HPLC

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HPLC by Ravi Pratap Pulla

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HPLC

  1. 1. HPLC I/II, Ist Semester M.Pharmacy Dept . Of Pharmaceutical Analysis, JNTUH Lecture by: RAVI PRATAP PULLA M.Pharm., Ph.D Asso.Professor, SSJ College of Pharmacy, V.N.Pally, Gandipet, Hyderabad-75. SSJCP, Department of Pharmaceutical Analysis 1
  2. 2. HPLC – THE DEVELOPMENT OF A NAME PERFORMANCE PRESSURE Price Prestige Peak Profit Propaganda High Promise Chromatography Philosophy Polite Problem Ph (F) antasy Liquid Pragmatic Pleasure Passion SSJCP, Department of Pharmaceutical Analysis 2
  3. 3. Introduction to Liquid Chromatography Columns System Components Applications Troubleshooting SSJCP, Department of Pharmaceutical Analysis 3
  4. 4. A Brief History of Chromatography  1903: Russian botanist Mikhail Tswett separated plant pigments  1938: Russian scientists Izmailov and Shraiber use “drop chromatography”.  Later perfected as Thin Layer Chromatography (TLC) by Kirchner in the U.S.  1952: Martin and Synge receive Nobel Prize for “invention of partition chromatography” or plate theory to describe column efficiency. SSJCP, Department of Pharmaceutical Analysis 4
  5. 5.  1966: HPLC was first named by Horvath at Yale University but HPLC didn’t “catch on” until the 1970s  1978: W.C. Stills introduced “flash chromatography”, where solvent is forced through a packed column with positive pressure. SSJCP, Department of Pharmaceutical Analysis 5
  6. 6. Modern HPLC  Late 1970s/early 1980s ►Instrumentation developed for high pressure solvent delivery: pumps, autosamplers, diode array detectors ► More uniform packing material produced columns for  Last 20 years ► Nothing really “new”, but by returning to the basic theory of chromatography, even better columns are on the market: smaller particle sizes which yield faster separations, but require hardware to withstand higher pressures. SSJCP, Department of Pharmaceutical Analysis 6
  7. 7. What is Chromatography?  Separation of a mixture into individual components.  The separation uses a Column (stationary phase) and Solvent (mobile phase).  The components are separated from each other based on differences in affinity for the mobile or stationary phase.  The goal of the separation is to have the best RESOLUTION possible between components. SSJCP, Department of Pharmaceutical Analysis 7
  8. 8. CHROMATOGRAPHY IS INCOMPLETE WITHOUT LEARNING FEW BASIC TERMINOLOGIES For any further clarification or details of the below content(s) feel free to mail me : ravipratappulla@gmail.com SSJCP, Department of Pharmaceutical Analysis 8
  9. 9. Absorption Bonded phase Additive Breakthrough volume Adsorbent Capillary column Adsorption Capillary LC Adsorption isotherm Cartridge column Affinity chromatography Cation exchange chromatography Agarose Channeling Alumina Chemisorption Amphoteric ion-exchange phase Chiral stationary phase Analyte Chlorosilane Anion exchange chromatography Co-ion Bed volume Column back pressure BET (Brunauer, Emmet & Teller) Column chromatography method SSJCP, Department of Pharmaceutical Analysis
  10. 10. Column plate number Eluite Column switching Elute Column volume Elution Competing base Exclusion chromatography (Size) Counterion Extra column effects Coverage Fast protein LC (FPLC) Cross-links Frontal chromatography Dead time (to / tm ) Displacement chromatography Gel filtration chromatography (GFC) Gradient elution Dynamic coating Graphitized carbon packing Effluent Guard column Eluate Heart cutting Eluent Hold-up volume ( VM or tM ) Degassing SSJCP, Department of Pharmaceutical Analysis
  11. 11. Hydrophobic interaction chromatography (HIC) Ion exclusion Ion chromatography Ion moderated partioning chromatography (IMPC) Imprinted phases Ion pair chromatography (IPC) Indirect detection Linear chromatography Injector (sample) Linear velocity Inlet Liquid chromatography In-line filter Mobile phase velocity Interparticle porosity (ee) Open tubular column Interstitial volume Partition chromatography Intraparticle porosity (ei) Packed column Intraparticle volume Peak Ion exchange chromatography Peak area Ion chromatography (IC) Peak maximum SSJCP, Department of Pharmaceutical Analysis 11
  12. 12. Peak width Retention factor (k) Phase ratio Retention volume (VR or tR) Plate height (H) Separation factor (a) Plate number (N) Solid support Pressure drop Solute Reduced mobile phase velocity (n) Stationary phase Resolution (Peak) [ Rs ]/ Resolution(R) Tailing Reduced plate height (h) Void volume Relative Retention time (RRT) Retention time (tR ) Interparticle time (tZ) Capacity factor (k’) Dead Volume(Vd) Selectivity factor (α) SSJCP, Department of Pharmaceutical Analysis 12
  13. 13. Activity Adsorption chromatography Asymmetry Back pressure Back flushing Band spacing Baseline Baseline noise Baseline resolved peak Breakthrough volume Buffer Calibration standard Capacity factor Chain length Channeling Chromatogram Chromatographic conditions Chromatographic resolution Chromatographic system Column performance Dead volume (Vm) Dead time (tm) Detection Detector Detection threshold Detector linearity SSJCP, Department of Pharmaceutical Analysis 13
  14. 14. Detector sensitivity Differential Refractive Index( RI) Electrochemical detector Elution order Elution chromatography Eluotropic sequence Elution volume Extra column volume External standard Flow rate Fluorescence detector Frit Fronting HETP Hydrophilic Hydrophobic Internal standard Integrator Interstitial particle volume Ion exchanger Ion suppression Isocratic analysis Isothermal chromatography Ligand Loading matrix SSJCP, Department of Pharmaceutical Analysis 14
  15. 15. Organic modifier Overload Partially resolved peaks Particle size (medium) Particle size distribution Peak broadening Peak area Peak base Peak height Peak identification Peak Quantitation Peak shape Phase system Polarity Pore diameter Pore volume Post column derivatization Pre column Pulsating flow Recycling Regeneration Retention Retention time Retention volume Sample Sample capacity SSJCP, Department of Pharmaceutical Analysis 15
  16. 16. Sample preparation Separation capacity silanization Silanol groups Sorbent S.P chemically bonded S.P Surface modification Specific surface SFC( supercritical fluid chromatography) Vacancy chromatogram Void Void time SSJCP, Department of Pharmaceutical Analysis 16
  17. 17. IUPAC RECOMMENDATIONS & FREQUENTLY USED SYMBOLS IN PARAMETER SYMBOL CHROMATOGRAPHY α Separation factor Selectivity factor (up to 1993 A.D) Area α a/A Diameter de Diffusion coefficient d ε / εt Porosity Flow rate (volumetric) f Plate height h Viscosity η Equilibrium distribution constant k Rate constant k Retention factor k SSJCP, Department of Pharmaceutical Analysis 17
  18. 18. PARAMETER Capacity factor SYMBOL k’ Length of the column l / L Plate number /number of theoretical plates n / N Density ρ Pressure p / P Pressure (relative) p Radius r Temperature (absolute) Time t /T t Retention time tr / tR Velocity (linear) u Volume v SSJCP, Department of Pharmaceutical Analysis 18
  19. 19. PARAMETER SYMBOL Retention volume vr Mass (Weight) w Peak width w Difference ∆ Partial diameter dp Flow F Height equivalent of a theoretical plate(HETP) H Internal diameter of the column I.D Wavelength λ Iso electric point pKa Resolution R Death time tm / t0 SSJCP, Department of Pharmaceutical Analysis 19
  20. 20. PARAMETER SYMBOL Gradient time tG Net retention time tR' Linear velocity μ Dead volume of apparatus Vd Pore volume Vp For any further clarification or details of the above content(s) feel free to mail me : ravipratappulla@gmail.com SSJCP, Department of Pharmaceutical Analysis 20
  21. 21. The Most Basic Explanation of Chromatography Ever SSJCP, Department of Pharmaceutical Analysis 21
  22. 22.  Drugs in multi component dosage forms, analyzed by HPLC method because of the several advantages like:  Improved resolution of the separated substances  Faster separation times  The improved accuracy, precision, & sensitivity with which the separated substances may be quantified. SSJCP, Department of Pharmaceutical Analysis 22
  23. 23. How Do You Get Separation?  Hardware: pumps, injector, detector  Column: particle diameter, column size, packing materials  Our seminar will focus on the contribution of each factor to perform separations. SSJCP, Department of Pharmaceutical Analysis 23
  24. 24.  Column Considerations ► Theory (including, well...you know) ► Different Stationary Phases  Hardware Components ► Pumps, Injectors, Detectors, etc. ► Examples of Application-Specific Configurations  Applications ► Pharmaceuticals and Proteomics ► Food and Beverage, Environmental ► Research and Method Development SSJCP, Department of Pharmaceutical Analysis 24
  25. 25.  System Troubleshooting Leaks, Reproducibility, Column Care, and More  Chromatography Software Method and Sequence Setup Calibration Curves and Reporting  Chromatography Hardware Modular LC-20 Prominence Integrated LC-2010HT, Empower 2 SSJCP, Department of Pharmaceutical Analysis 25
  26. 26. Modern HPLC v/s Traditional LC Methods  Classical open-column LC.  Thin-Layer Chromatography (TLC) and paper chromatography.  In modern HPLC the columns and packings are, in general, highly refined, high in resolving capacity, and are reusable. SSJCP, Department of Pharmaceutical Analysis 26
  27. 27. HPLC and Pre-HPLC Techniques SSJCP, Department of Pharmaceutical Analysis 27
  28. 28. MODES OF SEPARATION IN HPLC  There are different modes of separation in HPLC: ►Normal phase mode ► Reversed phase mode ► RP - Ion pair chromatography ► Affinity/Bioaffinity chromatography ► Size exclusion chromatography ► Displacement chromatography SSJCP, Department of Pharmaceutical Analysis 28
  29. 29.  Based on mode of chromatography ► Normal phase mode ► Reverse phase mode  Based on principle of separation ► Adsorption chromatography ► Ion exchange chromatography ► Ion pair chromatography ► Size exclusion chromatography ► Affinity chromatography SSJCP, Department of Pharmaceutical Analysis 29
  30. 30.  Based on elution technique ► Isocratic separation ► Gradient separation  Based on the scale of operation ► Analytical HPLC ► Preparative HPLC  Based on the type of analysis ► Qualitative analysis ► Quantitative analysis SSJCP, Department of Pharmaceutical Analysis 30
  31. 31. COLUMN TYPES Normal Phase LC  Polar - stationary phase: Silica  Nonpolar - mobile phase: Hexane, Ethyl acetate  The LEAST polar compound comes out first  Generally used for separation of non polar compounds. SSJCP, Department of Pharmaceutical Analysis 31
  32. 32. Normal Phase HPLC Columns Cyano : `Rugged, moderate polarity, general use -OH (Diol) : More polar and retentive Amino : Highly polar, less stable Silica : Very rugged, low cost, adsorbent & Unbonded NOTE: The cyano column with a low polarity mobile phase (hydrocarbon with a small amount of another solvent) will act as a normal phase column. SSJCP, Department of Pharmaceutical Analysis 32
  33. 33.  this method separates analytes based on their affinity for a polar stationary surface such as silica  based on analyte ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface.  Adsorption strengths increase with increased analyte polarity  interaction strength depends on the functional groups present in the structure of the analyte molecule, but also on steric factors SSJCP, Department of Pharmaceutical Analysis 33
  34. 34.  more polar solvents in the mobile phase will decrease the retention time of analytes  hydrophobic solvents tend to induce slower elution (increased retention times)  traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention.   governed mechanism almost exclusively by an SSJCP, Department of Pharmaceutical Analysis adsorptive 34
  35. 35. Reversed-Phase LC  Nonpolar - stationary phase: C8, C18  Polar - mobile phase: Water, ACN, Methanol  The MOST polar compound comes out first  Generally used for separation of polar compounds SSJCP, Department of Pharmaceutical Analysis 35
  36. 36. C18, C8 RP-HPLC Columns : Rugged, general purpose, highly retentive C3, C4 : Less retentive, used mostly for peptides & proteins Phenyl : Greater selectivity than alkyl-bonded Cyano : Moderate retention, normal & rev. phase Amino : Weak retention, good for carbohydrates NOTE : The cyano column with a high polarity mobile phase (Water/MeOH) will act as a RP- Column. SSJCP, Department of Pharmaceutical Analysis 36
  37. 37.  stationary phase is a silica which has been surfacemodified with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17.  retention time is longer for molecules which are less polar, while polar molecules elute more readily   can increase retention times by adding more water to the mobile phase  the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase SSJCP, Department of Pharmaceutical Analysis 37
  38. 38.  decrease retention time by adding more organic solvent to the eluent  RP-HPLC operates on the principle of hydrophobic interactions  RP-HPLC allows the measurement of these interactive forces.   The binding of the analyte to the stationary phase is proportional to the contact surface area around the nonpolar segment of the analyte molecule upon association with the ligand on the stationary phase. SSJCP, Department of Pharmaceutical Analysis 38
  39. 39.  solvophobic effect is dominated by the force of water for "cavity-reduction" around the analyte and the C18-chain versus the complex of both.  The retention can be decreased by adding a less polar solvent (methanol, acetonitrile) into the mobile phase to reduce the surface tension of water.   Gradient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis.  Structural properties of the analyte molecule play an important role in its retention characteristics. SSJCP, Department of Pharmaceutical Analysis 39
  40. 40.  an analyte with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) is retained longer because it is non-interacting with the water structure.  analytes with higher polar surface area (conferred by the presence of polar groups, such as -OH, -NH2, COO– or -NH3+ in their structure) are less retained as they are better integrated into water.  interactions are subject to steric effects in that very large molecules may have only restricted access to the pores of the stationary phase, where the interactions with surface ligands (alkyl chains) take place. SSJCP, Department of Pharmaceutical Analysis 40
  41. 41.  surface hindrance typically results in less retention.  Retention time increases with hydrophobic (non-polar) surface area.  Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased.  organic compounds with single C-C-bonds elute later than those with a C=C or C-C-triple bond, as the double or triple bond is shorter than a single C-C-bond. SSJCP, Department of Pharmaceutical Analysis 41
  42. 42.  mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention.  entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time.  mobile phase pH can change the hydrophobic character of the analyte.  For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH.  SSJCP, Department of Pharmaceutical Analysis 42
  43. 43.  Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of analyte-ammonium adducts.  volatile organic acid such as acetic acid, or formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column effluent.  Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is a fairly strong organic acid.  SSJCP, Department of Pharmaceutical Analysis 43
  44. 44.  Reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases as these will destroy the underlying silica particle.  Can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment.  A good test for the metal content of a column is to inject a sample which is a mixture of 2,2'- and 4,4'- bipyridine.  Because the 2,2'-bipy can chelate the metal, the shape of the peak for the 2,2'-bipy will be distorted (tailed) when metal ions are present on the surface of the silica. SSJCP, Department of Pharmaceutical Analysis 44
  45. 45. TYPICAL COLUMN SIZES SSJCP, Department of Pharmaceutical Analysis 45
  46. 46. SSJCP, Department of Pharmaceutical Analysis 46
  47. 47.  Particle size: 5 µm, 3 µm, and smaller  Mono dispersed means particles are the same size  Very important for stable pressure and flow  Smaller particles produce higher system pressure ► Pore size: 100-120 A is typical ► Surface area: 300-350 m2/g ► Carbon load: 9-12% for C8, 16-20% for C18  Higher carbon load = better resolution but longer run times  Lower carbon load = shorter run times, but may change selectivity v/s higher carbon load SSJCP, Department of Pharmaceutical Analysis 47
  48. 48. RP-HPLC MECHANISM      Synthesis of RP Packing RP Column Properties RP Retention Mechanisms Important RP parameters RP Optimization SSJCP, Department of Pharmaceutical Analysis 48
  49. 49. Synthesis of RP Packing SSJCP, Department of Pharmaceutical Analysis 49
  50. 50. RP COLUMN PREPARATION SSJCP, Department of Pharmaceutical Analysis 50
  51. 51. COMMON RP PACKING SSJCP, Department of Pharmaceutical Analysis 51
  52. 52. RP COLUMN PROPERTIES ► Hydrophobic Surface ► Particle Size and Shape ► Particle Size Distribution ► Porosity, Pore Size and Surface Area SSJCP, Department of Pharmaceutical Analysis 52
  53. 53. PARTICLE SIZE ► Columns have a distribution of particle sizes ► Reported “particle diameter” is an average ► Broader distribution ---> broader peaks SSJCP, Department of Pharmaceutical Analysis 53
  54. 54. Particle Size Distribution of several column batches Copyrights: Neue, HPLC Columns Theory, Technology and Practice, Wiley, 1997, p.82 SSJCP, Department of Pharmaceutical Analysis 54
  55. 55. RP MECHANISM (SIMPLE) SSJCP, Department of Pharmaceutical Analysis 55
  56. 56. RP Mechanism (Advanced)  Classical measures of retention ► capacity factors ► partition coefficients ► Van’t Hoff Plots  Give bulk properties only ► do not give molecular view of separation process SSJCP, Department of Pharmaceutical Analysis 56
  57. 57. PROPOSED RP MECHANISMS ► Hydrophobic Theory ► Partition Theory ► Adsorption Theory SSJCP, Department of Pharmaceutical Analysis 57
  58. 58. HYDROPHOBIC THEORY  Chromatography of “cavities” in solvent created by hydrophobic portion of analyte molecule  Surface Tension  Interaction of polar functions with solvent  Stationary phase is passive SSJCP, Department of Pharmaceutical Analysis 58
  59. 59. PARTITION THEORY  Analyte distributes between aqueous mobile phase and organic stationary phase  Correlation between log P and retention “organic” phase is attached on one end  Does not explain shape selectivity effects SSJCP, Department of Pharmaceutical Analysis 59
  60. 60. ADSORPTION THEORY  Analytes “land” on surface - do not penetrate  Non-polar interactions between analyte hydrophobic portion and bonded phase  Weak interactions ► dipole-dipole ► dipole-induced dipole ► induced dipole-induced dipole SSJCP, Department of Pharmaceutical Analysis 60
  61. 61. None of the above can completely explain all of the observed retention in RP-HPLC SSJCP, Department of Pharmaceutical Analysis 61
  62. 62. IMPORTANT REVERSED PHASE PARAMETERS  Solvent (mobile phase ) Strength  Choice of Solvent  Mobile Phase pH  Silanol Activity SSJCP, Department of Pharmaceutical Analysis 62
  63. 63. SOLVENT STRENGTH  Water is “weak” solvent  Increased organic ---> decreased retention  Organic must be miscible with water SSJCP, Department of Pharmaceutical Analysis 63
  64. 64. EFFECT OF SOLVENT SSJCP, Department of Pharmaceutical Analysis 64
  65. 65. SOLVENT STRENGTH COPYRIGHTS:Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 286. SSJCP, Department of Pharmaceutical Analysis 65
  66. 66. VARYING SELECTIVITY 30% MeCN 45% MeOH 70% Water 55% Water 30x0.46 cm C-18, 1.5 mL.min,254 nm, 10 mg each COPYRIGHTS:Snyder and Kirkland, introduction to Modern Liquid Chromatography, Wiley, 1979, p. 287. SSJCP, Department of Pharmaceutical Analysis 66
  67. 67. pH  Affects ionizable compounds ► organic acids ► organic bases  In reversed phase we need to suppress ionization as much as possible  May need very precise pH control SSJCP, Department of Pharmaceutical Analysis 67
  68. 68. pH Effect on Retention 1. Salicylic acid 2. Phenobarbitone 3. Phenacetin 4. Nicotine 5. Methylampohetamine 30x0.4 cm C-18, 10 mm, 2 mL/min, UV 220 nm COPYRIGHTS: Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 288. SSJCP, Department of Pharmaceutical Analysis 68
  69. 69. Use of Buffers  0.1 pH unit ---> significant effect on retention  Buffer mobile phase for pH reproducibility  pH of buffer should be within 1 pH unit of pKa of acid (best at pH = pKa)  Buffers weak (100 mM or less)  Check solubility SSJCP, Department of Pharmaceutical Analysis 69
  70. 70. Common buffers Buffer pKa Values Phosphate 2, 7 Acetate 4.75 Citrate 3.08, 4.77, 6.40 Useful buffering between pH 2-8. SSJCP, Department of Pharmaceutical Analysis 70
  71. 71. Silanol Activity  RP ligands occupy about 50% of silanols  Others are “active”  Weak acids SSJCP, Department of Pharmaceutical Analysis 71
  72. 72. Silica Surface SSJCP, Department of Pharmaceutical Analysis 72
  73. 73. Dealing with Residual Silanols  Silanols cause peak tailing and excessive retention  Endcapping ► bond a smaller group (helps a little)  Pre-treatment of silica ► fully hydroxylated best ► high purity best SSJCP, Department of Pharmaceutical Analysis 73
  74. 74. Silanol Interactions  Hydrogen bonding  Dipole-dipole  Ion exchange  Low pH --> silanols protonated  Add basic modifier (TEA) to compete for sties SSJCP, Department of Pharmaceutical Analysis 74
  75. 75. pH Effect on Tailing Neue, p196 SSJCP, Department of Pharmaceutical Analysis
  76. 76. RP Optimization SSJCP, Department of Pharmaceutical Analysis 76
  77. 77. SSJCP, Department of Pharmaceutical Analysis 77
  78. 78. IDEALIZED HPLC SEPARATION SSJCP, Department of Pharmaceutical Analysis 78
  79. 79. VOID VOLUME  The void volume is the amount of “dead” volume in the column that is not taken up by the particles of stationary phase.  In general, there is approximately 0.1 mL of void volume for each cm of column length, for columns with a 4.6 mm i.d. and 5 µm particles Vm ≈ 0.5dc2L Where, Vm is the column volume in mL, L is the column length in cm, and dc is the inner diameter in cm SSJCP, Department of Pharmaceutical Analysis 79
  80. 80.  The void volume is exactly determined by injecting a compound that is completely unretained, then using the chromatogram to calculate void volume.  void volume = Elution time x flow rate SSJCP, Department of Pharmaceutical Analysis 80
  81. 81. FACTORS INFLUENCING RESOLUTION  Capacity Factor, k’  Selectivity Factor, α  Efficiency, N SSJCP, Department of Pharmaceutical Analysis 81
  82. 82. RESOLUTION  For closely eluting or adjacent peaks, the resolution equation may be expressed as: Rs = 1 / 4[(α − 1) / α ] N [k ' /(1 + k ' )]  The terms of capacity factor (k’), selectivity (α), and efficiency (N) all contribute to resolution SSJCP, Department of Pharmaceutical Analysis 82
  83. 83. THE RESOLUTION EQUATION  Resolution is defined as the completeness of separation from one analyte to another  In general, resolution may be expressed as: Rs = 2(Vrb - Vra)/(Wa + Wb) = 2(trb - tra)/ (Wa + Wb) Where, Vra/b = retention volume of peak a/b t a/b = retention time of peak a/b SSJCP, rDepartment of Pharmaceutical Analysis 83
  84. 84. CAPACITY FACTOR, k ’  The relative degree to which an analyte component is delayed as it is eluted through a given system (retentivity). k’ = (V r - V 0 )/V 0 = (t r - t 0 )/t 0 Where, Vr = peak retention volume V0 = column void volume tr = peak retention time t0 = peak void time  The larger the k’, the later the analyte elutes after the void. SSJCP, Department of Pharmaceutical Analysis 84
  85. 85. EFFECT OF k’ ON OVERALL RESOLUTION  As k’ grows larger, its effect reaches a limit at a value of about 10.  Since k’ depends on retention time, longer columns eventually have a diminished effect on resolution. SSJCP, Department of Pharmaceutical Analysis 85
  86. 86. INFLUENCING THE CAPACITY FACTOR (k’)  Retentivity (k’) decreases 2 - 3 fold for each 10% increase in mobile phase strength.  Which of these is easiest to change?? ► Mobile Phase Strength As per the rule of thumb, altering the mobile phase strength also alters the retention of the analytes. ► Bonded Phase Functionality (RP) As the bonded phase hydrophobicity increases (increasing alkyl chain length, etc.) so will the retention of the analytes. ► Temperature As temperature increases, the retention time decreases. This does not necessarily result in poorer separation because of the other factors in the resolution equation. SSJCP, Department of Pharmaceutical Analysis 86
  87. 87. Mobile Phase Strength v/s k 4.6 mm ID Column, 1 mL/min, Changing MeOH % vs Water 0.079 100% 100% 90% 90% ’ Capacity Factor for Butyl Paraben (Peak 4) 0.212 0.472 80% 80% 70% 70% 1.127 2.813 60% 60% 7.666 50% 50% SSJCP, Department of Pharmaceutical Analysis 87
  88. 88. Temperature Effect on k 50°C ’ 2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH/Water 45°C 40°C 35°C 30°C 25°C 20°C SSJCP, Department of Pharmaceutical Analysis 88
  89. 89. Summary of k Effects ’  A larger value of k’ means better resolution...to a certain extent (k’ = 10 maximum)  Increasing the mobile phase strength decreases k’  Increasing the temperature decreases k’, but may not result in a “bad” separation based on the other factors affecting resolution. SSJCP, Department of Pharmaceutical Analysis 89
  90. 90. Selectivity Factor, α  The selectivity or separation factor represents the ratio of any two adjacent k’ values, there by describing the relative separation of adjacent peaks.  This relationship is expressed as: α = k’b/k’a  If α = 1, two components are perfectly overlapping  For early eluting peaks you want α to be large for good resolution.  For later eluting peaks, α can be smaller and still have acceptable separation. SSJCP, Department of Pharmaceutical Analysis 90
  91. 91. Effect of α on Overall Resolution  Remember the resolution equation? Rs = 1 / 4[(α − 1) / α ] N [k ' /(1 + k ' )]  Let’s only look at the part involving α Rs = 1 / 4[(α −1) / α]  And see how much resolution will improve with small changes in α SSJCP, Department of Pharmaceutical Analysis 91
  92. 92.  For an α value of 1.1, the contribution of the selectivity term is (1.1 – 1) / 1.1 = 0.09  For an α value of 1.4, the contribution of the selectivity term is (1.4 – 1) / 1.4 = 0.29  So, a very small change in α leads to a more than THREE-FOLD increase in the contribution to resolution. SSJCP, Department of Pharmaceutical Analysis 92
  93. 93.  As α grows larger, its effect reaches a limit at a value of about 5.  Since α depends on components’ retention factor k’, longer columns eventually have a diminished effect on resolution. SSJCP, Department of Pharmaceutical Analysis 93
  94. 94. Influencing the Selectivity Factor α  Which of these is easiest to change?? ► Mobile Phase Type The importance of the type of interactions between the mobile phase and analytes is critical to the optimization of the selectivity of a system. ► Column Type The bonded phase functionality can be selected by its chemical nature to provide better selectivity in an analytical method. ► Temperature Selective interactions between analyte molecules and the stationary phase may not become evident until a critical temperature is attained. SSJCP, Department of Pharmaceutical Analysis 94
  95. 95. Summary of α Effects  Since α is the ratio of two k’ values, the same general statements apply: ► Increasing the mobile phase strength decreases individual values of k’, but their ratio (α) may affect resolution ► Increasing the temperature decreases individual values of k’, but their ratio (α) may significantly affect resolution.  A small increase in α leads to a large increase in resolution SSJCP, Department of Pharmaceutical Analysis 95
  96. 96. Column Efficiency, N  The column efficiency is defined as the degree to which a column and/or other system components can physically and chemically affect the separation of analytes.  As column efficiency increases, analyte components will elute in a smaller volume of the mobile phase, usually observed as narrower or “sharper” peak shapes.  Column efficiency is generally expressed in terms of theoretical plate number. SSJCP, Department of Pharmaceutical Analysis 96
  97. 97. Calculation of Theoretical Plates N = A(tr /W)2 W A 4 Wh 5.54 Wi Method Width measured at Inflection point (60.7% of peak height) ½ Height 50% of peak height W3s 3s 32.4% of peak height W4s 16 4s 13.4% of peak height W5s 25 Wb 9 5s 4.4% of peak height 16 Tangent Baseline, following tangent drawing Constants A are different at each peak width, assuming a perfect Gaussian shape. Real-world peaks often have tailing, so widths measured at the lower part of the peak more accurately reflect the tailing when calculating N. SSJCP, Department of Pharmaceutical Analysis 97
  98. 98. Calculation of Efficiency, N Width measured at the baseline after tangent lines are drawn on the peak. Used when tailing is minimal. Width measured at 4.4% of peak height, no tangents drawn. Used when tailing is significant. SSJCP, Department of Pharmaceutical Analysis 98
  99. 99. Effect of N on Overall Resolution  Do you STILL remember the resolution equation? Rs let’s/lookαat − 1)part ]involving/(1 + k ' )] = 1 4[( the / α N [k ' N  Now Rs = 1 / 4 N  And see how much resolution will improve with changes in N SSJCP, Department of Pharmaceutical Analysis 99
  100. 100.  Since the contribution of N to resolution is a square root, doubling N from 5000 to 10,000 only increases the contribution to resolution by 41%.  To double the effect on resolution coming from N, we have to increase the value of N by a factor of 4 PLATE Plates 5000 5,000 10,000 10,000 20,000 20,000 √N CONTRIBUTION 141.4 100% √N Contribution 70.7 70.7 - - - - ----100 100 41% 41% 141.4 100% SSJCP, Department of Pharmaceutical Analysis 100
  101. 101. Effect of N on Overall Resolution  Note that there is no flattening of the curve like with k ’ and α.  Resolution will continue to increase as theoretical plates increase. SSJCP, Department of Pharmaceutical Analysis 101
  102. 102. Influencing the Efficiency, N  Particle Size and Size Distribution The smaller the particle size and the narrower the range of the particle size distribution, the more efficient the column.  Packing Type Totally porous particles will also have greater efficiency than solid or pellicular-shaped packing's, due to the additional surface area attributable to the pores.  Mobile Phase Viscosity As mobile phase viscosity increases, molecular movement through the mobile phase is inhibited.  Temperature For reverse phase chromatography, an increase in efficiency, N, may be realized as column temperature is increased. SSJCP, Department of Pharmaceutical Analysis 102
  103. 103. Effect of Particle Size on N  Smaller particle sizes result in higher numbers of theoretical plates Column Diameter (mm) Column Length (cm) Particle Size (µm) 4σ Peak Width (µL) Theoretical Plates per centimeter 10 25 10 1118 333 4.6 25 10 237 333 4.6 25 5 167 667 4.6 10 5 106 667 4.6 10 3 82 1111 4.6 3 3 45 1111 3 10 5 45 667 2 25 10 45 333 2 25 5 32 667 2 10 5 20 667 2 10 3 15 1111 1 25 10 11 333 1 25 5 8 667 1 25 3 6 1111 1 10 5 5 667 1 10 3 4 1111 SSJCP, Department of Pharmaceutical Analysis 103
  104. 104. Relative Influence of All Factors on Resolution Parameter Change N k’ α Rs Standard +10% N -25% N -50% N -60% N -75% N +10% k’ +10% α 10,000 11,000 7,500 5,000 4,000 2,500 10,000 10,000 2 2 2 2 2 2 2.2 2 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.2 1.52 1.59 1.31 1.07 0.96 0.76 1.56 2.78 Note that changing α a very small amount has the biggest effect SSJCP, Department of Pharmaceutical Analysis 104
  105. 105. Review of Factors PARAMTER INFLUENCED BY TARGET VALUE Efficiency, N Column, system flow path, configuration Minimum of 400 theoretical plates /cm Capacity factor, k’ MP strength 1.0 - 10 Selectivity, α M.P & S.P type 1.1 - 2 All of the above 1.3 – 1.5 or greater Resolution, Rs SSJCP, Department of Pharmaceutical Analysis 105
  106. 106. Normal Phase v/s Reversed Phase PARAMETER NP RP Polarity of packing Medium to high Low to medium Polarity of solvent Low to medium Medium to high Elution sequence Low polarity first High polarity first Increase solvent polarity Faster elution Slower elution SSJCP, Department of Pharmaceutical Analysis 106
  107. 107. ION EXCHANGE CHROMATOGRAPHY SSJCP, Department of Pharmaceutical Analysis 107
  108. 108.  based on the attraction between solute ions and charged sites bound to the stationary phase.  The stationary phase contains ionic groups like NR⁺з, SO⁻з which interact with the ionic groups of the sample molecules.  This method is suitable for the separation of charged molecules only.  Solute ions of the same charge as the charged sites on the column are excluded from binding  solute ions of the opposite charge of the charged sites of the column are retained on the column.  Strong acids & basic compounds may be separated by RP mode by forming ion pairs with suitable counter ions. SSJCP, Department of Pharmaceutical Analysis 108
  109. 109.  Solute ions that are retained on the column can be eluted from the column by changing the solvent conditions  They include: ► increasing the ion effect of the solvent system ► by increasing the salt concentration of the solution ► increasing the column temperature ► changing the pH of the solvent  SSJCP, Department of Pharmaceutical Analysis 109
  110. 110.  ion exchangers favor the binding of ions of higher charge and smaller radius.  increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time.  decrease in pH reduces the retention time in cation exchange while an increase in pH reduces the retention time in anion exchange.   lowering the pH of the solvent in a cation exchange column, more hydrogen ions are available to compete for positions on the anionic stationary phase, thereby eluting weakly bound cations. SSJCP, Department of Pharmaceutical Analysis 110
  111. 111. TYPES OF ION EXCHANGERS  Polystyrene resins ►These allow cross linkage which increases the stability of the chain. ►Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity.  Cellulose and dextran ion exchangers (gels) ► These possess larger pore sizes and low charge densities making them suitable for protein separation.  Controlled-pore glass or porous silica SSJCP, Department of Pharmaceutical Analysis 111
  112. 112. Examples  Stationary phase contains charged groups  SAX (Strong Anion Exchange): NH3+  WAX (Weak Anion Exchange): NR2H+(DEAE) [Di Ethyl Amino Ethanol]  SCX (Strong Cation Exchange): SO3-  WCX (Weak Cation Exchange): CarboxyMethyl (CM)  More highly charged analytes have stronger retention  More “bulky” stationary phases have weaker retention SSJCP, Department of Pharmaceutical Analysis 112
  113. 113.  IEC is widely used in the following applications: ►water purification ► preconcentration of trace components ► ligand-exchange chromatography ► ion-exchange chromatography of proteins ► high-pH anion-exchange chromatography of carbohydrates and oligosaccharides SSJCP, Department of Pharmaceutical Analysis 113
  114. 114. AFFINITY/ BIOAFFINITY CHROMATOGRAPHY SSJCP, Department of Pharmaceutical Analysis 114
  115. 115. AFFINITY CHROMATOGRAPHY  It uses highly specific biochemical interactions for separations.  The stationary phase contains specific groups of molecules which can absorb the sample if certain steric & charge related conditions are satisfied.  This technique can be used to isolate proteins, enzymes, receptors , ligands as well as antibodies from complex mixture. SSJCP, Department of Pharmaceutical Analysis 115
  116. 116. Affinity chromatography can be used to:  Purify and concentrate a substance from a mixture into a buffering solution  Reduce the amount of a substance in a mixture  Discern what biological compounds bind to a particular substance  Purify and concentrate an enzyme solution. SSJCP, Department of Pharmaceutical Analysis 116
  117. 117. Size Exclusion LC (or) Gel Permeation (or) Gel filtration SSJCP, Department of Pharmaceutical Analysis 117
  118. 118.  Stationary phase is a polymer (polystyrene-divinyl benzene or acrylamide) with a defined pore size  Large compounds cannot fit into the pores and elute first  Used to determine molecular weight distribution of polymers  Separates molecules according to their molecular mass.  Largest molecules are eluted first and smaller molecules last.  useful for determining the tertiary structure andquaternary structure of purified proteins.  used primarily for the analysis of large molecules such as proteins or polymers.  SEC works by trapping these smaller molecules in the pores of a particle.   widely used for the molecular weight determination of polysaccharides. SSJCP, Department of Pharmaceutical Analysis 118
  119. 119.  larger molecules simply pass by the pores as they are too large to enter the pores.  Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.  separates particles on the basis of molecular size (actually by a particle's Stokes radius or Stokes-Einstein radius, or hydrodynamic radius (RH).  named after George Gabriel Stokes is the radius of a hard sphere that diffuses at the same rate as the molecule.  generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of the purification. SSJCP, Department of Pharmaceutical Analysis
  120. 120.  The main application of gel-filtration chromatography: ► fractionation of proteins and other water-soluble polymers ► while gel permeation chromatography is used to analyze the molecular weight distribution of organicsoluble polymers. ► Either technique should not be confused with gel electrophoresis, where an electric field is used to "pull" or "push" molecules through the gel depending on their electrical charges. SSJCP, Department of Pharmaceutical Analysis 120
  121. 121. DISPLACEMENT CHROMATOGRAPHY  A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities  displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”.   because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentration. SSJCP, Department of Pharmaceutical Analysis 121
  122. 122. Aqueous Normal-Phase Chromatography (ANP)  ANP is a chromatographic technique which encompasses the mobile phase region between RPC and organic normal phase chromatography (ONPC).  This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reversed-phase solvents. SSJCP, Department of Pharmaceutical Analysis 122
  123. 123. ISOCRATIC & GRADIENT ELUTION  A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic (constant composition).  Word was coined by Csaba Horvath  A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution  In isocratic elution, peak width increases with retention time linearly   leads to the disadvantage that late-eluting peaks get very flat and broad.  SSJCP, Department of Pharmaceutical Analysis 123
  124. 124.  Gradient elution decreases the retention of the latereluting components so that they elute faster, giving narrower (and taller) peaks for most components  improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward.  increases the peak height (the peak looks "sharper")  may include sudden "step" increases in the percentage of the organic component, or different slopes at different times. SSJCP, Department of Pharmaceutical Analysis 124
  125. 125.  In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change  In gradient elution, the elution order may change as the dimensions or flow rate change  The driving force in RPC originates in the high order of the water structure.  The role of the organic component of the mobile phase is to reduce this high order and thus reduce the retarding strength of the aqueous component. SSJCP, Department of Pharmaceutical Analysis 125
  126. 126. ISOCRATIC SYSTEM  Same mobile phase concentration throughout the separation  Use 1 pump and pre-mix solvents  Use 1 pump and a valve for 4 different solvents  Use 2 pumps and vary the amount coming from each pump SSJCP, Department of Pharmaceutical Analysis 126
  127. 127. ISOCRATIC SEPARATION  1 pump and premixing  4.6 mm ID Column, 1 mL/min, Changing MeOH % vs Water SSJCP, Department of Pharmaceutical Analysis 127
  128. 128.  1 pump with valve and premixing To Column To Column A = 80% Methanol, 20% Water B = 70% Methanol, 30% Water ABCD C = 60% Methanol, 40% Water D = 50% Methanol, 50% Water SSJCP, Department of Pharmaceutical Analysis 128
  129. 129.  1 pump with mixer – let the pump do the work! To Column To Column Method 1: A.CONC = 20%, B.CONC = 80% Method 2: A.CONC = 30%, B.CONC = 70% Method 3: A.CONC = 40%, B.CONC = 60% ABCD Method 4: A.CONC = 50%, B.CONC = 50% SSJCP, Department of Pharmaceutical Analysis 129
  130. 130. LOW PRESSURE GRADIENT 1 Pump, solvents are mixed before the pump Requires degassing To Column To Column ABCD SSJCP, Department of Pharmaceutical Analysis 130
  131. 131. HIGH PRESSURE GRADIENT Binary Gradient 2 Pumps and Mixer Ternary Gradient 3 Pumps and Mixer ………. ………. ………. ………. ………. ………. ………. ………. ………. SSJCP, Department of Pharmaceutical Analysis 131
  132. 132. HIGH v/s LOW PRESSURE GRADIENT  High Pressure Gradient ► Multiple pumps are used with a mixer after the pumps  Low Pressure Gradient ► Solvents are mixed before the pump SSJCP, Department of Pharmaceutical Analysis 132
  133. 133. Gradient v/s Isocratic Conditions: Summarized  Isocratic ► mobile phase solvent composition remains constant with time ► Best for simple separations ► Often used in quality control applications that support and are in close proximity to a manufacturing process  Gradient ► mobile phase solvent (“B”) composition increases with time ► Best for the analysis of complex samples ► Often used in method development for unknown mixtures ► Linear gradients are most popular (for example, the “gradient” shown at right) SSJCP, Department of Pharmaceutical Analysis 133
  134. 134. PRINCIPLE OF SEPARATION  The principle of separation is Adsorption.  Separation of components takes place because of the difference in affinity of compounds towards stationary phase. SSJCP, Department of Pharmaceutical Analysis 134
  135. 135. The principle of separation in normal phase mode and reverse phase mode is adsorption.  The component which has more affinity towards the adsorbent, travels slower.  The component which has less affinity towards the stationary phase travels faster.  Since no two components have the same affinity towards the stationary phase, the components are separated. 1 Stronger interaction 2 Weaker interaction SSJCP, Department of Pharmaceutical Analysis 135
  136. 136. PRESENT CHALLENGES  Analysis of matrices like pharmaceutical dosage forms and biological samples will always be challenging, due to their great diversity, intricacy and complexity.  Analyzing complex samples like biological products and biological fluids is a significant challenge even with today’s advanced and sophisticated instrumentation.  Quality assurance & quality control of pharmaceuticals and formulations play a vital role in ensuring the availability of safe & effective drug products to the population.  Quantitative estimation of the chemical entity of a drug substance is pivotal to its quality assurance and control. SSJCP, Department of Pharmaceutical Analysis 136
  137. 137.  The problem may be a simple one when one is dealing with a pure and single substance.  But, during the process of formulation, the original drug substance of high purity is often diluted and mixed with other additives.  This may lead to interferences of the additives in the method of estimation.  The overall aim of our research is to develop new methods for quantitative determination of novel drugs in pharmaceutical dosage forms.  The emphasis is to find new principles for separations using liquid chromatography (HPLC) and to understand the mechanisms behind. SSJCP, Department of Pharmaceutical Analysis 137
  138. 138. INSTRUMENTATION SSJCP, Department of Pharmaceutical Analysis 138
  139. 139. SCHEMATIC REPRESENTATION OF AN HPLC UNIT 1.Solvent reservoirs 2. Solvent degasser 3. Gradient valve 4. Mixing vessel for delivery of the mobile phase 5. Highpressure pump 6.Switching valve in "inject position” & 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 SSJCP, Department of Pharmaceutical Analysis 139
  140. 140. BASIC FLOW CHART OF A HPLC SYSTEM SETUP SSJCP, Department of Pharmaceutical Analysis 140
  141. 141. HPLC System Components  Pumps ► Micro to Analytical to Preparative Flow Rates ► Isocratic and Gradient Configurations  Degasser ► How it Affects Pumping and Sample Injection  Valves ► Solvent Selection and Flow Selection SSJCP, Department of Pharmaceutical Analysis 141
  142. 142.  Sample Injection ► Manual Injector or Autosampler  Oven ► How Temperature Affects Separation ► Valves for Column Switching  Detectors ► UV-VIS ► Diode Array ► Fluorescence ► Light Scattering ► Refractive Index ► Conductivity ► Mass Spectrometer  Recorders and Integrators SSJCP, Department of Pharmaceutical Analysis 142
  143. 143.  Fraction Collector ► Isolate Specific Sample Components ► Purify Compounds for Multi-Step Synthesis  Column ► Types of Packing Material ► Factors Affecting Separation ► Particle Size and Column Length ► Flow Rate and Temperature SSJCP, Department of Pharmaceutical Analysis 143
  144. 144. A SOLVENT DELIVERY SYSTEM  A mobile phase is pumped under pressure from one or several reservoir and flows through the column at a constant rate.  For NP separation eluting power increases with increasing polarity of the solvent but for reversed phase separation, eluting power decreases with increasing polarity.  A degasser is needed to remove dissolved air and other gases from the solvent. SSJCP, Department of Pharmaceutical Analysis 144
  145. 145. HPLC DEGASSING  Degassing removes dissolved air that interferes with check valve operation  Refluxing ► not practicable  Ultrasonic degassing ► ineffective & applicable for ACN/ Water  Helium sparge ► Gas line from the tank directly in the solvent bottle  Vacuum degassing ► Sonicate before connecting to the system ► Online with a degassing unit SSJCP, Department of Pharmaceutical Analysis 145
  146. 146. Various solvent delivery systems SSJCP, Department of Pharmaceutical Analysis 146
  147. 147. PUMP MODULES Types:  Isocratic pump ► delivers constant mobile phase composition; ► solvent must be pre-mixed; ► lowest cost pump  Gradient pump ► delivers variable mobile phase composition; ► can be used to mix and deliver an isocratic mobile phase or a gradient mobile phase  Binary gradient pump ► delivers two solvents  Quaternary gradient pump ► four solvents SSJCP, Department of Pharmaceutical Analysis 147
  148. 148.  The pump is one of the most important component of HPLC, since its performance directly affects retention time, reproducibility and detector sensitivity.  Three main types of pumps are used in HPLC. ►Displacement pump ► Reciprocating pump ► Pneumatic (or) constant pressure pump SSJCP, Department of Pharmaceutical Analysis 148
  149. 149.  DISPLACEMENT PUMP: It produce a flow that tends to independent of viscosity and back pressure and also output is pulse free but possesses limited capacity (250ml).  RECIPROCATING PUMP: It has small internal volume (35400µl), their high output pressure(up to 10,000psi) and their constant flow rates. But it produces a pulsed flow.  PNEUMATIC (OR) CONSTANT PRESSURE PUMP: ► They are pulse free . ► Suffer from limited capacity as well as a dependence of flow rate on solvent viscosity and column back pressure. ► They are limited to pressure less than 2000 psi. SSJCP, Department of Pharmaceutical Analysis 149
  150. 150. HPLC PUMPS – TWO BASIC TYPES  Tandem piston ► Two pistons with different volumes (48 and 24 µL) ► During each stroke, 24 µL of liquid is delivered ► Best for higher analytical flow rates, up to 10 mL/min ► Some pulsation is observed, and pulse dampeners are available ► Not recommended for pulse-sensitive detectors like RID and CDD SSJCP, Department of Pharmaceutical Analysis 150
  151. 151. TANDEM PISTON PUMP Secondary Piston ↓ ← Primary Piston SSJCP, Department of Pharmaceutical Analysis 151
  152. 152. DUAL PISTON  Two pistons with equal volume (10 µL each)  During each stroke, 10 µL is delivered  Best for low flow rates (< 1 mL/min)  Little to NO pulsation  So it’s ideal for pulse sensitive detectors like RID and CDD SSJCP, Department of Pharmaceutical Analysis 152
  153. 153. DUAL PISTON SSJCP, Department of Pharmaceutical Analysis 153
  154. 154. OTHER PUMP COMPONENTS  Check Valves ► Control liquid movement in and out of the pump head SSJCP, Department of Pharmaceutical Analysis 154
  155. 155.  Piston/plunger seal ► Prevents solvent leakage out of pump head  Inline filter ► Removes solvent particulates SSJCP, Department of Pharmaceutical Analysis 155
  156. 156. VALVES USED WITH PUMPS  Solvent Selection – 2 Solvents Per Pump ► Use for solvent switching SSJCP, Department of Pharmaceutical Analysis 156
  157. 157.  Solvent Selection – 2 Solvents Per Pump ► Use for pump loading of large sample volumes SSJCP, Department of Pharmaceutical Analysis 157
  158. 158.  Solvent Selection – 4 Solvents Per Pump ► Use for low pressure gradient formation SSJCP, Department of Pharmaceutical Analysis 158
  159. 159.  Solvent Selection – 4 Solvents Per Pump ► Use for different gradients in method development SSJCP, Department of Pharmaceutical Analysis 159
  160. 160. SAMPLE INJECTION SYSTEM  There are three important ways of introducing the sample in to the injection port. ► Loop injection : in which a fixed amount of volume is introduced by making use of fixed volume loop injector. ► Valve injection: in which, a variable volume is introduced by making use of an injection valve. ► On column injection: in which, a variable volume is introduced by means of a syringe through a septum. SSJCP, Department of Pharmaceutical Analysis 160
  161. 161. SSJCP, Department of Pharmaceutical Analysis 161
  162. 162. SAMPLE INJECTION – MANUAL  Manual Injector with Syringe ► Fixed loop of varying sizes (1 to 20 mL or more) ► Fill with syringes of varying sizes ► Can include a switch to start a data system SSJCP, Department of Pharmaceutical Analysis 162
  163. 163. SAMPLE INJECTION – AUTOMATIC  Fixed-Loop Auto sampler ► Loop is installed on the valve and can be changed for different injection volumes ► External syringe draws sample and fills loop  Advantages: ► low cost ► rugged ► few moving parts  Disadvantages: ► Poor performance for low volume injections higher carryover ► always some sample loss SSJCP, Department of Pharmaceutical Analysis 163
  164. 164. Sample Injection… how is a sample actually put into an LC system  Manual Injector: 1. User manually loads sample into the injector using a syringe and then turns the handle to inject sample into the flowing mobile phase which transports the sample into the beginning (head) of the column, which is at high pressure  Autosampler: 1. User loads vials filled with sample solution into the autosampler tray (100 samples) and the autosampler automatically : 2. measures the appropriate sample volume, 3. injects the sample, 4. then flushes the injector to be ready for the next sample, etc., until all sample vials are processed for unattended automatic operation SSJCP, Department of Pharmaceutical Analysis 164
  165. 165. SAMPLE INJECTION – FIXED LOOP  External syringe draws sample, then fills the fixed-volume loop attached to the valve. SSJCP, Department of Pharmaceutical Analysis 165
  166. 166.  Needle-in-the-flow path auto sampler ► Sample loop and needle are a single piece of tubing ► Loop and needle are cleaned during the run ► Metering pump draws sample very precisely  Advantages: ► no sample loss, ► low carryover  Disadvantages: ► higher cost ► more delay volume for gradient SSJCP, Department of Pharmaceutical Analysis 166
  167. 167. SAMPLE INJECTION TO FLOW PATH Sample Loading SSJCP, Department of Pharmaceutical Analysis 167
  168. 168. RINSING AFTER INJECTION Rinse liquid flows through ports 5 and 6 of the high pressure valve. Sample aspiration uses port 5. If air is present around port 5, injection reproducibility will be low. Rinse liquid degassed! SSJCP, Department of Pharmaceutical Analysis MUST be 168
  169. 169. %A {H2O} %B %C {MeOH} Flow Rate Pressure (mL/min) (atmos.) to column load Ready inject Rheodyne Injector Varian 9010 Solvent Delivery System to injector through pulse dampener Ternary Pump A Column through pump C B SSJCP, Department of Pharmaceutical Analysis from solvent reservoir to det ect or 169
  170. 170. CHROMATOGRAPHIC COLUMN  The column is usually made up of heavy glass or stainless steel tubule to withstand high pressure  The columns are usually 10-30cm long and 4-10mm inside diameter containing stationary phase at particle diameter of 25µm or less  Column with internal diameter of 5mm give good results because of compromise between efficiency, sample capacity, and the amount of packaging and solvent required SSJCP, Department of Pharmaceutical Analysis 170
  171. 171.  Within the Column is where separation occurs  Key Point – Proper choice of column is critical for success in HPLC  Types of columns in HPLC: ►Analytical [internal diameter (i.d.) 1.0 - 4.6-mm; lengths 15 – 250 mm] ► Preparative (i.d. > 4.6 mm; lengths 50 – 250 mm) ► Capillary (i.d. 0.1 - 1.0 mm; various lengths) ► Nano (i.d. < 0.1 mm, or sometimes stated as < 100 µm)  Materials of construction for the tubing ► Stainless steel (the most popular; gives high pressure capabilities) ► Glass (mostly for biomolecules) ► PEEK polymer (biocompatible and chemically inert to most solvents) SSJCP, Department of Pharmaceutical Analysis 171
  172. 172. HPLC Columns Packing Materials  Columns are packed with small diameter porous particles.  The most popular sizes are: 5-μ m, 3.5- μ m and 1.8-μ m  Columns are packed using high-pressure to ensure that they are stable during use. Most users purchase pre-packed columns to use in their liquid chromatographs  These porous particles in the column usually have a chemically bonded phase on their surface which interacts with the sample components to separate them from one another for example, C18 is a popular bonded phase  The process of retention of the sample components (often called analytes) is determined by the choice of column packing and the selection of the mobile phase to push the analytes through the packed column. SSJCP, Department of Pharmaceutical Analysis 172
  173. 173. HPLC COLUMN OVENS  Block heater with solvent preheater ► Column is housed between 2 metal plates ► Mobile phase is plumbed into the block for preheating  Forced air ► Column is in a large chamber with air circulation ► Better temperature equilibration ► Room for column switching valves SSJCP, Department of Pharmaceutical Analysis 173
  174. 174. Why Use a Column Oven?  Retention times decrease & higher flow rates possible 2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH/Water 50°C 45°C 40°C 35°C 30°C 25°C 20°C SSJCP, Department of Pharmaceutical Analysis 174
  175. 175. DETECTORS  The function of detector in HPLC is to monitor the mobile phase as it merges from the column.  Detectors are usually of two types: ► Bulk property detectors: It compares overall changes in a physical property of the mobile phase with and without an eluting solute e.g. refractive index ,dielectric constant or density. ► Solute property detectors: It responds to a physical property of the solute which is not exbited by the pure mobile phase.e.g.UV absorbance,fluoroscence or diffusion current. SSJCP, Department of Pharmaceutical Analysis 171 175
  176. 176. TYPES OF DETECTORS There are mainly 4 types of detectors are used in HPLC:  Photometric detectors. ► Single wavelength detectors. ► Multi wavelength detectors. ► Variable wavelength detectors. ► Programmable detectors. ► Diode array detectors .  Fluorescence detectors.  Refractive index detectors.  Electrochemical detectors.  Evaporative light scattering detectors  IR detectors  UV detectors SSJCP, Department of Pharmaceutical Analysis 176
  177. 177. PHOTOMETRIC DETECTORS  These normally operate in the ultra violet region of the spectrum .  Most extensively used in pharmaceutical analysis. SSJCP, Department of Pharmaceutical Analysis 177
  178. 178. SINGLE WAVELENGTH DETECTORS  Equipped with a low pressure mercury discharge lamp.  The absorbance is measured at the wavelength of mercury at 254 nm. SSJCP, Department of Pharmaceutical Analysis 178
  179. 179. MULTI WAVELENGTH DETECTORS  Employ mercury and other discharge sources.  When used in combination with interference filters allow a no of monochromatic wavelengths to be selected e.g. 206, 226, 280 , 313, 340 or 365 nm. SSJCP, Department of Pharmaceutical Analysis 179
  180. 180. Multi-wavelength UV-Vis Absorption Detector Deuterium Lamp SSJCP, Department of Pharmaceutical Analysis Photodiode Array 180
  181. 181. VARIABLE WAVELENGTH DETECTORS  Use a deuterium light source.  A grating monochromator to allow selection of any wavelength in deuterium continuum (190-360 nm). SSJCP, Department of Pharmaceutical Analysis 177 181
  182. 182. UV-VISIBLE DETECTOR  UV-Visible ► Wavelength range 190-700 nm ► D2 and W lamps  Most common HPLC detector for a variety of samples ► Proteins and peptides ► Organic molecules ► Pharmaceuticals  Monitor two wavelengths at one time SSJCP, Department of Pharmaceutical Analysis 182
  183. 183. UV-Visible Detector SSJCP, Department of Pharmaceutical Analysis 183
  184. 184. Variable wavelength detector SSJCP, Department of Pharmaceutical Analysis 184
  185. 185. Variable UV/Vis Detector ABS AUFS λ RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min Ready SSJCP, Department of Pharmaceutical Analysis 185
  186. 186. PROGRAMMABLE DETECTORS  Allow the automatic change of wavelength between and during the chromatographic analysis. SSJCP, Department of Pharmaceutical Analysis 186
  187. 187. DIODE ARRAY DETECTORS  They are microprocessor – controlled photodiode array spectrophotometers in which light from an UV source passes through the flow cell into a polychromator which disperses the beam so that the full spectrum falls on the array of diodes. SSJCP, Department of Pharmaceutical Analysis 183 187
  188. 188. DIODE ARRAY DETECTOR  Wavelength range 190-900 nm  D2 and W lamps  Spectral information about sample  Create compound libraries to identify unknowns  Monitor an entire wavelength range at one time – up to 790 wavelengths vs. only 2 with a UV detector SSJCP, Department of Pharmaceutical Analysis 188
  189. 189. DIODE ARRAY DETECTOR SSJCP, Department of Pharmaceutical Analysis 189
  190. 190. FLUORESCENCE DETECTOR  These are essentially filter fluorimeter or spectro -fluorimeters equipped with grating monochromators, and micro flow cell.  Their sensitivity depends on the fluorescence properties of the components in the elute. SSJCP, Department of Pharmaceutical Analysis 190
  191. 191.  Fluorescence detector ► Xenon lamp for light source ► Excitation wavelength range: 200-650 nm ► Emission wavelength range: up to 900 nm depending on photomultiplier installed  Used primarily for amino acid analysis ► Derivatize samples before (pre-column) or after separation( post-column) SSJCP, Department of Pharmaceutical Analysis 191
  192. 192. Fluorescence Detector SSJCP, Department of Pharmaceutical Analysis 192
  193. 193. REFRACTIVE INDEX DETECTORS  Which respond to the change in the bulk property of the refractive index of the solution of the component in the mobile solvent system.  The sensitivity of the refractive index detector is much less than that of specific solute property detectors, they are useful for the detection of substances(e.g ,carbohydrates & alcohols) which do not exhibit other properties that can be used as the basis for specific detection. SSJCP, Department of Pharmaceutical Analysis 193
  194. 194. Refractive Index Detector  For samples with little or no UV Absorption  Alcohols, sugars, saccharides, fatty acids, polymers  Best results when RI of samples is very different from RI of mobile phase  Flow cell is temperature controlled with a double insulated heating block  Requires isocratic separations  Requires low pulsation pumps SSJCP, Department of Pharmaceutical Analysis 194
  195. 195. RI BALANCE  Fill sample and reference cell with mobile phase SSJCP, Department of Pharmaceutical Analysis 195
  196. 196. RI ANALYZE  Mobile phase flows through sample side only  As the refractive index changes, the image on the photodiode is deflected or “unbalanced”, and the difference in current to the photodiode is measured. SSJCP, Department of Pharmaceutical Analysis 196
  197. 197. Refractive Index Detector SSJCP, Department of Pharmaceutical Analysis 197
  198. 198. ELECTROCHEMICAL DETECTORS  These are based on standard electrochemical principles involving amperometry,voltametryand polarography.  These detectors are very sensitive for substances that are electroactive ,i.e. those that undergo oxidation or reduction .  They have found particular application in the assay of low levels of endogenous catecholamines in biological tissues,pesticides,tryptophan derivatives and many drugs. SSJCP, Department of Pharmaceutical Analysis 198
  199. 199. Electrochemical Detector SSJCP, Department of Pharmaceutical Analysis 199
  200. 200. EVAPORATIVE LIGHT SCATTERING (ELSD)  Also for low or no UV absorbing compounds  Sometimes called a “Universal” detector  Requires NO equilibration (unlike RID)  Can be used with gradients and volatile buffers (unlike RID)  Semi-volatile compounds can be detected at low temperatures SSJCP, Department of Pharmaceutical Analysis 200
  201. 201. ELSD OPERATION Column Effluent Nebulizer Nebulizer Gas (Air or Nitrogen) Nebulization Chamber Analyte Drift Tube (Heated Zone Evaporation Area) PMT Light Source Amplifier Light Scattering Cell SSJCP, Department of Pharmaceutical Analysis Signal Output 201
  202. 202. ELSD v/s OTHER DETECTORS  ELSD has higher sensitivity than UV and RID  ELSD can be used with gradients, unlike RID SSJCP, Department of Pharmaceutical Analysis 202
  203. 203. CONDUCTIVITY DETECTOR  Flow cell contains 2 electrodes  Measure ion amounts in sample  REQUIRES low pulsation pumps  Flow cell must be placed in a column oven SSJCP, Department of Pharmaceutical Analysis 203
  204. 204.  Use in Environmental and water testing ► Fl-, Cl- NO3-, PO43-, SO42► Li+, Na+, K+, Mg2+, Cu2+, M-CN complexes  Determine organic acids in fruit juice ► Oxalic, Maleic, Malic, Succinic, Citric  Analyze surfactants ► Sulfonates, long/short chain ammonium SSJCP, Department of Pharmaceutical Analysis 204
  205. 205. Mass Spectrometer Detector  Separate sample components as ions according to their mass to charge (m/z) ratio  Three stages to detection  Vaporization: liquid from HPLC column converted to an aerosol  Ionization: neutral molecules converted to charged species (either positive or negative)  Mass Analysis: filter ions by m/z ratio SSJCP, Department of Pharmaceutical Analysis 205
  206. 206. TWO IONIZIZATION TYPES  APCI: Atmospheric Pressure Chemical Ionization ► For molecules up to 1000 Da ► Singly charges ions ► Best for analysis of non-polar molecules  ESI: Electrospray Ionization ► Can be used for large biopolymers ► Forms multiply charged ions ► Best for the analysis of polar molecules, especially pharmaceutical products and proteins SSJCP, Department of Pharmaceutical Analysis 206
  207. 207. MS DETECTOR Heated capillary Q-array Orthogonal source geometry Octapole Electron Multiplier Detector Quadrupole mass analyser SSJCP, Department of Pharmaceutical Analysis 207
  208. 208. FRACTION COLLECTOR  Purify raw materials or compounds from synthesis  Collect by slope, level, time, volume  Isolate single peaks per tube, or divide peaks into small “slices” for extra purity SSJCP, Department of Pharmaceutical Analysis 208
  209. 209. Temperature Control in HPLC: Why is it needed?  Reproducibility ► Retention in HPLC is temperature-dependent ► If temperature varies, then it is difficult to assign “peaks” to specific compounds in the chromatogram and the peak areas/heights may vary  Solubility ► Certain chemical compounds may have low solubility in the HPLC mobile phase ► If they are injected into the flow stream they may precipitate or other difficulties may arise  Stability ► Certain chemical compounds, especially biological compounds such as enzymes or proteins, may not be stable at room temperature or higher ► The temperature needs to be much lower down to 4°C SSJCP, Department of Pharmaceutical Analysis 209
  210. 210. How is Temperature Control Achieved?  Three (3) ways the temperature of a column could be controlled, use: ► Oven ► Heater Block ► Water bath SSJCP, Department of Pharmaceutical Analysis 210
  211. 211. What is HPLC used for?  Separation and analysis of non-volatile or thermally-unstable compounds  HPLC is optimum for the separation of chemical and biological compounds that are non-volatile  NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline, etc.), gas chromatography is a better separation technique. SSJCP, Department of Pharmaceutical Analysis 211
  212. 212.  Typical non-volatile compounds are: ► Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol) ► Salts like sodium chloride and potassium phosphate ► Proteins like egg white or blood protein ► Organic chemicals like polymers (e.g. polystyrene, polyethylene) ► Heavy hydrocarbons like asphalt or motor oil ► Many natural products such as ginseng, herbal medicines, plant extracts ► Thermally unstable compounds such as trinitrotoluene (TNT), enzymes etc…. SSJCP, Department of Pharmaceutical Analysis 212
  213. 213. FOR QUALITATIVE ANALYSIS  The identification(ID) of individual compounds in the sample; ► the most common parameter for compound ID is its retention time (the time it takes for that specific compound to elute from the column after injection); ► depending on the detector used, compound ID is also based on the chemical structure, molecular weight or some other molecular parameter. SSJCP, Department of Pharmaceutical Analysis 213
  214. 214. FOR QUANTITATIVE ANALYSIS  The measurement of the amount of a compound in a sample (concentration); meaning, how much is there?  There are two main ways to interpret a chromatogram (i.e. perform quantification): ► determination of the peak height of a chromatographic peak as measured from the baseline; ► determination of the peak area (see figure below);  In order to make a quantitative assessment of the compound, a sample with a known amount of the compound of interest is injected and its peak height or peak area is measured.  In many cases, there is a linear relationship between the height or area and the amount of sample. SSJCP, Department of Pharmaceutical Analysis 214
  215. 215. Preparation of Pure Compound(s)  By collecting the chromatographic peaks at the exit of the detector  and concentrating the compound (analyte) by removing/evaporating the solvent  a pure substance can be prepared for later use (e.g. organic synthesis, clinical studies, toxicology studies, etc….).  This methodology is called preparative chromatography. SSJCP, Department of Pharmaceutical Analysis 215
  216. 216. Trace analysis  A trace compound is a compound that is of interest to the analyst but it’s concentration is very low, usually less than 1% by weight, often parts per million (ppm) or lower;  the determination of trace compounds is very important in pharmaceutical, biological, toxicology, and environmental studies since even a trace substance can be harmful or poisonous;  in a chromatogram trace substances can be difficult to separate or detect;  high resolution separations and very sensitive detectors are required SSJCP, Department of Pharmaceutical Analysis 216
  217. 217. SSJCP, Department of Pharmaceutical Analysis 217
  218. 218. SEPARATION TECHNIQUES IN HPLC METHOD DEVELOPMENT GOAL COMMENT Resolution Precise and rugged quantitative analysis requires that Rs be greater than 1.5 Separation time 3-10 min is desirable for routine procedures Quantitation ≤2% for assays; ≤ 5% for less-demanding analyses; ≤ 15% for trace analyses Peak Height Narrow peaks are signal/noise ratios Solvent composition Minimum mobile-phase use per run is desirable desirable SSJCP, Department of Pharmaceutical Analysis for large 218
  219. 219. THE VALIDATION PROCESS ► It consists of four distinct steps: Software validation Hardware (instrumentation) validation/qualification Method validation System suitability SSJCP, Department of Pharmaceutical Analysis 219
  220. 220. HPLC SYSTEM QUALIFICATION SSJCP, Department of Pharmaceutical Analysis 220
  221. 221. GOALS FOR AN IMPROVED ANALYTICAL METHOD DEVELOPMENT ► Qualitative identification - structural information, retention time, color change, pH etc ► Quantitative determination - accurate, precise and reproducible in any laboratory settings ► Ease of use, viability to be automated, high sample throughput, and rapid sample turnaround time. ► Decreased cost per analysis - using simple quality assurance and quality control procedures SSJCP, Department of Pharmaceutical Analysis 221
  222. 222. ► Sample preparation minimizing - time, effort, materials, and volume of sample consumed ► Direct output of qualitative or quantitative data evaluations, interpretation, printing out and transmission OPTIMIZATION & ANALYTICAL FIGURES OF MERIT ► initial sets of conditions - resolution, peak shape, plate counts, asymmetry, capacity, elution time, detection limits ► quantifying the specific analyte of interest, accuracy and precision of Quantitation and specificity must be defined. SSJCP, Department of Pharmaceutical Analysis 222
  223. 223. ► Chromatographic resolution adequate ► Limits of detections are lower ► Calibration plots are linear ► Sample throughout is increased ► Sample preparation before analysis is minimized ► Interference is minimized and identified ► Data acquisition - translated, interpreted, printed & stored ► Reproducibility of analytical figures of merit & Cost per analysis is minimized SSJCP, Department of Pharmaceutical Analysis 223
  224. 224. METHOD VALIDATION APPROACHES ► Samples of the given analyte ► Concentration in the matrix ► High degree of accuracy and precision ► Zero, Single and Double –Blind spiking methods ► Inter laboratory collaborative studies ► Comparison with a currently accepted compendium method SSJCP, Department of Pharmaceutical Analysis 224
  225. 225. STEP-BY-STEP HPLC METHOD DEVELOPMENT, OPTIMIZATION AND VALIDATION: AN OUTLINE ► Analyte Standard Characterization ► Method Requirements ► Literature Search and Prior Methodology ► Choosing a Method ► Instrument Setup and Initial Studies ► Optimization ► Demonstration of Analytical Figures of Merit with Standards SSJCP, Department of Pharmaceutical Analysis 225
  226. 226. ► Evaluation of Method Development with Actual Samples and Derivation of Figures of Merit ► Validation of Figures of Merit ► Determination of Percent Recovery of Actual Sample and Demonstration of Quantitative Sample Analysis ► Method Validation ► Preparation of Written Protocols and Procedures ► Transfer of Method Technology to Outside Laboratories and Interlaboratory Collaborative Studies SSJCP, Department of Pharmaceutical Analysis 226
  227. 227. ► Comparison of Interlaboratory Collaborative Studies ► Preparation of Summary Report on Overall Method Validation Results ► Summary Report of Final Method and Validation Procedures and Results and also Preparation of Journal Article for Submission THE OUTLINE PROTOCOL OF HPLC METHOD SSJCP, Department of Pharmaceutical Analysis 227
  228. 228. STEPS FOR HPLC METHOD DEVELOPMENT Information on sample, define separation goals Validate method for release to routine laboratory Need for special procedure sample pretreatment, etc Quantitative calibration Choose detector and detector settings Choose LC method; preliminary run; estimate the best separation conditions Check for problems or requirement for special procedure Optimize separation conditions SSJCP, Department of Pharmaceutical Analysis
  229. 229. PARAMETERS USED IN METHOD VALIDATION SSJCP, Department of Pharmaceutical Analysis 229
  230. 230. SPECIFICITY ► It is the ability to measure accurately and specifically the analyte of interest in the presence of other components that may be expected to be present in the sample matrix ► Specificity is also measured and documented in a separation by the resolution, plate count (efficiency) and tailing factor ► Blank solution to show no interference with excipients or degradation products or impurities ► Placebo to demonstrate the lack of interference from excipients ► Spiked samples to show that all known related substances are resolved from each other SSJCP, Department of Pharmaceutical Analysis 230
  231. 231. LINEARITY AND RANGE ► It is the ability of the method to elicit test results that are directly proportional to analyte concentration within a given range ► Reported as the variance of the slope of the regression line ► ICH guidelines specify a minimum of five concentration levels ► Assay : 80-120% of the theoretical content of active Content Uniformity: 70-130% ► Dissolution: ±20% of limits; e.g if limits cover from 20% to 90% l.c. (controlled release), linearity should cover 0-110% of l.c. SSJCP, Department of Pharmaceutical Analysis 231
  232. 232. ► Impurities: reporting level to 120% of shelf life limit ► Assay/Purity by a single method: reporting level of the impurities to 120% of assay limit ► Correlation coefficient (r) = API: ≥ 0.998 & Impurities: ≥ 0.99 ► y-intercept and slope should be indicated together with plot of the data SSJCP, Department of Pharmaceutical Analysis 232
  233. 233. ACCURACY ► Measure of exactness of an analytical method or closeness of agreement between the measured value and the value that is accepted either as a conventional, true value or an accepted reference value ► Measured as percentage of analyte recovered by assay, by spiking samples in a blind study ► API (Active Pharmaceutical Ingredient): against an RS (Reference Standard) of known purity, or via an alternate method of known accuracy; analysis in triplicate ► FPP (Finished Pharmaceutical Product): samples/placeboes spiked with API, across the range of 80-120% of the target concentration, 3 concentrations, in triplicate each SSJCP, Department of Pharmaceutical Analysis 233
  234. 234. ► Report % recovery (mean result and RSD): 100±2% ► Impurities: API/FPP spiked with known impurities ► Across the range of LOQ-150% of the target concentration (shelf life limit), 3-5 concentrations, in triplicate each. (LOQ, 50%, 100%, 150%) ► % recovery: in general, within 80-120%, depends on the level of limit ► ICH Q2 states: accuracy may be inferred once precision, linearity and specificity SSJCP, Department of Pharmaceutical Analysis 234
  235. 235. LOD / LOQ ► LOD: the lowest concentration of an analyte in a sample that can be detected though not necessarily quantitated. ► LOQ: the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method ► signal to noise ratio: LOD = 3:1 , LOQ = 10:1 ● May vary with lamp aging, model/manufacturer of detector, column ► standard deviation of the response and the slope of the calibration curve at levels approximating the LOD /LOQ ● σ = the standard deviation of the response, based on the standard deviation of the blank & the calibration curve & S = Slope SSJCP, Department of Pharmaceutical Analysis
  236. 236. ► should be validated by analysis of samples at the limits ► LOD: below the reporting threshold ► LOQ: at or below the specified limit ► Not required for assay/dissolution methods ► Applicant should provide ● the method of determination ● the limits ● chromotograms SSJCP, Department of Pharmaceutical Analysis 236
  237. 237. ROBUSTNESS / RUGGEDNESS ► Robustness: capacity of a method to remain unaffected by small deliberate variations in the method parameters ► Ruggedness: degree of reproducibility of the results obtained under a variety of conditions, expressed as % RSD ► Evaluated by varying method parameters such as percent organic solvent, pH, ionic strength or temperature , determining the effect on the results of the method, columns, laboratories, analysts, instruments, reagents and experimental periods. SSJCP, Department of Pharmaceutical Analysis 237
  238. 238. SYSTEM SUITABILITY TESTING (SST) ► used to verify resolution, column efficiency, and repeatability of the analysis system to ensure its adequacy for performing the intended application on a daily basis. ►Parameters: ● Number of theoretical plates (efficiency) ● Capacity factor ● Separation (relative retention) ● Resolution ● Tailing factor ● Relative Standard Deviation (Precision) SSJCP, Department of Pharmaceutical Analysis 238
  239. 239. Center for Drug Evaluation and Research (CDER) Limits for SST SSJCP, Department of Pharmaceutical Analysis 239
  240. 240. CHARACTERISTICS TO BE VALIDATED IN HPLC CHARACTERISTICS ACCEPTANCE CRITERIA Accuracy/trueness Recovery 98-102% with 80, 100 & 120% spiked sample Repeatability RSD < 2% Intermediate precision RSD < 2% Specificity/selectivity No interference Detection limit S/N > 2 or 3 Quantitation limit S/N > 10 Linearity Correlation coefficient r > 0.999 Range 80 – 120% Stability of sample solution > 24 hours or > 12 hours SSJCP, Department of Pharmaceutical Analysis 240
  241. 241. TYPICAL HPLC INSTRUMENT VERIFICATION REPORT TEST ITEM USER LIMIT ACTUAL LIMIT DAD noise < 5 X 10-5 AU 1 X 10-5 AU Baseline drift < 2x 10-3 AU/hour 1.5 X 10-4 AU/hour DAD WL calibration ± 1 nm ± 1 nm DAD linearity 1.5 AU 2.2 AU Pump performance < 0.3% RSD RT 0.15% RSD RT Temperature stability ± 0.15° C (column heater) ± 0.15° C Precision of peak area 0.09% RSD 0.5% RSD SSJCP, Department of Pharmaceutical Analysis 241
  242. 242. METHOD VALIDATION PROTOCOL 1. On day 1, a linearity test over 5 levels for both the drug substance (bulk) and dosage form is performed 2. Comparison of the results between the drug substance and dosage form fulfills the accuracy requirement 3. At the end of day 1, 6 repetitions are performed at 100% of the drug substance for repeatability 4. Steps 1 and 2 are repeated over 2 additional days for intermediate precision 5. LOQ is evaluated by analyzing the drug substance over 5 levels, plus 6 repetitions for precision 6. Baseline noise is evaluated over 6 repetitions of blank injections for the determination of LOD. SSJCP, Department of Pharmaceutical Analysis 242
  243. 243. TROUBLE SHOOTING (TIPS & FACTS) ASK PULLA For any further clarification or details of the below content(s) feel free to mail me : ravipratappulla@gmail.com SSJCP, Department of Pharmaceutical Analysis 243
  244. 244. 1.What is HPLC anyway? 2. How to become friendly with your HPLC equipment? 3. How to get started? 4. Which column do I have to install in the HPLC instrument? 5. How do I prepare a mobile phase? 6. What is the requirement of equilibrating the system before the advent of sample preparation. 7. What do I have to pay attention to before starting a measurement? 8. How do I start working with the HPLC equipment? 9. What's the reason for quitting your HPLC system? SSJCP, Department of Pharmaceutical Analysis 244
  245. 245. SIMPLE TESTS & DECISION CRITERIA 10.What does the name of a column tell us? 11. Is this C18 column the right choice for my sample? 12. Why are polar solutes well separated with one C18 column and hardly at all with another? 13. How can I clean the RP Phase quickly? 14. How best do I degas my mobile phase? 15. Methanol or Acetonitrile? Best choice of solvent…..? 16. The pH of the mobile phase too high or too low. What can I do? 17.What is the right ionic strength of the buffer? 18.How to make sense of the dead volume of an isocratic apparatus? 19.Producing a gradient chromatogram – influence of instrumentation? 20. Does the pump work correctly, precisely or accurately? SSJCP, Department of Pharmaceutical Analysis 245
  246. 246. 21. How to test an HPLC instrument and its modules? 22. Injections of solutes as an aqueous solutions? 23. What is the largest tolerable injection volume? 24 . How critical are the temperature changes? 25. How to choose HPLC equipment and a supplier? 26. Is the current method a robust one? PROBLEMS & THEIR SOLUTIONS 27. Sample preparation – how critical are which mistakes? 28. Flushing of an HPLC equipment? 29. Dirt in the UV detection cell? 30. The lamp is new – what happened to the peak? 31. What are the causes of pressure changes or deviations? SSJCP, Department of Pharmaceutical Analysis 246
  247. 247. 32. Is the right or the left pump head defective? 33. Baseline noise and damping? 34. The retention times increase- is it the pump or the M.P ? 35. Which buffer is right for which pH? 36. An interesting alternative for the separation of acids & bases with a buffer….. 37. What can be the reasons for a change in retention times? 38. I use up a lot of RP columns; what should I do? 39. Why does my NP system not work any more? 40. Chemical tailing at the presence of metal ions? 41. How to avoid memory effects? 42. How do the default values on my PC affect the resolution? SSJCP, Department of Pharmaceutical Analysis 247
  248. 248. TIPS TO OPTIMIZE THE SEPARATION 43. Which is the right injection techniques to get sharper peaks? 44. My peaks appear too early – how can I move them in an RP system to later retention times? 45. How can I increase the plate number? 46. Limit of detection: How can I see more? 47. How can I speed up a separation? 48. How can I optimize a separation? 49. Dead volume capacity, capacity factor, selectivity – how can I use them in everyday life? 50. Which flow is optimal for me? 51. How can I optimize a gradient elution? 52. Separation of ionic solutes? What works out best –end capped phases, inert phases, phosphate buffer or ion pairing reagents? SSJCP, Department of Pharmaceutical Analysis 248
  249. 249. SITUATION/SYMPTOM/CAUSE EQULIBRATION 53. SLOW COLUMN  RP- Ion pairing long chain 54.VARYING / VARIABLE RETENTION TIMES        gradient insufficient column regeneration time ion pairing insufficient equilibration time isocratic insufficient equilibration time irregular column equilibration time Leak change in M.P composition air trapped in pump SSJCP, Department of Pharmaceutical Analysis 249
  250. 250.  buffer capacity insufficient  contamination buildup  equilibration time insufficient for gradient run or changes in isocratic M.P  first few injections – active sites  inconsistent online M.P mixing or delivery  selective evaporation of M.P component  varying column temperature  check valve malfunctioning  pump cavitations, phase collapse (de-wetting process)  Column temperature fluctuations  First few injections adsorption on active sites  column overloading  sample solvent incompatible with M.P SSJCP, Department of Pharmaceutical Analysis 250
  251. 251.  Column problem  improper M.P  column aging 55. INCREASED RETENTION TIME          decreasing flow rate, changing M.P composition, loss of bonded S.P, active sites on column packing Low M.P flow rate Column temperature low Improper gradient setting Column activity increasing System not equilibrated SSJCP, Department of Pharmaceutical Analysis 251
  252. 252.      M.P removing water from LSC column Incorrect M.P Loss of bonded S.P M.P composition changing Active sites on silica packing 56. DECREASED RETENTION TIME        column overloaded with sample increasing flow rates loss of bonded S.P or base silica from column column aging, basic compounds – pH too low High M.P flow rate Column temperature high SSJCP, Department of Pharmaceutical Analysis 252
  253. 253.       Improper gradient Incorrect M.P Column activity decreased System not equilibrated Deactivation by strongly retained garbage Too strong sample solvent 57. RETENTION BEYOND TOTAL PERMEATION VOLUMES  SEC – solute interaction with S.P. SSJCP, Department of Pharmaceutical Analysis 253
  254. 254. 58. LOSS OF RESOLUTION           M.P contaminated/deteriorated Obstructed guard or analytical column Column overload with sample Degraded column Column not fully equilibrated Loss of S.P from the column Dirty column Loss of column liquid phase Distorted column bed Wrong column or M.P SSJCP, Department of Pharmaceutical Analysis 254
  255. 255. SENSITIVITY 59. Lack of sensitivity  auto sampler flow lines blocked  detector attenuation set too high  first few samples injections  sample adsorption in injector sample loop or column  injector sample loop under filled  not enough sample injected  peak signals are outside  detector’s linear range  sample losses during sample preparation  sample losses on column peak too broad SSJCP, Department of Pharmaceutical Analysis 255
  256. 256. BASELINE 60. Distribution At Void  air bubbles in M.P  positive-negative differences in RI of injection solvent & M.P 61. BASELINE DRIFT  Column temperature fluctuations  Non homogeneous M.P  Contaminant or air buildup in detector, sample or reference cell  Plugged outlet line after detector  M.P mixing problem or change in flow rate  Slow column equilibration when changing M.P  M.P contaminated or deteriorated or not prepared from high quality chemicals SSJCP, Department of Pharmaceutical Analysis 256
  257. 257.  Strongly retained materials in sample can elute as very broad peaks and appear to be a rising baseline  Detector not set at absorbance maximum but at slope of curve  M.P or sample vaporizing  Failing detector source  Detector problem  Solvent immiscibility  Contamination bleed in system  Solvent demixing  Slow change in pump output  Partial plugging of injection port or sample valve or column inlet by particulate matter  Contaminated or bleed column  Contamination in detector cell SSJCP, Department of Pharmaceutical Analysis 257
  258. 258.       Change in detector temperature Malfunction of detector source Contamination in solvent reservoir Previous M.P not removed Negative direction Positive direction 62. BASELINE NOISE (REGULAR)        Air in M.P or detector cell or pump Pump pulsations Incomplete M.P mixing Temperature effect Other electronic equipment on same line Leak or partial blockage of loop injector valve or detector lamp problem Dirty flow cell SSJCP, Department of Pharmaceutical Analysis 258
  259. 259. 63. BASELINE NOISE (IRREGULAR)  Leak  M.P contaminated or deteriorated or prepared from low quality materials  Detector or recorder electronics  Air trapped in system  Air bubbles in detector  Detector cell contaminated  Weak detector lamp  Column leaking silica or packing material or column packing passing through detector  Continuous detector lamp problem or dirty in the flow cell  gradient or isocratic proportioning - lack of solvent mixing & malfunctioning proportioning valves SSJCP, Department of Pharmaceutical Analysis 259
  260. 260.  occasional sharp spikes,  external electric interferences,  periodic pump pulse,  random contamination buildup,  spikes – bubble in detector & column temperature higher than B.P of solvent RECOVERY 64. POOR SAMPLE RECOVERY  absorption or adsorption of proteins  adsorption on column packing  absorption on tubing and other hardware components  chemisorption on column packing  hydrophobic interactions between S.P & biomolecules SSJCP, Department of Pharmaceutical Analysis 260
  261. 261.  less than 90% yield for acidic compounds irreversible adsorption on active sites  less than 90% yield for basic compounds irreversible adsorption on active sites LEAKS 65. LEAKY FITTING      A loose fitting Stripped fitting Over tighten fitting Dirty fitting Mismatched part/fitting SSJCP, Department of Pharmaceutical Analysis 261
  262. 262. 66. LEAKS AT PUMP       Loose check valve Mixer seal failure Pump seal failure Pressure transducer failure Pulse damper failure Proportioning valve failure 67. INJECTOR LEAKS       Rotor seal failure Blocked loop Loose injection port seal Improper syringe needle diameter Waste line siphoning Waste line blockage SSJCP, Department of Pharmaceutical Analysis 262
  263. 263. 68. COLUMN LEAKS  Loose end fittings  Column packing in ferrule  Improper frit thickness 69. DETECTOR     LEAKS Cell gasket failure Cracked cell window Leaky fittings Blocked waste line SSJCP, Department of Pharmaceutical Analysis 263
  264. 264. PROBLEMS DETECTED BY SMELL, SIGHT & SOUND 70. SOLVENT SMELL  Leak  Spill 71. HOT SMELL  Overheating 72. ABNORMAL METER READING  Pressure abnormality  Column oven  Detector lamp failing 73. WARNING LAMP  Pressure limits exceeded  Other warning signals SSJCP, Department of Pharmaceutical Analysis 264

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