Column chromatography 2019

The present slide gives us an insight into the different aspects of application of columnn chromatrography the principle behind it and at the same time its recent advances.

1. COLUMN CHROMATOGRAPHY Presented by : Anand Sagar Tiwari M. Pharm (First Semester) Dept. of Pharmacology Guided by : Dr. S.M.Malipatil

2. Chromatography Tswett (1906) stated that chromatography is a method in which the components of a mixture are separated on adsorbent column in a flowing system. IUPAC definition (1993): Chromatography is a physical method of separation in which the component to be separated are distributed between two phases one of which is stationary while the other moves in a definite direction. Stationary phase may be a solid or a liquid supported on a solid or gel , the mobile phase may be either a gas or a liquid .

3. Column Chromatography Introduction : Column chromatography is a separation technique ion which a column of stationary phase is used. The stationary phase used in column chromatography may be a solid or liquid. Based on the type of stationary phase used column chromatography may be divided into two types :- Adsorption Column C hromatography where solid is stationary phase. Partition Column Chromatography where liquid is stationary phase.

4. PRACTICAL REQUIREMENTS Stationary phase Mobile phase Column characteristics Preparation of column Introduction of sample Development of technique Detection of components Recovery of components

5. Stationary Phase An adsorbent used in column chromatography should meet the following criteria :- The particles should have uniform size distribution and have spherical shape. Particle size: 60-200µ. Should have high mechanical stability. Should be inert and should not react with the solute or other components. Insoluble in the solvents or mobile phases used. It should be colorless to facilitate observation of zones and recovery of components. Should allow free flow of mobile phase and should be capable of separating for wide variety compounds . Most commonly used adsorbent is 80-100 mesh or 100-200 mesh size.

6. Selection of Stationary phase Removal of impurities No. of components to be separated [ More components to be separated a strong adsorbent is used] Affinity differences between components Length of the column used Quantity of adsorbent used [ Adsorbate : Adsorbent ratio= 1:20 or 1:30]

7. Mobile phase Mobile phase is very important and they serve several functions. They act as a solvent, developer and as a eluent. The functions of a mobile phase are ; To introduce the mixture into the column- As solvent To develop the zones for separation – As developing agent To remove pure component out of the column- As eluent e.g. Petroleum ether, Carbon tetrachloride, Water etc.

8. Column characteristics The material of the column is mostly good quality neutral glass since it should not be affected by solvents, acids and alkalies . The column dimensions are important for effective column separations. The effective length : diameter ratio ranges from 10:1 to 30:1 . The length of the column depends upon :- Affinity of compounds towards the adsorbent used. Number of compounds to be separated. Types of adsorbents used. Quantity of sample

9. Resolution The resolution of a quantitative measure of how well two elution peaks can be differentiated in a chromatographic separation. It is defined as the difference in retention times between the two peaks, divided by the combined widths of the elution peaks. A resolution value of 1.5 or greater between the two peaks ensure that the sample components are well separated to a degree where the area or height of each p eak can/may be accurately measured. The equation depends on selectivity(separation factor) , efficiency and retention(capacity factor).

10. Column efficiency It is expressed by the no. of theoretical plates. The no. 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. It is expressed by the equation: Where, tr = retention time measured from the instant of injection and W= peak width

11. Contd…… The column is considered as being made of theoretical plates which are parallel layers one above the other. Sample being introduced into the column the components distribute themselves in between the stationary phase and mobile phase according to their partition coefficients. The rate of movement of mobile phase is assumed to be in dynamic equilibrium with stationary phase and the components move down the column at a rate given by the equation:- R= Rate of movement of component Rate of movement of mobile phase

12. The thickness of the theoretical plate is called the height equivalent to a theoretical plate(HETP). HETP = Length of Column (L) Number of theoretical plates (N) The smaller the HETP the greater is the number of theoretical plates and greater is the efficiency of the column. Thus the efficiency of a column is measured by its number of theoretical plates .

13. HETP value can also be determined by the Van Beemter equation . The equation of HETP is often used to describe the efficiency of column . An efficient column would have a minimum HETP value .

14. Factors affecting column efficiency Nature of solvents : T he rate of flow of a solvent is inversely proportional to its viscosity . Hence the solvents must have less viscosity for effective separation at faster rate and also f or proper elution strength. Dimension of column : The efficiency can be increased by means of increasing the length/width ratio . The range is observed to be 20:1 to 100:1 depending upon the efficiency required. Particle size of the adsorbent : The adsorbent activity is proportional to the surface area of the adsorbent i.e. adsorbent activity increases with the increase in particle size. Efficiency can be increased by decrease in particle size .

15. Contd……. Temperature of the column : The rate of elution can be increased by increasing the temperature of column , but, it decreases the adsorbent power of the column . Hence a compromise is made in between speed of elution and adsorbent power. As a result difficult soluble samples are separated at high temperature while others are separated at room temperatures. Pressure : Application of high pressure above the column and lower pressure below the column, the efficiency of the column can be increased. High pressure above the column is achieved by maintaining a column of liquid on the top of the column or by using pressure devices (pumps). Pressure below the column is decreased by applying vacuum , using vacuum pump .

16. Preparation of the column The bottom portion of the column is packed with cotton wool or glass wool above which the column of adsorbent is packed. After packing the column with the adsorbent a similar paper disc is kept on the top, so that the adsorbent layer is not disturbed during the introduction of sample or mobile phase. Disturbance in the layer of adsorbent will lead to irregular bands in separation. There are two types of preparing the column called as packing techniques. They are:- Dry packing technique Wet packing technique

17. Dry Packing Technique : Adsorbent is packed in the column in dry form. Solvent is filled till equilibrium is reached. Demerit : Air bubbles are entrapped in between M.P. and S.P.----- cracks appear in the adsorbent layer. Wet Packing Technique : Ideal and common technique. Adsorbent + M.P. in a beaker and poured into the column. S.P. settles uniformly and no crack in the column of adsorbent .

18. Introduction of sample The sample which is usually a mixture of components is dissolved in minimum quantity of the mobile phase used for preparing the column or a solvent of minimum polarity. The entire sample is introduced into the column at once and gets adsorbed on to the top portion of the column. From this zone the individual samples can be separated by a process of elution.

19. Development technique There are mainly three types of Development technique:- Frontal Analysis Displacement Analysis Elution Analysis

20. Frontal Analysis This technique was developed by Tisellius. Column is filled with a known amount of adsorbent and the sample mixture is continuously added to the column. If any mixture is added the less adsorbed component leaves the column in a pure form. Thus this method can be used for separation of readily eluted component from others with greater affinity and is also termed as preparative method. Used to separate relatively smaller qualities of unwanted substances which are strongly adsorbed than that of desired components from a given sample.

21. Displacement Analysis Here the sample mixture is introduced first into the column. The components in the mixture are separated by running a solution which consists of a substance more strongly adsorbed than any of the components in the sample mixture. The substance used to separate the components of mixture is called as displacing agent . Displacing agent is adsorbed strongly at the top of the column and spreads downwards forcing out the components of the sample mixture from adsorption sites . Components leave the column based on their degree of adsorptivities .

22. Elution Analysis In this method the sample components are separated by means of eluting the column with fresh solvent. A small quantity of mixture is introduced into the column . Fresh solvent having lesser affinity for stationary phase than sample components is allowed to run down towards the column. Partition coefficients of the components in the sample if different from the mixture they separate out into bands and migrate down towards the column at different rates and are separated. The components can be separated by means of elution analysis by employing two different methods.

23. Isocratic Elution Technique : (Iso means same or similar) In this elution technique , the same solvent composition or solvent of same polarity is used throughout the process of separation. e.g. Chloroform only; Pet.ether : Benzene = 1:1 only etc. Gradient Elution Technique : (Gradient = gradually) In this process solvents of gradually increasing polarity or increasing elution strength are used during the process of separation. Initially low polar solvent is used followed by gradually increasing the polarity to a more polar solvent. e.g. Initially benzene, then chloroform, then ethyl acetate, then to methanol etc.

24. Detection of Components The detection of colored components can be done visually . Different colored bands are seen moving down the column which can be collected separately. But for colorless compounds the technique depends upon the properties of the components. Different properties which can be used are:- Absorption of light using (UV/Vis)- using UV/Vis detector Flame ionization detector Evaporation of solvent and weighing the residue By monitoring the fractions by TLC .

25. Recovery of components Earlier recovery of the components were done by cutting the column into several distinct zones . Later extrusion of the column into zones were done by using plunger . The best technique is to recover the components by a process called as elution . The recovered fractions are detected by using the techniques discussed earlier. If a fraction still contains several components it can be resolved by using another column.

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

27. Contd…. Separation of Geometrical isomer : Winterstein reported the first chromatographic separation of cis/trans isomers of bixin and crocetin dimethyl ether . Later Zechmeister separated cis/trans isomers carotenoids on calcium carbonate, aluminum oxide and other adsorbents. The separation is based on steric factors . Isomers whose functional group can approach the surface of the adsorbent more easily and more strongly adsorbed.

28. Contd….. Separation of diastereomers : A derivative having an optically active partner can’t be separated from the latter. Such separations can be carried out by Column Chromatography on various adsorbents . e.g. The separation of diastereomeric 7-chloro-azibicyclo(4:1:0)- heptane has been done on silica gel using pentane diethyl-ether as a solvent. Separation of tautomeric mixtures : Separation of tautomeric mixtures can be carried at a high temperature by column chromatography. e.g. The keto and enol forms of the p-hydroxyl-phenyl pyruvic acid and indole pyruvic acid can be separated in the liquid phase .

29. Column Partition Chromatography Introduction: Partition chromatography is based on the differences in partition coefficients of a mixture between a liquid stationary phase and a gaseous or liquid mobile phase. Principle : Some substance shaken with a mixture of two immiscible liquids distribute themselves between the immiscible liquids such that the concentration in each liquid is proportional to the partition coefficient . Thus solvents having larger difference in their partition coefficients can be employed to separate the required components from a mixture.

30. Theory In partition chromatography the liquid stationary phase is supported in the column by means of a solid material which is polar in character. Silica gel and cellulose powder are frequently used as solid supports. The liquid phase should be chosen such that it is immiscible with the liquid stationary phase .

31. Application It is used for separation of wide range of biological compounds. Used for the separation of closely related substances such as amino acids formed in the hydrolysis of proteins. Also used in separation of closely related aliphatic alcohols, sugar derivatives and carboxylic acids .

32. Bonded Phase Chromatography

33. In this technique the stationary phase may be bonded with the support material. This type of partition chromatography in which both the monomeric as well as polymeric phases have been bonded to a large no of support materials is called as Bonded Phase Chromatography . Polymeric bonded stationary phases can be produced by using di- or tri- chlorosilanes in the presence of moisture as a result of which a polymeric layer is formed at silica surface . The main advantage of this technique is to prevent loss of stationary phase from the separation column and used for the separation of bioactive molecules .