HPLC-Chromatography by Dr. Anurag Yadav

chromatography
column liquid chromatography
HPLC technique

1. Presenter : Dr.ANURAG YADAV Moderator: Dr.AVINASH.S.S C hroma to graphy -III HPLC

2. Contents HPLC: Definition Basic principle Instrumentation Practical consideration Applications Advantages & disadvantages of HPLC

3. HPLC (High-performance liquid chromatography) Type of liquid chromatography Conducted in a column Characterized by the use of high pressure & small particle size to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution.

4. Basic principle Based on distribution of solute between a liquid mobile phase and a stationary phase. The small diameter particles are used as stationary phase support.

5. The table shows relation between various parameters of HPLC. Trendline: Stationary phase have small particulate size and high surface areas. Columns: 20 cm or less Mobile phase pumped at high pressures of 40 0Bar , 6000 psi. Flow rates: 1-3 cm 3 per min Column length No. of theoretical plates per unit area Resolving power Column length Particle size Surface area Basic principle

6. Components of HPLC Solvent Reservoir Pumps Sample Injection System Columns Detectors Computer Waste collector

7. Solvent Reservoir (mobile phase) Inert container → mobile phase Must be free of contaminants & bubble forming gases that get trapped in column or detector. Measures: microfilter , degasser Degassing : - Vacuum filtration - Warming - Stirring vigorously with magnetic stirrer - Sparge with inert gas (N 2 or He) - Ultrasonication

8. HPLC Pump To produce an appropriate pressure to push solvent & sample into the column. Ideal pump Deliver high pressure (upto 50MPa) Deliver pulse free flow Constant volume delivery Deliver high volumes (flow rates) of solvent (to 10 mL /min) Solvent replacement is easy

9. Types of HPLC Pump Constant Pressure A steady pump pressure (usually about 1000–2000 psi) is needed to ensure reproducibility & accuracy. Constant displacement Pump Reciprocating pump : constant flow rate through the column Slight cyclical variation in pressure→pulse dampeners.

10. Pumps in HPLC

11. HPLC Pump operating mode Isocratic elution: A separation that employs a single solvent or solvent mixture of constant composition with single pump throughout the run. Uses: Simple separation & compound with similar structures & retention time Gradient elution: Here two or more solvent systems that differ significantly in polarity with separate pumps. the ratio of the solvents is varied in a programmed way, sometimes continuously and sometimes in a series of steps. Separation efficiency is greatly enhanced by gradient elution. Uses : complex separations

12. Sample Injector 1) Manual Injector: Manually load sample into the injector using a syringe and then injected sample → into the flowing mobile phase →which transports the sample into the beginning (head) of the column, which is at high pressure

13. Sample Injector 2) Autosampler injector: User loads vials filled with sample solution into the autosampler tray (100 samples) Autosampler automatically Takes appropriate sample volume, injects the sample, then flushes the injector to be ready for the next sample, until all sample vials are processed.

14. 18 Manual Injectors Front View Inject Rear View Load - Inject Sample Loop

15. 19 Automatic Injectors Step 1 Step 2 Step 3

16. Control of column temperature as constant temp is required. Achieved by: Column chambers Water jackets Temperature controlled blankets Heating/cooling blooks Stable column temperature is required to generate reproducible retention time. Column Heater/Chillers

17. Guard column Repeated application of impure samples (sera, urine) → ↓column resolving power. Installed between injector & analyte column Column characterstics : short(1-2cm), same ID & packed with similar particles to that present in analytical column. Retains contaminating particles, can be replaced at regular intervals.

18. Column: • Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters. •The small particles inside the column are what cause the high back pressure at normal flow rates. •The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph. Analyte Columns in HPLC

19. Analyte Columns in HPLC Standard Column : 3-25cm long, ID(4.6 mm) ;optimum flow volume = 1 ml/min. Narrow Bore : 3 mm(ID); flow volume 0.6 ml/min Microbore /Open tubular : 25-50cm long, 1 mm(ID); 50 microliter /min.

20. Several Column Types ( can be classified as) Normal phase Reverse phase Size exclusion Ion exchange

21. Normal phase In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample

22. 26 STATIONARY PHASES (NORMAL POLARITY) Silica or alumina possess polar sites that interact with polar molecules. Most polar…….Least polar Components elute in increasing order of polarity. Polar Group silica

23. Reverse phase In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile -water . Common Reverse Phase Solvents – Methanol Acetonitrile Tetrahydrofuran Water CH 3 OH CH 3 CN H 2 O

24. 28 STATIONARY PHASES (REVERSE POLARITY) If the polar sites on silica or alumina are capped with non-polar groups, they interact strongly with non-polar molecules. Most non-polar…….Least non-polar Components elute in decreasing order of polarity. C 18 phase silica

25. Size exclusion In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size. Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules .

26. 30 STATIONARY PHASES (SIZE EXCLUSION) Size exclusion gels separate on the basis of molecular size. Individual gel beads have pores of set size, that restrict entry to molecules of a minium size. Larger molecules…….Smaller molecules Large molecules elute fast (restricted path), while small molecules elute slowly (long path length)

27. Ion exchange In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase. Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.

28. 32 STATIONARY PHASES (CATION EXCHANGE) Silica is substituted with anionic residues that interact strongly with cationic species (+ve charged) Most +ve…….Least +ve +ve charged species adhere to the support and are later eluted with acid (H + ) Cations exchange Na + silica

29. 33 STATIONARY PHASES (ANION EXCHANGE) Silica is substituted with cationic residues that interact strongly with anionic species (-ve charged) Most -ve…….Least -ve -ve charged species adhere to the support and are later eluted with acid (H + ) Anions exchange Cl - silica

30. HPLC Columns Within the Column is where separation occurs. Key Point –Proper choice of column is critical for success in HPLC 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 Packing material: The packing material is prepared from SILICA particle, ALUMINA particle and ion exchange RESIN. Porous plug of stainless steel or Teflon are used in the end of the columns to retain the packing material. According to the mode of HPLC , they are available in different size , diameters, pore size or they can have special materials attached ( such as antigen or antibody ) for immuno affinity chromatography.

31. 35 Modes of High Performance Liquid Chromatography 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

32. Types of columns in HPLC : Guard Column Fast Column 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 ) Analytical[internal diameter ( i.d .) 1.0 -4.6-mm; lengths 15 –250 mm]

33. Fast Column One of the primary reasons for using these column is to obtain improved sample output ( amount of compound per unit time). Fast column are designed to decrease the time of chromatographic analysis Here internal diameter is same but length is short and packed with smaller particles , that are 3 μm diameter. Advantages- Increased sensitivity Decreased analysis time Decreased mobile phase usage Increase reproducibility

34. Capillary Column It is also known as micro columns It has a diameter much less than a millimeter and there 3 types: Open tubular Partially packed Tightly packed They allow the user to work with nanoliter sample volume , decreased flow rate and decreased solvent usage volume , led to cost effectiveness

35. Preparatory Column Used when objective is to prepare bulk ( milligrams) of sample for laboratory preparatory application. It has usually a large column diameter , which is designed to facilitate large volume injections into the HPLC system

36. Column packing Particulate packings : particle diameters 1.8 to 10 μ m. Bonded phase : stationary phase is bonded chemically to surface of silica particles through a silica ester or silicone polymeric linkage. Eg . In reverse phase HPLC, ODS bound to silica particles (C 18 column). Polymeric : eg . Polystyrene- polyvinylbenzene , stable at PH 2-13 Chiral : separation of enantiomers .

37. • The detector can see (detect) the individual molecules that come out (elute) from the column. •A detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sample components. •The detector provides an output to a recorder or computer that results in the liquid chromatogram(i.e., the graph of the detector response). Detectors in HPLC

38. 42 Common HPLC Detectors UV-VIS Diode Array Multiple Wavelength Variable Wavelength Mass Spectrometers Refractive Index Fluorescence Light Scattering Electrochemical Radioactivity Conductivity

39. 1. Variable wavelength (UV-VIS absorbance) detector UV-Visible spectrometry Measures absorbance of light as down to 190nm. Detection limit < 1 ng ADV: High sensitive Small amount of sample . Uses: amides, carboxylic acid, cholesterol.,

40. 2. Scanning wavelength detector Records complete absorption spectrum of analyte By using photodiode array technique Scans complete spectrum of analyte within 0.01 sec Adv : could analyse sample simultaneously at diff wavelength. Disadv : less sensitive than UV-VIS detector.

41. 3. Fluorescence detector Based on measurement of fluorescence Compared to UV-Vis detectors fluorescence detectors offer a higher sensitivity and selectivity that allows to quantify and identify compounds in complex matrices at extremely low concentration levels (trace level analysis). Detection limit: pg to ng Uses: amino acids and amines. Limitation: only fluorescent analytes measured.

42. 4 .Electrochemical detector Electroactive analytes Electrochemically measures Oxidation reaction: hydrocarbon, amines, phenols, catecholamines Reduction reaction: alkenes, esters, ketones Detection limit: pg to ng .

43. 5. Refractive Index (RI) Detection The refractive index (RI) detector uses a monochromator and is one of the least sensitive LC detectors. This detector is extremely useful for detecting those compounds that are non-ionic, do not absorb ultraviolet light and do not fluoresce. e.g. sugar, alcohol, fatty acid and polymers.

44. LC-MS HPLC column eluted solutes introduced into a ion source of a MS, blasted with electrons, which cause them to turn into positively charged molecular ions and fragmented ions (ion source). When these charged particles passed through filter → separated according to m/e ratio → ions collected. TIC the current generated by all such ions from analytes is measured, which would be proportional to the concentration of analyte .

45. HPLC Detectors

46. Computer Electronic signals generated by detectors are recorded in the form of chromatograghic peak at varied function of time Peak Area, height, retention time, base width of chromatograghic peak is measured to compute analyte concentration of each peak.

47. 51 How can We Analyze the Sample? For example: Carbohydrates 1. fructose 2. Glucose 3. Saccharose 4. Palatinose 5. Trehalulose 6. isomaltose 1 2 3 4 5 mAU time 6

48. 52 Separations Separation in based upon differential migration between the stationary and mobile phases . Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica Mobile Phase - carries the sample through the stationary phase as it moves through the column. Injector Detector Column Solvents Mixer Pumps High Performance Liquid Chromatograph Waste

49. 53 Separations Injector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time High Performance Liquid Chromatograph

50. 54 Separations Injector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time

51. 55 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

52. 56 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

53. 57 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

54. 58 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

55. 59 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

56. 60 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

57. 61 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

58. 62 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

59. 63 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

60. 64 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

61. 65 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

62. 66 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

63. 67 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

64. 68 Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time

65. 69 The Chromatogram Injection t o t R mAU time t R t o - elution time of unretained peak t R - retention time - determines sample identity Area or height is proportional to the quantity of analyte.

66. Preparation for HPLC Sample preparation : Sample purification and Sample Derivatization . Before HPLC analysis , solutes are chemically derivatized before or after chromatographic separation to increase their ability to be detected . Eg . Derivatization of amino acids with O- Phthalaldehyde OR Dansyl chloride. Degassing

67. Factors which influence the HPLC performance Internal diameter of column - the smaller in diameter, the higher in sensitivity 2. Pump pressure - the higher in pressure, the higher in separation 3. Sample size 4. The polarity of sample, solvent. 5. Temperature - the higher in temperature, the higher in separation

68. CALIBRATION Calibration of HPLC is done to check the performance of its instrument. 1.flowrate(pump calibration) 2. Detector and injector linearity 3.System precision 4.Column oven temperature 5.Detector wavelength accuracy

69. 1.Pump calibration Disconnect the column and connect the inlet and outlet tubing’s with a union. Prime all the lines at 5 ml/min flow rate with water and ensure that flow line is free from air bubbles. Set the flow rate at 1ml / min and collect the mobile phase (water) in a dry preweighed beaker and collect the mobile phase for 10 min. weigh the beaker to get the weight of mobile phase. Calculate the flow rate by dividing the weight obtained with weight per ml and 10 (run time). Calculate the corresponding flow rate. Carry out the experiment in duplicate.

70. Acceptance criteria 0.05ml for small quantities 0.1ml for larger quantities 2.Detector and Injector linearity Column : ODS or C18 Mobile phase : milli Q water and acetonitrile (80:20) Flow rate: 1ml/min Temperature: 40°Centigrade Detector wavelength:272 nm Runtime:10min

71. Preparation of stock solution: Take 50mg of caffeine and transfer into 50ml of flask and make it up to the mark with the diluent Caffeine is used a it gives a multiwavelength response and is stable Prepare solutions of different PPM (100-600) Inject them and calculate the area Correlation coefficient r is used to check the detector linearity and cannot be less than .999 r = NƩXY-ƩXƩY/ √ [NƩX²-( Ʃx ) ²][NƩY²-(ƩY)²] The graph obtained between concentration and area is linear

72. 3.System precision From the stock solution pipette out 1ml of stock solution and transfer into 10ml flask and make it upto the mark with diluent for 100PPM conc. Calculate the percentage of RSD of areas of 5 different injections % RSD = standard deviation/average value of above 5 injection areas × 100 Where SD = √ Ʃ(X-M) ²/n-1 N= # of injections M=average area X=area For system precision % of RSD is not more than 1

73. 4.Column oven temperature Set the column oven temperature to 30° and leave it for 30 minutes Open the door of the column oven and keep the thermometer and leave it for 30 min now note down the reading in the thermometer Similarly change the column oven temperature to 40°C and 50°C and repeat the above procedure Acceptance criteria ±2°c

74. 5.Detector wavelength accuracy Column : C 18 Mobile phase: HPLC grade methanal Flow rate:1ml/min Retention time:5 minutes Wavelength:272nm Inject the methanol and record blank Inject 20 ml of standard solution at 268nm Similarly inject the standard solution by increasing the nm upto 278nm(increasing it 1 at a time) Acceptance criteria 273nm ± 1

75. MAINTAINANCE 1.RESERVOIR Possible Cause Preventive Maintenance 1. Blocked inlet frit 1. a. Replace (3–6 months)   b. Filter mobile phase with 0.4 - 0.5 µm filter 2 . Gas bubbles 2 . Degas mobile phase, sonification

76. 2. PUMP Possible Cause Preventive Maintenance 1. Air bubbles 1. Degas mobile phase , do not change mobile phase during run 2. Pump seal failure 2. Replace (3 months ),clean with 1 N acid 3. Check valve failure 3. Filter mobile phase; use inlet-line frit ; keep spare   4.Improper cleaning 4.Clean with Isopropyl alcohol , mobile phase container must be cleaned with mobile phase and other sections with solvent

77. 3.INJECTOR Possible Cause Preventive Maintenance 1.Washing 1.wash before and after use 2. Rotor seal wear 2. a. Do not overtighten   3.Syringe b. Filter samples 3.Sterilize when fresh sample is used C. Injector

78. 4.Column Possible Cause Preventive Maintenance 1. Number of injections 1. 2000 or less 2. Blocked frit 2.. a. Filter mobile phase   b. Filter samples   c. Use in-line filter and/or guard column 3. Void at head of column 3.. a. Avoid mobile phase pH >8   b. Use guard column   c. Use precolumn (saturator column D. Column

79. 5.Detector Possible Cause Preventive Maintenance 1. Lamp failure; decreased detector 1. Replace (6 months) or keep spare lamp   response; increased detector noise 2. Bubbles in cell 2. a. Keep cell clean   b. Use restrictor after cell   c. Degas mobile phase

80. 6.Software Update frequently , around 6-12 months

81. TROUBLESHOOTING Identifying problem using a chromatogram 1.The above pattern occurs when the mobile phase is not suitable

82. 2.Baseline noise I. Blank base(normal) II. Noisy : occurs when there is contamination of mobile phase improper cleaning

83. 3.Drift Temperature is unstable Contamination of column

84. 4.Peak tailing Improper mixing of mobile phase(90% of the time) Problems with column(30% of the time)

85. After adjusting PH with buffer Good resolution

86. 5.Peak fronting Low temperature Overloading of sample Flow rate Overfilling of column Insufficient mixing of mobile phase

87. 6.High concentration of sample

88. RT=4.0 7.Retention time variation Occurs due to problems with mobile phase RT=3.0 RT=3.5

89. 8.Communication error Due to problem in communication between detector and the software

90. 9.Peak splitting Occurs when column lifetime is diminished

91. 10.Power fluctuation

92. 11.Variation in area Problem with injector Fluctuation in flow rate Error in detection A=3010 A=3215 A=3516

93. 12. Ghost peaks Contamination of mobile phase Upset equilibrium Production not finished

94. 13.Detector lifetime diminished

95. Applications of HPLC Therapeutic & diagnostic uses In clinical diagnosis : detection and estimation of amino acids, metabolites, sugars in physiological samples, Mucopolysaccharides in urine and blood –useful for screening and diagnosis of inborn metabolic disorders Clinical diagnosis of Aminoacidurias , hemoglobinopathies , mucopolysaccharidoses , etc. Separation & identificaton of lipids, carbohydrates & proteins.

96. Applications of HPLC Therapeutic & diagnostic uses Measurement of drugs & other metabolites in biological fluids Monitoring of cirrhosis pt through aquaporin-2 in urine

97. Applications of HPLC Forensic analysis Forensic analysis of blood and urine alcohol levels Forensic analysis of blood and urine levels of drug abuse and steroids. Used for toxiclogical analysis of biological fluid – op poisoning

98. Applications of HPLC Pharmaceuticals industry uses Quantity of drug determination from pharmaceutical dosage forms, ex. Paracetamol determination in panadol tablet Quantity of drug determination from biological fluids. Analysis of natural contamination - Phenol & Mercury from sea water Food and essence manufacture - sweetener analysis in the fruit juice - preservative analysis in sausage.

99. Advantages of HPLC Needs a small sample with a high accuracy and precision Non-destruction of sample

100. Disadvantages Need a skill to run the instruments Solvents consuming Maintenance .

101. Advantages & disadvantages HPLC Both volatile & non- volitile analytes can be measured without degradation. Time of separation is very less bec high pressure pump is used. Length of column is small Non-explosive Non-expensive GC Only volatile analytes can be measured. More time Bigger coloumn than HPLC Explosive Expensive

102. Ultra-performance liquid chromatography (UPLC) As pointed earlier, the resolution of a mixture of analytes increases as the particle size of stationary phase decreased. But such a decrease leads to a high back-pressure from the eluent flow. Solution to this is represented by HPLC with new sationary phase less than 2 μ m diameter by the waters corporation (1.7 μ m diameter)- made of Bridged Ethylsiloxane silica Hybrid (BEH) TM This can sustain of back-pressure of 150MPa.

103. The instrument available under the trade name of ACQUITY TM & the term UPLC is registered under Waters. this operates upto 10 times faster than the conventional HPLC, & can complete in less than 5mins The peaks may last for only 1s, so the detectors should respond ultra fast to respond to the peaks.

104. Perfusion chromatography The high resolution achieved by the HPLC si based on use of small diameter particles. However, same can be achieved at low cost by generating high flow rate without high pressure. Perfusion chromatography comes with this, with particle size around 10-50 μ m diameter, that have channels of around 1 μ m diameter running through them. The high flow rate result in small plate heights & hence high resolution in very short separation times.

105. The particles are made of polystyrene- divinylbenzene & are available under the name of POROS.

106. References Keith wilson -technique text book. Tietz –clinical chemistry text book. Kaplan-technique text book. Upadhyay - biophysical chemistry.

107. 114 UV-Vis Detectors b c Detector Flow Cell I I Log I = A = abc I Principles : The fraction of light transmitted through the detector cell is related to the solute concentration according to Beer’s Law. Characteristics : Specific, Concentration Sensitive, good stability, gradient capability. Special : UV-Vis Spectral capability (Diode Array Technology ).

108. 115 Fluorescence Detection