column chromatography and ultra performance liquid chromatography

analysis

1. COLUMN CHROMATOGRAPHY AND ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY MQA101T Guided by : Presented by: Miss. Shraddha J. Parmar Henisha Patel Department of pharmaceutical science, Quality Assurance 1

2. Introduction Definition Principle Terminology Classification of column chromatography Requirements for column chromatography Column efficiency parameters Advantages Disadvantages Application Conclusion Recent advancement in column chromatography techniques CONTENTS 2

3. INTRODUCTION Column chromatography was first developed by the American chemist TSWETT in 1906. When chromatography is carried out in column it is called column chromatography. Otherwise known as gravity chromatography. 3

4. Column chromatography is a separation technique in which components of mixture is separated by using a glass column packed with stationary phase and the liquid mobile phase flowing continuously through the column. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column. DEFINITION 4

5. When a mixture of mobile phase and sample to be separated are introduced from top of the column, the individual components of mixture move with different rates. Those with lower affinity and adsorption to stationary phase move faster and eluted out first while those with greater adsorption affinity move or travel slower and get eluted out last. The solute molecules adsorb to the column in a reversible manner. The rate of the movement of the components is given as follows R= Rate of movement of a component / Rate of movement of mobile phase. i.e. it is the ratio of distance moved by solute to the distance moved by solvent. PRINCIPLE 5

6. TERMINOLOGY Stationary phase : called adsorbent It is a solid. Mobile phase : It is also known as solvent & eluent. It is a liquid. Sample : It is known as adsorbate. Which get adsorbs. Elution : It is a process of removing the components from column. Eluate : separated component. 6

7. Chromatographic methods can be classified according to the nature of the stationary and mobile phases. Adsorption chromatography Partition chromatography Ion exchange chromatography Size exclusion or gel permeation chromatography The modern instrumental techniques of GLC and HPLC provide excellent separation and allow accurate assay of very low concentrations of wide variety of substance in complex mixtures. CLASSIFICATION OF COLUMN CHROMATOGRAPHY 7

8. Adsorption chromatography In adsorption chromatography, the mobile phase containing the dissolved solutes passes over the surface of the stationary phase. Retention of the component and their consequent separation depends on the ability of the atoms on the surface to remove the solutes from the mobile phase and adsorb them temporarily by means of electrostatic forces. Usually silica or alumina is utilized as the adsorbent with relatively non polar solvents such as hexane as the mobile phase in normal phase adsorption whereas in reversed phase adsorption non polymer beds with relative polar solvents such as water, acetonitrile methanol as mobile phase. 8

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10. Partition chromatography In partition chromatography an inert solid material such as silica gel or diatomaceous earth serves to support a thin layer of liquid which is the effective stationary phase . As the mobile phase containing the solutes passes in close proximity to this liquid phase, retention and separation occur due to the solubility of the analytes in the two fluids as determined by their partition coefficients. The method suffers from disadvantage due to some solubility of stationary phase in the mobile phase. 10

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12. Ion exchange chromatography In ion exchange chromatography, the stationary phase consists of a polymeric resin matrix on the surface of which ionic functional groups, e.g., carboxylic acids or quaternary amines , have been bonded chemically. As the mobile phase passes over this surface, ionic solutes are retained by forming electrostatic chemical bonds with the functional groups. The mobile phase used in this type is always liquid. 12

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14. Size exclusion or gel permeation chromatography In size exclusion chromatography the stationary phase is a polymeric substance containing numerous pores of molecular dimensions. Solutes whose molecular size is sufficiently small will leave the mobile phase to diffuse into the pores. Large molecules which will not fit into the pores remain in the mobile phase and are not retained. This method is mostly suitable for the separation of mixtures in which the solutes vary considerably in molecular size. The mobile phase in this type may be either liquid or gaseous. 14

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16. 1. Column characteristics & selection 2. Stationary phases 3. Mobile phases 4. Preparation of column 5. Introduction of sample 6. Development of column 7. Detection & recovery of components REQUIREMENTS OF COLUMN CHROMATOGRAPHY 16

17. (1) COLUMN CHARACTERITIC & SELECTION: Table:1: selecting a suitable column dimension Materials of construction Good quality neutral glass, plastic or nylon Adsorbent(stationary phase)/adsorbate (mixture) weight ratio 30:1   Column length to diameter ratio(cm) 10-15:1 or 30-100:1 Multi component system is present Long column is used Components with similar affinities for adsorbent are present Long column is used Components with different affinities for adsorbent are present Short column is used 17

18. (2) STATIONARY PHASE Stationary phases used in column adsorption chromatography are also known as adsorbents. Requirements of an ideal stationary phase: 1.They should be insoluble in solvents or mobile phases. 2.chemically inert. 3.colourless to facilitate observation of zones. 4.should have reproducible properties from batch to batch. 5.The particle should have uniform size distribution and have spherical 6. particle size :60-200µ 18

19. Type of mesh Size in microns 60/120 mesh 120-250 micron 100/200 mesh 75-150 micron 70/230 mesh 63-200 micron 230/400 mesh 37-63 micron 70/325 mesh 45-200 micron The most commonly used chromatographic adsorbent is silica or silicic acid or silica gel (80-100 mesh or 100-200 mesh size, which has a particle size of 63-200µm). 19

20. Table 2: Adsorbents used in column chromatography Weak activity Medium activity Strong activity Sucrose Calcium carbonate Activate magnesium silicate Starch Calcium phosphate Activated alumina Insulin Magnesium carbonate Activate charcoal Talc Magnesium oxide Activated magnesia Sodium carbonate Calcium oxide Activated silica 20

21. (3) MOBILE PHASE OR SOLVENTS OR ELUENTS Mobile phase act as a solvent to introduce the mixture into the column as developer to develop the zones for separation and as an eluent to remove the pure component out to the column. Requirements of an ideal mobile phase: The choice of solvent is depend on the solubility characteristic of the mixture. It can be used in either pure form or as mixture of solvents. Polarity as seen the most important factor. The solvents should also have sufficiently low boiling points to permit ready recovery of eluted material. 21

22. (4) COLUMN PREPARATION A column is prepared by packing a solid absorbent into a cylindrical glass or plastic tube. The size will depend on the amount of compound being isolated. The base of the tube contains a filter, either a cotton or glass wool plug, or glass frit to hold the solid phase in place. A solvent reservoir may be attached at the top of the column. Two methods are generally used to prepare a column: The dry method and The wet method   22

23. 1. DRY METHOD In this method the dry adsorbent is poured to the column directly Vibration is applied to get rid of air bubbles then the mobile phase is passed through the adsorbent. The demerit with this method is that air bubbles are entrapped between the solvent and the stationary phase. This method cannot be applied in gel chromatography. 23

24. 2. WET METHOD The adsorbent is suspended in the mobile phase and stirred very well to remove all air bubbles. The resulted slurry is then poured in to the column At the bottom portion of the column a piece of glass wool or cotton/ whattman filter paper disc must be added before the slurry application. The top of the silica should be flat, and the top of the silica can be protected by a layer of sand. After slurry application, the column must be allowed to settle overnight. This is the ideal method of column packing. 24

25. (5) INTRODUCTION OF SAMPLE A)Wet application: Dissolve the sample in the initial mobile phase and apply by pipette to the top of the column. This is very good method but in most of cases the samples are not soluble in the initial mobile phase. B) Dry loading: Dissolve sample in any volatile solvent. The sample solution is then adsorbed an small weight of adsorbent and the solvent is allowed to evaporate. The dry adsorbent loaded with the sample is then applied to the column. 25

26. Light petroleum ether (petroleum) ether, hexane, heptane Carbon tetra chloride Cyclohexane Carbon disulphide Benzene Toluene Chloroform Ethyl acetate Alcohols Water Pyridine Organic acid Increasing eluting power 26

27. (6) DEVELOPMENT OF COLUMN Removal of individual components from a column is called development of column. Normal phase : Stationary phase (Polar) Mobile phase (Non-polar) Non polar Compounds Elutes first. Reverse phase : Stationary Phase (Non-polar) Mobile phase (Polar) Polar compounds Elutes first. In most of the analysis, Reverse Phase is used as many of the drugs are polar in nature. 27

28. Frontal analysis: This technique was developed by Tiselius in 1940 In this method, the solution of sample mixture is added continuously on the column. No mobile phase (solvent) is used for development of column. A mixture containing A,B,C is added on the column. If component A is least adsorbed, component B is adsorbed to intermediate extent and component C most strongly to the column adsorbent material. The mixture flows through the column, the least adsorbed A runs down the column fast, component B to intermediate extent while ‘c’ is retained at the top of the column. A plot of amount of substance against volume of eluate is gives a chromatogram.   28

29. Displacemental Analysis: In this method, a small volume of mixture is added to the column and elution is carried out by a solvent containing a solute which has adsorptivity for column material. The adsorbed constituents of mixture are displayed by the solute from mobile phase. Each solute in the mixture in turn displaces another substance solute which is less firmly adsorbed. The least adsorbed constituent is pushed out of the column. The substance used in mobile phase is called as displacer. Displacement analysis technique is mainly used in preparative work and is not suitable for analysis since some overlapping may occur. The plot of amount of substance (conc. In eluate) against volume of eluate is gives a chromatogram. 29

30. Elution analysis: It is a common method used in column chromatography. In this method a small volume of mixture to be separated is added on the top of column & mobile phase is allowed to flow through the column. The mixture introduced on the column gets separated into individual as the components of mixture are adsorbed to the column material to different extent. On other phase of mobile phase, each component of mixture is eluted out as separated components (called eluate). 30

31. A plot of amount of substance per ml of eluate against volume of eluate will gives the following chromatogram. a) Isocratic elution: (Iso means same or similar) In this elution technique, the same solvent composition or solvent of sample polarity is used throughout the process of separation. e.g.(chloroform only as a solvent or CHCL3:MeOH=1:1) b) Gradient elution: (gradient means gradual) In this elution technique, solvents of gradually increasing polarity or increasing elution length are used during the process of separation. 31

32. (7) DETECTION & RECOVERY OF COMPONENTS Fractions are collected by elution analysis Each fraction is examined by TLC using suitable experimental conditions Those fractions which give same Rf values in TLC are added as a common fraction From the column fraction, solvent is evaporated, dried & the materials collected in container After spectral analysis (NMR, MS, X-RD etc.)the compound is identified. 32

33. 1.Column efficiency 2.Resolution 3.Retention time 4.Retention volume 5.Separation factor 6.HETP 7.Asymetric factor COLUMN EFFICIENCY PARAMETERS 33

34. 1.Column efficiency: It is expressed by the number of theoretical plates It is determined by the formula: The number of theoretical plates is a measure of the “goodness” of the column If the retention time is high and peak width is narrow then it shows excellent chromatograms. 34

35. Where, tr is the retention time measured from the instant of injection w is the peak width 35

36. 2.Resolution Resolution is the ability to separate two signals In chromatography it’s the ability to separate two peaks.ie separation of constituents Where, tr1 and tr2 and w1 and w2 are the times and widths, respectively, of the two immediately adjacent peaks. If the peaks are sufficiently close w is nearly the same for both peaks and resolution may be expressed as 36

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38. 3.Retention time The rates of migration of substances in chromatographic procedures depend on the relative affinity of the substances for the stationary and the mobile phases It’s the difference in time between the point of injection and the time of emergence of separation of component from the column. It is actually the time required for 50% of the component to get eluted. It is measure in minutes or seconds 38

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40. 4.Retention volume It is the volume of carrier gas required to elute components from the column to the time the peak maximum is obtained. Retention volume depends upon flow rate and retention time 40

41. 5.Separation factor It is the ratio of partition coefficient of two components to be separated. 41

42. 6.HETP HETP is numerically equal to the column length divided by the number of theoretical plates in the column It varies from to one column to another as well as one solute to other The more efficient the column the better the resolution and the smaller the HETP. HETP=Length of column / no of theoretical plate 42

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44. 7.Asymetric factor A chromatographic peak should be symmetrical about its centre to follow gaussian distribution Asymetric factor is the measure of peak tailing or fronting. It is defined as the distance from the centre line of the peak to the back slope divided by the distance from the centre line of the peak to the front slope. 44

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46. Any type of mixture can be separated by column chromatography. Any quantity of the mixture can also be separated. Wider choice of mobile phase. In preparative type, the sample can be separated and reused. Automation is possible. ADVANTAGES 46

47. Time consuming method. More amount of solvents are required which are expensive. Automation makes the technique more complicated and expensive. DISADVANTAGES 47

48. In the separation of the mixtures into the pure individual components. Removal of impurities and in the purification of compounds. Determination of the homogeneity of chemical substances. Identification of unknown compounds. Used in the separation of geometrical isomers, diastereomers, racemates and tautomers. In the separation and identification of inorganic anions and cations. The concentrated of substance from dilute solutions such as those obtained when natural products are extracted with large volumes of the solvents from the leaves of plants, trees, roots or barks. APPLICATION 48

49. Column chromatography is a conventional tool for separation of phytochemicals, removal of impurities and purification of drugs. Effective separation of constituents from different sources in preparative scale (milligram to gram) can be achieved by column chromatography. Availability of wide range of stationary phases makes the technique to be used for different kinds of mechanisms. Understanding the basic principles of column chromatography enables us to find solutions for current research problems. CONCLUSION 49

50. ADVANCEMENT IN COLUMN CHROMATOGRAPHY TECHNIQUES LIKE Flash chromatography, High Performance Liquid Chromatography, Gas Chromatography, Ultra-Performance Liquid Chromatography and Ultra Performance Convergence Chromatography (Super Critical chromatography). The aim of this is to specify the changes that the scientists have made in each and every step to improve the technique (column chromatography). Now over 60% of chemical analysis worldwide is currently done with chromatography. 50

51.   ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) 51

52. Introduction Principle Advantages Disadvantages Comparison between HPLC & UPLC Instrumentation Common UPLC chromatographic conditions Application Advancement in UPLC References CONTENTS 52

53. Ultraperformance liquid chromatography (UPLC) is a recent technique in liquid chromatography, which enables significant reductions in separation time and solvent consumption. It improves in three areas: 1.Chromatographic resolution 2.Speed 3.Sensitivity INTRODUCTION 53

54. UPLC is a rising chromatographic separation technique whose packing materials have smaller particle size lesser than 2.5µm. Reducing these separation times without reducing the quality of the separation would mean that important analytical information could be generated more quickly. The technology takes full advantage of chromatographic principles to run separations using columns packed with smaller particles and high flow rates. 54

55. The principle of UPLC is based on Van Deemter equation which describes the relationship between flow rate and HETP or column efficiency H=A+B/v + Cv Where, A = Eddy diffusion B = Longitudinal diffusion C = Equilibrium mass transfer v = flow rate van Deemter equation, that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency) PRINCIPLE 55

56. Decreases run time and increases sensitivity. Reducing analysis time so that more product can be produced with existing resources. Provides the selectivity, sensitivity, and dynamic range of LC analysis Maintains resolution performance. Fast resolving power quickly quantifies related and unrelated compounds. Operation cost is reduced. Less solvent consumption. ADVANTAGES 56

57. Due to increased pressure requires more maintenance and reduces the life of the columns of this type.   DISADVANTAGES 57

58. Comparison between HPLC & UPLC 58

59. INSTRUMENTATION 59

60. COMPONENTS: 60

61. Pumping system Achieving small particle, high peak capacity separations requires a greater pressure range. Both the gradient and isocratic separation modes are used. The binary solvent manager uses two individual serial flow pumps to deliver a parallel binary gradient. There are built-in solvent select valves to choose from up to four solvents. There is a 15,000-psi pressure limit (about 1000 bar) to take full advantage of the sub-2μm particles 61

62. 2.Sample injection In UPLC, sample introduction is critical. Conventional injection valves, either automated or manual, are not designed and hardened to work at extreme pressure. To protect the column from extreme pressure fluctuations, the injection process must be relatively pulse-free and the swept volume of the device also needs to be minimal to reduce potential band spreading. Low volume injections with minimal carry over required to increase sensitivity. 62

63. 3.UPLC columns Resolution is increased in a 1.7 μm particle packed column because efficiency is better. Separation of the components of a sample requires a bonded phase that provides both retention and selectivity. Four bonded phases are available for UPLC separations: 1. ACQUITY UPLCTM BEH C18 & C8 (straight chain alkyl columns), 2. ACQUITY UPLC BEH Shield RP18 (embedded polar group column) 3. ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl functionality with a C6 alkyl) 4. ACQUITY UPLC BEH Amide columns (trifunctionally bonded amide phase). 63

64. 1.ACQUITY UPLCTM BEH C18 & C8 These are considered as the universal columns of choice for most UPLC separations by providing the widest pH range. They incorporate trifunctional ligand bonding chemistries which produce superior low pH stability. This low pH stability is combined with the high pH stability of the 1.7μm BEH particle to deliver the widest usable pH operating range. 2.ACQUITY UPLC BEH Shield RP 18 These are designed to provide selectivity's that complement the ACQUITY UPLC BEH T M C18 and C8 Columns. 64

65. 3.ACQUITY UPLC BEH Phenyl columns These utilize a trifunctional C6 alkyl ethyl between the phenyl ring. 4.ACQUITY UPLC BEH Amide columns 1. BEH particle technology, in combination with a trifunctionally bonded amide phase, provides exceptional column life time, thus improving assay robustness. 2. BEH Amide columns facilitate the use of a wide range of phase pH [2 –11]. 65

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67. Requirements of an ideal detector:- It should give quantitative response. It should have high sensitivity and low noise level. It should have a short response time. It should provide signal or response quantitative to wide spectrum of radiation received. It should generate sufficient signal or electrical current, which can be measured or easily amplified for detection by meter. 67

68. 1. UV detectors The majority of organic compounds can be analyzed by UV detectors, and almost 70% of published HPLC analyses were performed with UV detectors. Measures the ability of solutes to absorb light at a particular wavelength(s) in the ultraviolet (UV) or visible wavelength range. When light of certain wavelength is directed at flow cell, the substance inside the flow cell absorb the light. As a result the intensity of the light that leaves the flow cell is less than that of the light that enters it. An absorbance detector measure the extent to which the light intensity decreases ( i.e. the absorbance). 68

69. Three common 3 types of this detectors: Fixed wavelength detectors Variable wavelength detectors Photodiode array detectors Fixed wavelength detector absorbance of only one given wavelength is monitored by the system at all times ( usually 254nm) Simplest and cheapest of the UV/VIS detectors Limited in flexibility Limited in types of compounds that can be monitored. Variable wavelength detector a singal wavelength is monitored at any given time, but any wavelength in a wide spectral range can be selected Wavelength very from 190-900nm More expensive, requires more advanced optics 69

70. More versatile, used for a wide range of compounds More sensitive due to photomultiplier tube or amplification circuitry. Photodiode array detector operates by simultaneously monitoring absorbance of solutes at several different wavelength. Light from the broad emission source such as a deuterium lamp is collimated by an achromatic lens system so that the total light passes through the detector cell onto a holographic grating. In this way , the sample is subjected to light of all wavelengths generated by lamp. The dispersed light from the grating is allowed to fall on to a diode array . The array may contain many 100 of diodes and the output from is diode is regularly sampled by a computer and stored on a hard disc. 70

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72. 2. Fluorescent detector The light from an excitation source passes through a filter or monochromator, and strikes the sample. A proportion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted in all directions. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90° to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector. 72

73. Fluorescent detector 73

74. 3. Refractive index detector (ADD AFTER 73 NO. SLIDE) This detector based on the deflection principle of refractometry, where the deflection of a light beam is changed when the composition in the sample flow-cell changes in relation to the reference side (as eluting sample moves through the system). As sample elutes through one side, the changing angle of refraction moves the beam. This results in a change in the photon current falling on the detector which unbalances it. The extent of unbalance (which can be related to the sample concentration) is recorded on a strip chart recorder. 74

75. Refractive index detector 75

76. 4. Light scattering detector Universal , distractive Useful for large molecules and wide linear range. Analytes are de-solvated in the detector. The reduction in light intensity detected (due to scattering by the analytes) is measured. There are 3 steps involved in detection: Nebulization Mobile phase evaporation Detection The flow from the column is nebulized with a stream of inert gas. 76

77. The mobile phase, which must be volatile, is evaporated, leaving tiny particles of the analytes . The particles are passed through a laser beam and they scatter the laser light. The scattered light is measured at right angles to the laser beam by a photodiode detector. 77

78. 5.Electrochemical detector Most sensitive detector The level of current is directly proportional to the analyte concentration. Respond to substances that are oxidizable or reductable. 3 electrodes are employed Working electrode Auxiliary electrode Reference electrode 78

79. 6. Mass spectrometric detector It is a method that combines separation power of HPLC with detection power of mass spectrometry. Mass spectrometry (MS) is a powerful analytical tool that can supply both structural information about compounds and quantitative data relating to mass. 79

80. 1. Columns: ACQUITY UPLC BEM C18, BEH Shield RP18, BEH C8 OR BEH Phenyl Column. 2. Dimensions: 2.1 X 50mm 1.7μm. 3. Mobile Phase A1: 20mM NH4COOH in H2O, pH 3.0. 4. Mobile Phase A2: 20mM NH4HCOOH in H2O, pH 10.0. 5. Mobile Phase B1: Acetonitrile . 6. Mobile Phase B2: Methanol. 7. Flow rate: 0.5ml/min. 8. Injection Volume: 10.0μl. 9. Week needle wash: 3% methanol. 10. Strong needle wash: 90% acetonitrile. 11. Temperature: 30°C. 12. Detection: UV 254 nm. 13. Sampling rate: 20pts/sec Common UPLC chromatographic conditions 80

81. Analysis of natural products and traditional herbal medicine. Identification of metabolite. Study of metabonomics/metabolomics. Bio analysis/bioequivalence studies. Manufacturing/QA/QC Impurity profiling. Forced Degradation Studies. Dissolution Testing. Toxicity Studies. APPLICATION 81

82. A.H. BECKETT & J.B. STENLAKE, Practical pharmaceutical chemistry, 4th edition, part two, page no: 86-105. ASHUTOSH KAR, Pharmaceutical analysis – II, page no: 161-181.  DAVID G.WATSON, Pharmaceutical analysis, page no: 270 - 271. B.K. SHARMA, Instrumental methods of chemical analysis, page no : C-8 to C-15. Dr. A.V. KASTURE, Dr. K.R. MAHADIK, Dr. S.G. WADODKAR, Dr. H.N. MORE, Pharmaceutical analysis volume – II, page no: 10-17. VOGEL’S, Text book of quantitative analysis, page no:289- 314. G.DEVALA RAO, A text book of advanced pharmaceutical analysis, page no:332- 335 Review article -Ranjith Reddy Kondeti et al., World J Pharm Sci 2014; 2(9): 1375-1383 Lucie Novakova, Ludmila Matysova, Petr Solich. Advantages of application of UPLC in pharmaceutical analysis. Talanta: 2006. P. 908-918. H KAUR Instrumental methods of chemical analysis ninth edition 2013 ;1091-109 REFERENCES 82

83. ACKNOWLEDGEMENT Thank you for paying attention 83