  Internal standard method  Standard addition method
  25. 25. Calibration Curve for Absolute Calibration Curve Method 25 C1 C4 C3 C2 Concentration Area A1 A2 A3 A4 C1 C2 C3 C4 A1 A2 A3 A4 Concentration Peakarea Calibration curve
  26. 26. Calibration Curve for Internal Standard Method 26 C1 C4 C3 C2 Concentration Area A1 A2 A3 A4 C1/CIS C2 /CIS C3 /CIS C4 /CIS A1/AIS A2 /AIS A3 /AIS A4 /AIS Concentration of target substance / Concentration of internal standard Areafortargetsubstance/Areaforinternalstandard Calibration curve Target substance Internal standard CIS CIS CIS CIS AIS AIS AIS AIS
  27. 27. Advantages of Internal Standard Method (1)  Not affected by inconsistencies in injection volume. 27 10 µL injected 9 µL injected CX / CIS AX / AIS X IS X IS Same area ratio
  28. 28. Advantages of Internal Standard Method (2)  Not affected by the pretreatment recovery rate. 28 100% recovery rate 90% recovery rate CX / CIS AX/AIS X IS X IS Same area ratio
  29. 29. Selection Criteria for Internal Standard 29  It must have similar chemical properties to the target substance.  Its peak must appear relatively near that of the target substance.  It must not already be contained in the actual samples.  Its peak must be completely separated from those of other sample components.  It must be chemically stable.
  30. 30. 3) Separation of mixtures for later analysis –preparative HPLC In chromatography a small volume of a mixture of chemicals is passed through a column using a solvent and different molecules exit the column at different times – this is called a separation.  The separation of a compound involves its physical interaction with a stationary phase and a mobile phase.
  31. 31.  A small, high-surface-area stationary phase maximizes the interaction between the substance to be separated and the stationary phase, which results in better separation.
  32. 32. Chromatogram Parameters Methods for Expressing Separation and Column Performance 32
  33. 33. Retention Factor, k 33 tR t0 Strengthofdetectorsignal Time tR: Retention time t0: Non-retention time 0 0R t tt k  
  34. 34. Theoretical Plate Number, N 34 W W1/2 H1/2 H 2 . 21 R R / R W t W 2 2 2  545 16     Area Ht t N H L N 
  35. 35. Evaluation of Column Efficiency Based on Theoretical Plate Number 35  If the retention times are the same, the peak width is smaller for the one with the larger theoretical plate number.  If the peak width is the same, the retention time is longer for the one with the larger theoretical plate number. N: Large N: Small N: Small N: Large
  36. 36. 36 Peak asymmetry ( A ) : s
  37. 37. Separation Factor, a 37 Separation factor: Ratio of k’s of two peaks )( 12 1 2 kk k k   k1 k2 1. When calculating the selectivity factor, species 1 elutes faster than species 2. The selectivity factor is always greater than or equal to one ( 1). 2. In general, if the selectivity of two components is equal to 1, then there is no way to separate them by improving the column efficiency
  38. 38. Resolution, RS 38 2,2/11,2/1 RR 21 RR S 12 12 18.1 )( 2 1 hh WW tt WW tt R       tR1 tR2 W1 W2 W1/2h,1 W1/2h,2 h1/2
  39. 39. Resolution Required for Complete Separation 39 If the peaks are isosceles triangles, they are completely separated. tR2 - tR1 = W1 = W2 RS = 1 (tR2 - tR1) W1 W2 W1 W2 If the peaks are Gaussian distributions, RS > 1.5 is necessary for complete separation. tR2 - tR1 = W1 = W2 RS = 1 (tR2 - tR1)
  40. 40. Relationship Between Resolution and Other Parameters  The resolution is a function of the separation factor, the theoretical plate number, and the retention factor.  The separation can be improved by improving these 3 parameters! 40       1 1 4 1 )( 2 1 2 2 21 1R2R S k’ k’ N WW tt R  
  41. 41. Calculation of HPLC Resolution Factor (Rs) Defined as the amount of separation between two adjacent peaks
  42. 42. Contribution of Capacity Factor to Resolution  Increasing the capacity factor improves resolution  A capacity factor of around 3 to 10 is appropriate. Exceeding this just increases the analysis time. 42 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 15 20 Capacity factor Contributionratioforresolution
  43. 43. Contribution of Theoretical Plate Number to Resolution  The resolution increases in proportion to the square root of the theoretical plate number. 0.0 1.0 2.0 0 10000 20000 30000 Theoretical plate number Contributionfactorforresolution 43
  44. 44. To Improve Separation... 44 k’ increased N increased  increased Before adjustment Eluent replaced with one of lower elution strength. Column replaced with one of superior performance. Column lengthened. Column (packing material) replaced. Eluent composition changed. Column temperature changed.
  45. 45. Instrumentation of HPLC Mobile phase reservoir Solvent mixing valve Pump HPLC Chart Sample injection valve Recorder Waste Detector
  46. 46. HPLC Analysis Parameters 46 Mobile Phases Flow Rate Composition Injection Volume Column Oven Temperature Wavelength Time Constant
  47. 47. -Often the reservoirs contain a filtration system for filtering dust and particulate matters from the solvent to prevent these particles from damaging the pumps or injection valves or blocking the column. -The reservoirs are equipped with a degasser for removing dissolved gases- usually oxygen and nitrogen-that interfere by forming bubbles in the column and the detector. Mobile-Phase Reservoir Mobile phase reservoir Solvent mixing valve Pump Chart injection valve Recorder Waste Detector
  48. 48. The Function: The pump provide a flow of the mobile-phase through the HPLC injector, column, and detector. HPLC Pump Types of HPLC Pumps:  Constant-Pressure Pump.  Constant-Flow Pump. The requirements of standard HPLC pump include:  Generation of pressures up to 6000 Ibs/in2.  Pulse-free output.  Flow rate ranging from 0.1 to 10 ml/min.  Made of corrosion-resistant materials (stainless steel). Mobile phase reservoir Solvent mixing valve Pump Chart injection valve Recorder Waste Detector
  49. 49. Two types of pump operation 1) Isocratic Elution In which the composition of the mobile phase solvent remains constant with time  Best for simple preparation 2) Gradient Elution the composition changes during the separation process (mobile phase solvent composition increase with timed)  Best for Complex preparation
  50. 50. Aim of Gradient System  In isocratic mode 50 Long analysis time!! Poor separation!! CH3OH / H2O = 6 / 4 CH3OH / H2O = 8 / 2 (Column: ODS type)
  51. 51. Aim of Gradient System If the eluent composition is changed gradually during analysis... 51 95% 30% Concentrationofmethanolineluent
  52. 52. Four types of columns used in HPLC 1) High performance analytical columns The internal diameter 1.0 - 4.6 mm; lengths 15 –250 mm] - used mainly for qualitative and quantitative analysis 2) Preparative columns The Internal Diameter > 4.6 mm; lengths 50 –250 mm) – used mainly for preparative work 3) Capillary columns The internal Diameter 0.1 -1.0 mm; various lengths) 4) Nano columns the Internal diameter (< 0.1 mm) HPLC Column
  53. 53. PARAMETERS: 1- Internal diameter: A critical aspect that determines quantity of analyte that can be loaded onto the column and also influences sensitivity. 2- Particle size: Smaller particles generally provide more surface area and better separations. 54
  54. 54. 3- Pore size: Many stationary phases are porous to provide greater surface area. Small pores provide arger pore size has better kinetics. 55
  55. 55. The outer particle surface to its inner one is about 1:1000. 4-Pump pressure: The pump performance is measured on their ability to yield a consistent and reproducible pressure and flow rate. 56
  56. 56. Temperature Control in HPLC: To achieve 1-Reproducibility 2-Solubility 3-Stability 57 Temperature is controlled by: 1.Oven 2.Heater Block 3.Water bath
  57. 57. HPLC Detector The ideal characteristics: 1. Adequate sensitivity for the particular task. 2. Good stability and reproducibility. 3. Insensitive to changes in solvent, flow rate, and temperature. 4. High reliability and ease of use. 5. Non-destructive for the sample. Mobile phase reservoir Solvent mixing valve Pump Chart injection valve Recorder Waste Detector
  58. 58. Representative HPLC Detectors 59  UV-VIS absorbance detector  Photodiode array-type UV-VIS absorbance detector  Fluorescence detector  Refractive index detector  Evaporative light scattering detector  Electrical conductivity detector  Electrochemical detector  Mass spectrometer
  59. 59. Comparison of Detectors Selectivity Possibility of Gradient System Absorbance Light-absorbing substances Possible Fluorescence Fluorescent substances Possible Differential refractive index None Impossible Evaporative light scattering Nonvolatile substances Possible Electrical conductivity Ionic substances Partially possible Electrochemical Oxidizing / reducing substances Partially possible 60 Note: The above table indicates general characteristics. There are exceptions.
  60. 60. HPLC separation modes 1) Normal Phase Liquid Chromatography  Is a technique that uses columns packed with polar stationary phases ( e.g Silica gel) combined with nonpolar or moderately-polar mobile phases (e.g hexane) to separate the components of mixtures.  The rate at which individual solutes migrate through HPLC columns is primarily a function of their polarity.  Less polar solutes move the fastest and therefore exit the column and are detected first, followed by solutes of increasing polarity which move more slowly
  61. 61. Stationary Phase and Mobile Phase Used in Normal Phase Mode 62  Stationary Phase  Silica gel: -Si-OH  Cyano type: -Si-CH2CH2CH2CN  Amino type: -Si-CH2CH2CH2NH2  Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH  Mobile Phase  Basic solvents: Aliphatic hydrocarbons, aromatic hydrocarbons, etc.  Additional solvents: Alcohols, ethers, etc.
  62. 62. Relationship between Hydrogen Bonding and Retention Time in Normal Phase Mode 63 OH HO SiOH SiOH Strong Weak Steric hindrance Very weak
  63. 63. Relationship Between Eluent Polarity and Retention Time in Normal Phase Mode 64 100/0 Eluent: Hexane/methanol 95/5 98/2
  64. 64. Nonpolar (Hydrophobic) Functional Groups and Polar (Hydrophilic) Functional Groups 65  Nonpolar Functional Groups  -(CH2)nCH3  Alkyl groups  -C6H5  Phenyl groups  Polar Functional Groups  -COOH  Carboxyl groups  -NH2  Amino groups  -OH  Hydroxyl groups
  65. 65. 2) Reverse Phase Liquid Chromatography The mobile phase is polar and the stationary pahse is non polar The silica in the column is modified to make it non-polar (Silica C18 molecule), typically 8 or 18 carbons are added to the silica (C8 – C18) then the silica C18 is non polar. The non polar molecules binds/adsorbs to it and the polar molecules will pass more quickly through the stationary phase.
  66. 66. Relationship Between Retention Time and Polarity 67 C18 (ODS) CH3 Strong Weak OH
  67. 67. Basic Settings for Eluent Used in Reversed Phase Mode 68  Water (buffer solution) + water-soluble organic solvent  Water-soluble organic solvent: Methanol Acetonitrile Tetrahydrofuran etc.  The mixing ratio of the water (buffer solution) and organic solvent has the greatest influence on separation.  If a buffer solution is used, its pH value is an important separation parameter.
  68. 68. Relationship between Polarity of Eluent and Retention Time in Reversed Phase Mode 69 60/40 Eluent: Methanol / Water 80/20 70/30
  69. 69. Normal Phase / Reversed Phase Stationary phase Mobile phase Normal phase High polarity (hydrophilic) Low polarity (hydrophobic) Reversed phase Low polarity (hydrophobic) High polarity (hydrophilic) 70
  70. 70. Why the Reverse phase HPLC is more commonly used than Normal phase HPLC 1) Reverse phase is easier to use than normal phase 2) Reverse phase has hydrophobic stationary which can be applied to a wide range of molecules, it works well in retention time for most of the organic analytes. (70 – 80 % of common analytes can be measured by RP – HPLC) 3) Reverse phase has more options for chromatographer It also allows precise control of variables such as organic solvent type, concentration and pH
  71. 71. Comparison of Normal Phase and Reversed Phase 72  Normal Phase  Effective for separation of structural isomers  Offers separation selectivity not available with reversed phase  Stabilizes slowly and is prone to fluctuations in retention time  Eluents are expensive  Reversed Phase  Wide range of applications  Effective for separation of homologs  Stationary phase has long service life  Stabilizes quickly  Eluents are inexpensive and easy to use
  72. 72. (3)Ion Exchange Chromatography 73 N+ R R R SO3 - + + + ++ ++ + + + Electrostatic interaction (Coulomb force) Anion exchange Cation exchange Molecules with the higher charge density bind more strongly to the resin. The bound sample may be selectively removed from the stationary phase by changing the pH or salt concentration of the mobile phase
  73. 73. (4)Size Exclusion Chromatography 74  Separation is based on the size (bulkiness) of molecules.  The name varies with the application field!  Size Exclusion Chromatography (SEC)  Gel Permeation Chromatography (GPC)  Chemical industry fields, synthetic polymers, nonaqueous systems  Gel Filtration Chromatography (GFC)  Biochemical fields, biological macromolecules, aqueous systems
  74. 74. Principle of Size Exclusion Mode 75 Packing material The size of the solute molecules determines whether or not they can enter the pores.
  75. 75. Modes of High Performance Liquid Chromatography 76 Types of Compounds Mode Stationary Phase Mobile Phase Neutrals Weak Acids Weak Bases Reversed Phase C18, C8, C4 cyano, amino Water/Organic Modifiers Ionics, Bases, Acids Ion Pair C-18, C-8 Water/Organic Ion-Pair Reagent Compounds not soluble in water Normal Phase Silica, Amino, Cyano, Diol Organics Ionics Inorganic Ions Ion Exchange Anion or Cation Exchange Resin Aqueous/Buffer Counter Ion High Molecular Weight Compounds Polymers Size Exclusion Polystyrene Silica Gel Filtration- Aqueous Gel Permeation- Organic
  76. 76. Guidelines for Selecting Separation Mode Required Information 77  Soluble solvent  Molecular weight  Structural formula and chemical properties  Do the substances ionize?  Is there UV absorption or fluorescence?
  77. 77. Guidelines for Selecting Separation Mode 78  Reversed phase mode using an ODS column is the first choice!  Exceptions  Large molecular weight (> 2,000)  Size exclusion  Optical isomers  Chiral column  Stereoisomers, positional isomers  Normal phase / adsorption  Inorganic ions  Ion chromatography  Sugars, amino acids, short-chain fatty acids •  Special column: Amino acids: Cation exchange. • Short-chain fatty acids: Ion exclusion
  78. 78. Sample Pretreatment Tasks Performed Before Injection 79
  79. 79. Objectives of Pretreatment 80  To improve the accuracy of quantitative values  To improve sensitivity and selectivity  To protect and prevent the deterioration of columns and analytical instruments  To simplify measurement operations and procedures  To stabilize target substances
  80. 80. Substances That Must Not Be Injected into the Column 81  Insoluble substances (e.g., microscopic particles and precipitation)  Substances that are precipitated in the eluent  Substances that irreversibly adsorb to the packing material  Substances that dissolve, or chemically react, with the packing material
  81. 81. Filtration and Centrifugal Separation  In general, filter every sample before injection!  It is convenient to use a disposable filter with a pore diameter of approx. 0.45 µm.  Centrifugal separation is applicable for samples that are difficult to filter. 82 Filter Syringe
  82. 82. Solid Phase Extraction 83 (1) Conditioning (2) Sample addition (3) Rinsing (4) Elution Solvent with low elution strength Solvent with high elution strength Target component Unwanted components
  83. 83. HPLC Applications 84 Chemical Environmental Pharmaceuticals Consumer Products Clinical polystyrenes dyes phthalates tetracyclines corticosteroids antidepressants barbiturates amino acids vitamins homocysteine Bioscience proteins peptides nucleotides lipids antioxidants sugars polyaromatic hydrocarbons Inorganic ions herbicides
  84. 84.  Pharmaceutical Application  Assay  Analytical Method Validation  Stability Studies  Compound Identification Tablet dissolution study of pharamceutical dosages form Identification of active ingredients of dosage form Quality Control
  85. 85. High-performance liquid chromatography (HPLC) is a chromatographic technique used to split a mixture of compounds in the fields of analytical chemistry, biochemistry and industrial. The main purposes for using HPLC are for identifying, quantifying and purifying the individual components of the mixture. CONCLUSION
  86. 86. REFRENCES  V.R. Meyer, “Practical High – Performance Liquid Chromatography”, Wiley, 2010  Analysis of peak asymmetry in chromatography, Pápai Z1, Pap TL  Glajch J.L., Quarry M.A., Vasta J.F., and Snyder L.R. 1986. Separation of peptide mixtures by reversed-phase gradient elution. Use of flow rate changes for controlling band spacing and improving resolution. Anal. Chem. 58: 280–285.  Hancock W.S. and Sparrow J.T. 1983. The separation of proteins by reversed-phase high-performance liquid chromatography. In High-performance liquid chromatography. Advances and perspectives (ed. C. Horváth), vol. 3, pp. 50– 87. Academic Press, New York.  Liquid-solid sample preparation in drug analysis R. D. MCDOWALL*, J. C. PEARCE and G. S. MURKITT 