Column chromatography

This presentation contains all the topics related to column chromatography. That includes introduction, principle,apparatus, experimental aspects of column chromatography, application of column chromatography, advantage and disadvantage of column chromatography with reference.

1. COLUMN CHROMATOGRAPHY Submitted to : Dr. Prema Kumari . K. B. Asst. Professor, Department of Pharmaceutics, College of Pharmaceutical Sciences, DSU Dayananda sagar college of Pharmacy, Banglore . Presented by: Arpitha B M M Pharm (I SEM), Department of Pharmaceutics, College of Pharmaceutical Sciences, DSU Dayananda Sagar College of Pharmacy, Banglore .

2. Contents Introduction Principle Apparatus Experimental aspects of CC Detection of compounds Recovery of compounds Factors affecting Column efficiency Application Advantage and disadvantage 2

3. INTRODUCTION 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. It is a technique that enables the separation, identification and purification of the components of a mixture for qualitative and quantitative analysis. It was developed by the American petroleum chemist D. T. Day in 1900. In 1906, M. S. Tswett , Polish Botanist used the adsorption columns to detect the plant pigments. 3

4. 4 Martin and Synge introduced partition column chromatography in 1941 . CC may be defined as a separation process involving the uniform percolation of a liquid solute through a column packed with finely divided material. If solid is used as stationary phase then it is Adsorption CC (solid – liquid chromatography) If liquid is used as stationary phase then it is Partition CC (liquid – liquid chromatography)

5. Principle: Adsorption The rate of absorption varies with a given adsorbent for different materials. This principle of selective adsorption is used in column chromatography. 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. Thus the compounds are separated. 5

6. The various bands present in the column become more defined. The banded column of the adsorbent is termed as chromatogram . The portion of a column which is occupied by a particular substance is called as zone. The narrower the zones, the longer the number of substances which can be separated in a column of a definite length and the more concentrated are the elutes. This type of interaction between the stationary phase (adsorbent) and the solute is reversible in nature. 6

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8. 8 ADSORPTION COLUMN CHROMATOGRAPHY

9. 2. PARTITION In partition column chromatography it is based on the differences in partition coefficient of the individual components of a mixture. The technique is similar to column adsorption chromatography except the stationary phase is liquid. Stationary phase: liquid is supported in the column by means of a solid material. 9

10. Eg . silica gel and cellulose powder are frequently used as solid supports. Mobile phase: liquid. 10

11. PARTITION COLUMN CHROMATOGRAPHY 11

12. Experimental aspects of Column chromatography 1. 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) 12

13. 13 Various accessories are attached to the top and bottom of the column for maintenance of the elution process. The length of the column depends upon: Number of compounds to be separated Type of adsorbent used Quality 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. 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 14

15. 15 where tr is the retention time measured from the instant of injection w is the peak width W is determined by SD= σ ie w=4 σ

16. 2. ADSORBENT IDEAL PROPERTIES : Their particle should be spherical in shape and uniform in size. Their mechanical stability must be great enough to prevent the formation of fine dust which might be deposited in the channels of the packing. They should not react chemically, either with the eluting solvents or with the sample components. They should contain as small amount of soluble component as possible. They should be categorically inactive and as a rule, have neutral surface. Exceptions are ion exchangers. 16

17. It should be colorless to facilitate of zones and recovery of components. It should allow free flow of mobile phase. Useful for separating wide variety of compounds. The adsorbent is generally an active solid with a large surface area. Three types: 1. Weak – talc, sucrose, starch, inulin etc., 2. Intermediate – magnesia, CaCO 3 , MgCO 3 , Na 2 SO 4 , etc., 3. Strong – alumina, charcoal, bauxite, etc., Silica gel (acidic) may strongly retain basic compounds. Alumina (basic) should not be used for base sensitive compounds 17

18. GRADE SERIES OF ADSORBENTS: 1. Sucrose, Starch 8. Magnesium Carbonate 2. Inulin 9. Magnesium mixtures 3. Talc 10. Lime 4. Sodium carbonate 11. Activated Magnesium 5. Calcium carbonate 12. Silicate 6. Calcium Phosphate 13. Activated Alumina 7. Magnesium carbonate 14. Fuller’s earth Inulin 18

19. 3) 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 developing agent To remove pure components out of the column – as eluent 19

20. The choice of the solvent is depend on the solubility characteristics of the mixture. 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 are used: ( in increasing order of polarity) Petroleum ether, carbon tetrachloride, cyclohexane , ether, acetone, benzene, toluene, esters, water, etc 20

21. It can be used in either pure form or as mixture of solvents. More polar solvent move the molecules more efficiently Less polar move the molecules less efficiently separation occurs 21 Alkanes Toluene Diethyl ether Chloroform Acetone Ethyl acetate Ethanol Methanol (CH 3 OH) More polar Least polar Alkanes Toluene Diethyl ether Chloroform Acetone Ethyl acetate Ethanol Methanol (CH 3 OH)

22. SOLVENTS ELUTING POWER 1. Petroleum ether 1.90 2. Cyclohexane 2.02 3. Carbon tetra chloride 2.24 4. Trichloro ethylene 3.40 5. Benzene 2.28 6. Chloroform 4.81 7. Absolute alcohol 4.34 8. Ethyl acetate 6.11 9. Pyridine 12.40 10. Acetone 21.40 11 n- propanolol 21.80 12. Ethanol 25.80 13. Methanol 33.60 14. Water 80.40 15.Formamide More than 84.0 22

23. PREPARATION OF COLUMN The bottom portion of the column is packed with cotton wool or glass wool or asbestos pad, above which the column of adsorbent is packed. A Whatman filter paper disc can also be used. After packing the column with the adsorbent, a similar paper disc is kept on the top to avoid the disturbance of adsorbent layer during the introduction of sample or mobile phase. Disturbance in the layer of adsorbent will lead to irregular bands of separation. 23

24. There are two types of preparing the column, which are called as packing techniques. They are: 1. Dry packing technique 2. Wet packing technique Dry packing is the method of choice for a microscale column. There are two versions of dry packing; 24

25. Type 1: Begin by filling the column with a nonpolar solvent. Slowly add the powdered alumina or silica while gently tapping the side of the column with a pencil. The solid should “float” to the bottom of the column. Try to pack the column as evenly as possible; cracks, air bubbles, and channels will lead to a poor separation. 25

26. Type 2: The stationary phase is deposited in the column before the solvent. In this case fill the column to the intended height with the stationary phase and then slowly add the nonpolar solvent. The solvent should be added slowly as to avoid uneven channelling. This method is typically used with alumina only, since silica gel expands and does not pack well with this dry method. 26

27. Dry Packing Technique 27

28. The disadvantages of this technique are: Air bubbles are entrapped between the solvent & stationary phase. Column is not packed uniformly. Cracks appear in the adsorbent present in the column. 28

29. Wet packing method (slurry method) The slurry method is often used for macro scale separations. Combine the solid stationary phase with a small amount of nonpolar solvent in a beaker. Thoroughly mix the two until a consistent paste is formed, but is still capable of flowing. Pour this homogeneous mixture into the column which is clamped in a vertical position as carefully as possible using a spatula to scrape out the solid as you pour the liquid. 29

30. Never allow your column to run dry. It is allowed to settle under gravity until a column of the desired height is obtained. The tap at the lower end is then opened to allow the liquid to run out until it just covers the top of the medium. Wet packing is common with adsorbents like alumina and magnesia etc., The slurry method normally gives the best column packing, but is also a more difficult technique to master. 30

31. Whether the dry or slurry method is chosen, the most important aspect of packing the column is creating an evenly distributed and packed stationary phase. As mentioned, cracks, air bubbles and channelling will lead to a poor separation. 31

32. 32 Wet packing method

33. INTRODUCTION OF THE SAMPLE 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. Dry loading Dissolve sample in any volatile solvent. The sample solution is then adsorbed on small weight of adsorbent and the solvent is allowed to evaporate. 33

34. The dry adsorbent loaded with the sample is then applied to the column. 34

35. Developmental techniques Frontal analysis Elution analysis Displacement analysis 35

36. Frontal analysis It consists of passing the sample solution continuously through the adsorbent column. This makes the strongly adsorbed solutes are adsorbed at the upper part of the column whereas the less strongly adsorbed solutes are adsorbed down the column. The solvent comes out first followed by the least adsorbed solute. The other solutes emerge one after another depending upon their degree of adsorptivity . 36

37. For example: The continuous addition of sample mixture of A & B on to the column which is filled with a known amount of the adsorbent. After some time the mixture is adsorbed completely and the column gets saturated. Then on continous addition of the mixture, the less strongly adsorbed component A leaves the column in a pure form. Then it is eluted as a mixture with component B which gets adsorbed more strongly and consequently moves more slowly. Frontal analysis is a preparative method which is primarily used for the separation of one readily eluted component from others with greater affinity. 37

38. 38 E F D

39. ELUTION ANALYSIS ELUATE: The  eluate , or mobile phase is a combination of the mobile phase "carrier" and the  analyte material that emerge from the  chromatograph. It specifically includes both the analytes and solutes passing through the column. A small quantity of the sample solution is introduced at the top of the column followed by pure solvent which gives rise to differential migration of solutes in the mobile phase. 39

40. Depending upon the partition coefficient the solute comes out from the column. For example: A small amount of the mixture having components A & B at the top of the column and it is eluted with an eluant C which is having a lesser affinity for the stationary phase than the sample components. Depending upon their relative affinity for the stationary phase the components gets eluted in the order of their affinities. The components can be separated completely with a zone of the mobile phase that is eluant C. 40

41. Elution analysis Types The components can be separated by means of elution analysis by employing 3 different types of methods : 1.STEP-WISE ELUTION: In this method, a set of eluants with increasing eluting powers are used to separate components which are having greater affinity towards the stationary phase. Solvents of gradually increasing polarity or increasing elution strength is used. Example: initially benzene, then chloroform, then ethyl acetate, then to methanol. 41

42. 2.GRADIENT ELUTION: In this method components are separated by varying the composition of the eluants . In gradient elution, a modulator is often used in the mobile phase to adjust eluent strength for better results in chromatographic separations. Compared with isocratic elution, the modulator concentration in the mobile phase in gradient elution is increased or decreased continuously with time. Therefore, gradient elution can be used to separate components which have a wide range of retentivity with no loss of resolution. 42

43. Gradient elution is able to produce high peak heights in a shorter operation cycle compared with isocratic elution. For these reasons, gradient elution has been widely used in high performance liquid chromatography for analytical purposes. Increasingly, gradient elution is applied to preparative- and large-scale chromatography for the separation of various macromolecules, such as proteins. 3) Isocratic elution technique: Same solvent composition or solvent of same polarity is used throughout the process of separation 43

44. Displacement analysis A small quantity of a sample solution is first introduced at the top of the column. The components of the mixture are then separated by running a solution of a substance which is more strongly adsorbed that any of the components of the mixture. The substance is termed as displacing agent . The displacing agent is adsorbed by the column and forms a zone at the top of the column. This zone spreads downwards forcing out the components of the sample from adsorption sites. 44

45. The components in turn displace one another from the column. The least adsorbed component will leave the column first followed by others depending upon their degree of adsorptvities . Advantage of this technique is that a large amount of sample solution can be introduced into the column. Disadvantage is that the column is saturated with the displacing agent after a run. For example: The sample mixture containing A & B is first introduced at the top of the column where it adheres to the stationary phase. 45

46. The elution takes place when the displacing eluant containing C is passed through the column, displacing the components on the stationary phase. During the process the components undergo separation because of the differences in partition or adsorption properties. 46

47. DETECTION OF COMPOUNDS If the mixture to be separated contains colored compounds, then monitoring the column is very simple. The colored bands will move down the column along with the solvent and as they approach the end of the column, collect the colors in individual containers. However, most organic molecules are colorless . To detect the components which are colorless , several techniques depending on the properties like absorption of radiations, refractive index, TLC, fluorescence etc are used. 47

48. Eluting the sample: Components a, b and c separate as column progresses . Fractions can be collected in test tubes, vials, beakers, or Erlenmeyer flasks. Detection of components 48

49. RECOVERY OF COMPOUNDS Once you believe all the materials have been removed from the column, the colours of the materials results should indicate which fractions contain the compound(s) you are interested in isolating. Combine the like or same fractions and evaporate the solvent. The pure separated compound will be left behind. Recrystallization may be used to further purify a solid product. However, on a milligram scale, there is usually not enough material to do this. 49

50. Column chromatography resolution calculation The ultimate goal of chromatography is to separate different components from a solution mixture. The resolution express the extent of separation between the compounds from mixture. The higher the resolution of the chromatogram, the better the extent of separation of the samples the column gives. This data is good way of determining the column’s separation properties of that particular sample. 50

51. The resolution can be calculated from the chromatogram. The separate curves in the curve represent different sample elution concentration profiles column resin. To calculate resolution the retention time and curve width are required . Retention time: the time from the start of signal detection by the detector to the peak height of the elution concentrtion profile of each different sample . Curve width: The width of the concentration profile curve of the different samples in the chromatogram units of them. 51

52. The simplified method of calculating chromatogram resolution is to use the plate mode . The plate model assumes that the columns can be divided into a certain number of sections, or plates and divided into a certain nuber of sections, or plates and the mass balance can be calculated for each individual plate. This approach approximates atypical chromatogram curve as a gaussian distribution curve . By doing this, the curve width is estimated as 4 times the standard deviation of the curve, The retention time is the time from the start of signal detection to the time of the peak height of the gaussian curve. 52

53. From the variables in the figure above the resolution, plate number and plate height of the column plate model can be calculated using the equation 53

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55. Factors affecting column efficiency Nature of the solvents – Solvents of low viscosities are used for high efficiency separations because the rate of flow is inversely proportional to viscosity. Dimension of the column – For better separations, sample/column packing ratios should range from 1 : 20 to 1 : 100. Pore diameter of column packing – polar adsorbents should possess a pore diameter of ≤ 2nm. 55

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57. Particle size of adsorbent – Column efficiency can be increased by using the smaller particles (100 to 200 mesh) of adsorbents. It should be non-catalytic and fine enough to hold the substance and give relatively sharp bands. Pressure – High pressure above the column by maintaining a column of liquid on the top of the column. Low pressure below the column by applying vacuum using vacuum pump. Temperature – Speed of elution is increased at higher temperature, but adsorbent power is decreased. Hence a room temperature is used. 57

58. APPLICATION 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 , recemates and tautomers . 58

59. 6. In the separation and identification of inorganic anions and cations . 7. 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. 59

60. 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 60

61. REFERENCE Instrumental methods of chemical analysis by B. K. Sharma Page no: C 92-110 Instrumental methods of chemical analysis by Gurdeep R. Chatwal , S.K. Anand . Page no 2.646 -2.655 www:http :/Google/CC/methodology.com 61

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