column chromatography and ultra high performance liquid chromatography

column chromatography and ultra high performance liquid chromatography
M.PHARM
SEM-1
MORDEN PHARMACEUTICAL ANALYSIS TECHNIQUES

1. SEMINAR/ASSIGNMENT-I(MAT101T) COLUMN CHROMATOGRAPHY NAME :-VINIT NAI M.PHARM(PQA) SEM 1 SUBJECT :-MORDERN PHARMACEUTICAL ANALYSIS TECHNICS SUBJECT CODE :-MAT101T GUIDED BY- DR.SHAILESH KORADIA KRISHNA SCHOOL OF PHARMACY & RESEARCH(KSP) 1

2. CONTENTS Introduction History Principle Experimental aspects of column chromatography Packing techniques in C.C Development technique (Elution) Detection of components Factors affecting column efficiency Applications Advantages and disadvantages of C.C 2

3. CHROMATOGRAPHY Chromatography is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase. There are a number of different kinds of chromatography, which differ in the mobile and the stationary phase used. 3

4. HISTORY Column Chromatography was developed by the American chemist in 1901. Mikhail Tswett, the Polish botanist, in 1906 used adsorption columns in his investigations of plant pigments. 4

5. Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids. This is a solid - liquid technique in which the stationary phase is a solid & mobile phase is a liquid. 5

6. PRINCIPLE :- Adsorption Mixture of components dissolved in the M.P is introduced in to the column. Components moves depending upon their relative affinities. Adsorption column chromatography, the adsorbent, packed in a glass column, and a solvent, the mobile phase, that moves slowly through the packed column. A solvent used as a mobile phase is called an eluent. 6

7. A compound attracted more strongly by the mobile phase will move rapidly through the column, and elute from, or come off, the column dissolved in the eluent. In contrast, a compound more strongly attracted to the stationary phase will move slowly through the column. Partition:- The liquid is coted on the column the separation of components between two liquid phases viz original solvent and the film of solvent used in the column . 7

8. Experimental aspects of column chromatography:- Adsorbents :- The usual adsorbents employed in column chromatography are silica, alumina, calcium carbonate, calcium phosphate, magnesia, starch, etc. Alumina is generally suitable for chromatography of less polar compounds. Silica gel gives good results with compounds containing polar functional groups. 8

9. Adsorbent in C.C should meet following criteria:- Particles should be spherical in shape & uniform in size Mechanical stability must be high They shouldn’t react chemically It should be useful for separating for wide variety of compounds It should be freely available & inexpensive The particle size of the commercially available grade is in the range 50 – 200 µm. 9

10. Selection of Stationary Phase Success of chromatography depends upon proper selection of S.P, it depends on the following: Removal of impurities No. of components to be separated . Length of the column used Affinity differences b/w components Quantity of adsorbent used 10

11. Mobile Phase They act as solvent, developer & eluent. The function of a mobile phase are: As developing agent To introduce the mixture into the column – as solvent To remove pure components out of the column – as eluent The choice of the solvent is depend on the solubility characteristics of the mixture. 11

12. The solvents should also have sufficiently low boiling points to permit ready recovery of eluted material. However, polarity as seen the most important factor in adsorption chromatography. Different mobile phases used:(in increasing order of polarity) Petroleum ether, carbon tetrachloride, cyclohexane, ether, acetone, benzene, toluene, esters, water, etc It can be used in either pure form or as mixture of solvents 12

13. COLUMN CHARACTERISTICS The main function of all the columns is to support the stationary phase. The material of the column is mostly good quality neutral glass since it shouldn’t be affected by solvents. An ordinary burette can also be used as column for separation. Column dimensions - length & diameter ratio (10:1,30:1 or 100:1). Various accessories are attached to the top and bottom of the column for maintenance of the elution process. 13

14. The length of the column depends upon :- Number of compounds to be separated Type of adsorbent used Quantity of the sample Affinity of compounds towards the adsorbent used Better separation will be obtained with a long narrow column than short thick column because number of plates will be more. 14

15. PREPARATION OF THE COLUMN It consists of a glass tube with bottom portion of the column – packed with glass wool/cotton wool or may contain asbestos pad, Above which adsorbent is packed After packing a paper disc kept on the top, so that the adsorbent layer is not disturbed during the introduction of sample or mobile phase. 15

16. Packing techniques in C.C There are two types of preparing the column, they are… 1.Dry packing / dry filling 2.Wet packing / wet filling The column should be free from impurity, before using column, it should be washed properly and dry it. Before filling column with stationary phase, cotton/glass wool is kept It should be uniformly filled 16

17. DRY PACKING TECHNIQUE Adsorbent is packed in the column in dry form Fill the solvent, till equilibrium is reached After filling tapping can be done to remove void spaces. 17

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19. WET PACKING TECHNIQUE ideal & common technique The material is slurried with solvent and generally added to the column in portions. S.P settles uniformly & no crack in the column of adsorbent. solid settle down while the solvent remain upward. this solvent is removed then again cotton plug is placed. 19

20. Introduction of the Sample The sample which is usually a mixture of components is dissolved in minimum quantity of the mobile phase. The entire sample is introduced into the column at once and get adsorbed on the top portion of the column. From this zone, individual sample can be separated by a process of elution. 20

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22. Development technique (Elution) By elution technique, the individual components are separated out from the column. The two techniques are:- ( i ) Isocratic elution technique :- ( ii) Gradient elution techniques:- ( i ) Isocratic elution technique :- I n this elution technique , same solvent composition or solvent of same polarity is used throughout the process of separation. Example: chloroform only 22

23. ( ii) Gradient elution techniques:- Solvents of gradually ↑ polarity or ↑ elution strength are used during the process of separation. E.g. initially benzene, then chloroform, then ethyl acetate then chloroform 23

24. DETECTION OF COMPONENTS If the compounds separated in a column chromatography procedure are colored, the progress of the separation can simply be monitored visually. If the compounds to be isolated from column chromatography are colorless. In this case, small fractions of the eluent are collected sequentially in labelled tubes and the composition of each fraction is analyzed by TLC. 24

25. Eluting the sample:- Components a, b, and c separate as column progresses. Fractions can be collected in test tubes, vials, beakers, or Erlenmeyer flasks. 25

26. Analyzing the fractions: . Analyze the fractions by thin-layer chromatography 26

27. FACTORS AFFECTING COLUMN EFFICIENCY 1.Dimension of the column:- column efficiency has been improved by increasing length/width ratio of the column. 2. Particle size of column packing:- separation to be improved by decreasing the particle size of the adsorbent. 3. Temperature of the column :- The speed of the elution increases at higher temperatures. 4. Packing of the column 5. Quality of solvents:- solvents having low viscosities is giving better results. 27

28. APPLICATIONS Separation of mixture of compounds Purification process Isolation of active constituents Estimation of drugs in formulation Isolation of active constituents separation of diastereomers 28

29. • Advantages of C.C » Any type of mixture can be separated » Any quantity of mixture can be separated » Wider choice of Mobile Phase » Automation is possible • Disadvantages of C.C » Time consuming » more amount of Mobile Phase are required »Automation makes the techniques more complicated & expensive 29

30. Reference:- https://www.slideshare.net/shaisejacob/column-chromato https://www.slideshare.net/krakeshguptha/column-chromatography-26966949 S.RAVI SHANKAR, Text book of pharmaceutical analysis, Rx publications, 2001.Pg no : 13-4 to 13-13 30

31. ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (UHPLC) 31

32. CONTENTS INTRODUCTION PRINCIPLE COMPARISON INSTRUMENTATION ADVANTAGES DISADVANTAGES APPLICATION 32

33. INTRODUCTION:- UHPLC refers to ultra high performance liquid chromatography. It improves in three areas, chromatographic resolution, speed, sensitivity. UHPLC is a rising chromatographic separation technique whose packing materials have smaller particular size lesser than 2.5µm. The technology takes full advantage of chromatographic principles to run separation using column packed with smaller particles and higher flow rates. 33

34. It can withstand high system back-pressure. Special analytical columns UHPLC BEH C18 packed with The factor responsible for development of UHPLC technique was evolution of packing material used to effect the separation. The technology takes full advantage of chromatographic principles to run separations using columns packed with smaller particles. It decreases analysis time and solvent consumption 1.7µm particles are used in connection with system 34

35. PRINCIPLE:- The Principle of UHPLC is based on van deemeter equation which describes the relationship between flow rate and HETP or column efficiency. H=A + B/v + Cv when, A= Eddy diffusion B= Longitudinal diffusion C= equilibrium mass transfer v= flow rate Van deemeter equation that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency) 35

36. COMPARISON BETWEEN HPLC AND UHPLC 36 Parameters HPLC UHPLC Column XTerra,C18,50 × 4.6mm AQUITY UHPLC BEH C18,50 ×2.1mm Particle size 4µm particles 1.7µm particles Flow rate 3.0 ml per min 0.6 ml per min Injection volume 20 µl 3 µl partial loop fill or 5 µl full loop fill Total run time 10 min 1.5 min Theoretical Plate count 2000 7500 Column temperature 30 °C 65 °C

37. Parameters HPLC UHPLC Maximum back pressure 35-40 Mpa less 103.5 Mpa more Resolution Less High Analysis time More Less sensitivity Less Higher 37

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39. A completely new system design with advanced technology in the pump, auto sampler, detector, data system, and service diagnostics was required. Achieving small particle, high peak capacity separations requires a greater pressure range than that achievable by HPLC system. 39

40. INSTRUMENTATION:- SOLVENT RESERVOIR: The most common type of solvent reservoir is glass bottle. Most of manufacturers supply these bottles with special caps, tubing and filters to connect to the pump inlet and so the purge gas (helium) used to remove dissolved air 40

41. PUMP:- CONSTANT PRESSURE PUMP: Constant pressure is used only for column packing. CONSTANT FLOW PUMP: This type is mostly used in all common UPLC application. RECIPROCATING PISTON PUMP: DUAL PISTON PUMP 41

42. SAMPLE INJECTION:- In UHPLC , Sample introduction is critical. Conventional injection valves, either automated or manual, 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 carryover required to increase sensitivity. 42

43. UHPLC COLUMN: Resolution is increased in a 1.7µm particle packed column because 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 UHPLC separations: ACQUITY UHPLC BEH C18 and C8 (straight chain alkyl columns), ACQUITY UHPLC BEH shield RP 18 (embedded polar group column) ACQUITY UHPLC BEH (phenyl group tethered to the silyl functionality with a C6 alkyl) ACQUITY UHPLC BEH Amide columns 43

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45. CHEMISTRY OF SMALLPARTICLES 45 As the particle size decreases to less than 2.5µm, not only there is significant gain in efficiency, but the efficiency doesn't diminish at increased flow rates. By using smaller particles, speed and peak capacity (number of peaks resolved per unit time in gradient separation) can be extended to new limits, termed ultra performance liquid chromatography.

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47. D ETECTORS:- UV Detectors Fluorescent detector Refractive index detector Light scattering detector Electrochemical detector Mass spectrometric detector 47

48. ADVANTAGES:- 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. 48

49. DISADVANTAGES: Due to increased pressure requires more maintenance and reduces the life of the columns of this type. In addition, the phases of less than 2µm are generally non-regenerable and thus have limited use. 49

50. APPLICATION: Analysis of natural products and traditional herbal medicine. Identification of metabolite Study of metabonomics / metablomics Bio analysis/bioequivalence studies. Manufacturing/QA/QC Impurity profiling Forced degradation studies Dissolution testing Toxicity studies 50

51. It provides high speed, accuracy and reproducible results for analysis of drugs and their related substance. Thus method development time decrease. UPLC used for accurate, reliable and reproducible analysis of amino acids in area of protein characterization, cell culture monitoring and nutritional analysis of food. 51

52. Reference:- https://www.slideshare.net/pavi96/ultra-high-performance-liquid-chromatography https://www.slideshare.net/YendaManishankar/uhplc 52

53. Thank you 53