2. COLUMN
CHROMATOGRAPHY

3. INTRODUCTION OF CHROMATOGRAPHY

4. What is chromatography?
Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one which is stationary phase and other is mobile phase move in a definite direction.

5. Background of Chromatography
The term “chromatography’’ is derived from Greek, chroma meaning “color” and graphein meaning “to write”
Chromatography is a new technique which was first invested by Mikhail Tswett, in 1906 in Warsaw.
He was successful in doing the separation Of colored substances by percolating vegetable extract through a column of ca carbonate.
The Calcium carbonate act as adsorbent and different substances got absorbed to different extent and this give rise to color bands at different position on the column. Tswett termed this system of colored band as the chromatogram and the method as chromatography.

6. Theory of Chromatography
Plate theory
Rate theory

7. Classification of Chromatography
Adsorption chromatography
Partition chromatography
Ion exchange chromatography
Size exclusion chromatography
Affinity chromatography

8. Chromatography types are further Subdivided into
1- Adsorption Chromatography
Gas –solid chromatography
Thin layer chromatography
HPLC
2-Partition Chromatography
Gas liquid chromatography
Liquid -liquid chromatography
Paper chromatography
3-Ion exchange Chromatography
Ion exchange chromatography
4 - Size Exclusion Chromatography
5- Affinity Chromatography
(DNA affinity chromatography)

9. Column Chromatography
It was developed by the American petroleum Chemist D.T Day in 1900
M.S Tswett , the polish botanist in1906 used adsorption column in his investigation .
column chromatography is also known as adsorption chromatography .In which the solid stationary phase is packed in a tubular column and mobile phase is allowed to flow through the solid .
the column in which the stationary phase is packed consist of glass or Teflon tube, typically 10 to 50mm in diameter.

10. TYPES OF COLUMN CHROMATOGRAPHY

11. GENERAL TYPES OF COLUMN CHROMATOGRAPHY
Adsorption chromatography
Gel filtration chromatography
Ion exchange chromatography
Affinity chromatography
Gas chromatography
High performance liquid chromatography

12. TYPES OF COLUMN CHROMATOGRAPHY ON THE BASIS OF FLOW OF SOLVENT
Gravity column chromatography
Flash column chromatography

13. Gravity Column Chromatography
INTRODUCTION
This is the method employed by Mikhail Tswett in It is still used commonly in developing countries although the advent of faster more efficient variants has led to a decline in its use in developing countries.

14. DEFINITION
It is the type of column chromatography in which the mobile liquid is passed by gravity through the column of stationary phase.

15. ADVANTAGES OF GCC
The advantages of this technique is that it requires little in a way of special equipment and gives good results with a relative low level of experimental expertise. The amount of supervision required is much lesser compare to that from other techniques.

16. Flash Column Chromatography
INTRODUCTION
This method is developed by W. C. Stills in 1978, which involves application of positive pressure to the mobile phase solvent from the top of the column.

17. DEFINITION
Flash column chromatography is a specialized chromatography technique that uses compressed gas (such as nitrogen or air) or a pump to push solvent through the column.

18. APPLICATION OF FCC The main application of flash chromatography are:
Purification of synthetic products,
Isolation of target compounds from natural products,
The simplification of mixtures prior to high resolution
preparative (usually) liquid chromatography
The fractionation of complex mixtures into simpler
group for analysis.

19. DIFFERENCE BETWEEN GRAVITY AND FLASH COLUMN CHROMATOGRAPHY

20. Gravity column chromatography Flash column chromatography
It is the type of column chromatography in which the mobile liquid passed by gravity through the column of stationary phase
Flash column chromatography is a specialized chromatography technique that uses compressed gas (such as nitrogen or air) or a pump to push solvent through the column
The normal particle size range of silica gel in traditional gravity column chromatography is 63 – 200 micrometer.
The normal particle size range of silica gel in flash column chromatography is 40 – 63 micrometer

21. Gravity column chromatography Flash column chromatography
The mesh number of gravity column chromatography is
The mesh number of flash column chromatography is 250 – 400.
Large solvent and adsorbent consumption
Less solvent and adsorbent consumption
It requires long separation time period
It requires short separation time period

22. PRINCIPLE OF COLUMN CHROMATOGRAPHY

23. The basis of all forms of chromatography is the distribution or partition coefficients (kd), which describes the way in which a compound (analyte) distributes between two immiscible phases. In this a solid stationary phase and a liquid mobile phase is used and the principle of separation is adsorption.

24. It is basically based on the basic operations of adsorption, partition, ion exchange, ion pairing and moleculer exclusion.
When a mixture of components dissolved in the mobile phase is introduced into the column, the individual components move with different rates depending upon their relative affinities.

25. The compound with lesser affinity towards the stationary phase (adsorbent) moves faster and hence it is eluted out of the column first. The one with greater affinity towards the stationary phase (adsorbent) moves slower down the column and hence it’s eluted later. Thus, the compounds are separated.

26. The type of interaction between the stationary phase (adsorbent) and the solute is reversible in nature; the rate of movement of a component (Rf) is given as follows:
Rf = Rate of movement of a component
Rate of movement of a mobile phase
This equation can be simplified as follows:
Rf = Distance moved by the solute
Distance moved by the solvent

27. RETENTION TIME:
The time between sample injection and an analyte peak reaching a detector at the end of the column is termed as Retention time (tR). Each analyte in a sample will have a different retention time. A term called the retention factor is often used to describe the migration rate of an analyte through a column.

28. When the mixture of analyte is applied and the mobile phase, commonly referred to as the eluent is passed through the column. As the eluent flows through the column the analytes separate on the basis of their distribution coefficients and emerge individually in the eluate as it leaves the column.

29. INSTRUMENTATION OF COLUMN CHROMATOGRAPHY:

30. BASIC COMPONENTS: PUMP INJECTOR INJECTION VALVE COLUMN DETECTOR
FIVE MAJOR COMPONENTS:
PUMP
INJECTOR
INJECTION VALVE
COLUMN
DETECTOR

31. 1) PUMPS: A device designed to deliver the mobile phase at a
controlled flow-rate to the separation system. Pumps are
generally used in column chromatography.
MAJOR TYPES:
Syringe Pumps
Reciprocating Pumps
Pneumatic Pumps

32. SYRINGE PUMPS:

33. RECIPROCATING PUMPS:

34. PNEUMATIC PUMPS:

35. 2) INJECTOR: Direct injector Bypass injector On-column injector
A device by which a liquid, solid or gaseous sample is introduced into the mobile phase or the chromatographic bed. Main types are:
Direct injector
Bypass injector
On-column injector

36. 3) INJECTION VALVE: FUNCTION:
The injection valve allows a defined amount of sample to be
pumped onto the column. The sample and the sample's
injection into the system are the most critical factors in any
analytical process. Therefore, the quality, reproducibility, and
flexibility of the sample injection valve are important.
FUNCTION:
Injection valves are used for injecting liquid additives directly
into the column.

37. 4) COLUMN:
The column is where the actual separation takes place. It is usually a glass or metal tube of sufficient strength to withstand the pressures that may be applied across it. The column contains the stationary phase. The mobile phase runs through the column and is adsorbed onto the stationary phase. The columns available are simple glass tubes, varying in length and diameter . They usually have a stopcock attached to control the solvent flow, and may have a fritted plate to support the adsorbent.

38. MAJOR TYPES OF COLUMN: 1- Gravity Columns:
2- Flash Columns (Air or nitrogen pressure):

39. 3-Low and Medium Pressure Columns (pumped):
4-Vacuum Columns [Vacuum liquid chromatography (VLC)]: 5-High pressure Columns (HPLC):

40. COLUMN LENGTH AND DIAMETER:
As a general practice, 15m columns are used for fast screening, simple mixtures, or very high molecular weight compounds. The 30m length has become the most popular one for most analyses. Very long columns (50, 60 and 105m) are for extremely complex samples.
Increased diameter means more stationary phase, even with the same thickness, for greater sample capacity. When sample capacity is a major consideration, as with gases, very volatile samples, and purge and trap or headspace sampling, large id or even PLOT columns may be appropriate.

41. 5) DETECTORS: The UV detectors The electrical conductivity detectors
The fluorescence detectors
The refractive index detectors

42. WORKING OF COLUMN CHROMATOGRAPHY

43. GRAVITY COLUMN CHROMATOGRAPHY

44. Gravity Column Chromatography
It is a type of column chromatography which work under the force of gravity.
Used for large scale separation of components.
This method is a lot slower to run.
They also are more difficult to pack with adsorbent.

45. PROCEDURE Packing of the column Sample loading Elution
Detection of components
Isolation of compound

46. Detection of components
Steps involved
Packing of the column
Sample loading
Elution
Detection of components
Isolation of compound

47. 1. Packing Of The Column
There are two common methods of packing a gravity column:
The slurry method
The dry pack method

48. i. The Slurry Method
A slurry of adsorbent and solvent is made and poured into column.
Place a flask under the column, open the pinch clamp, and allow the liquid to drain into it.
Transfer the slurry to the column until all the stationary phase is added.
Drain the excess solvent.
Close the pinch clamp.
The column is now packed and ready for use.

49. ii. The Dry Pack Method Fill the column with solvent.
Using a funnel, sprinkle dry stationary phase into the solvent, allow solvent to drain.
Let the stationary phase settle and gently tap the column.
Drain the excess solvent.
The column is now packed and ready for use.

50. 2. Sample Loading
The analyte sample is dissolved in a very small amount of solvent (1-3ml) and added to the top of the column.
After this, a small layer of white sand is added to the top of the column.

51. 3. Elution i. Isocratic Elution Technique
The same solvent composition or solvent of same polarity is used throughout the process of separation.

52. ii. Gradient Elution Technique
Solvent of gradually increasing polarity is used during the process of separation.

53. 4. Detection of Components
For colored compounds, they can be seen visually to separate.
For colorless compounds, either Thin Layer Chromatography (TLC) or Gas Chromatography (GC) may be used to identify the compounds present in the different fractions.

54. 5. ISOLATION OF DESIRED COMPOUND
The solvent of compound containing fraction is evaporated through Rotary evaporator and the compound is isolated.
Recrystallization of compound may be used to further purify the product.

55. FLASH COLUMN CHROMATOGRAPHY

56. Step 1: Preparation of Column
Cotton wool at bottom and little amount of sand

57. OR
Addition of dry silica gel by scooping
Pouring through beaker

58. Step 2: Pre-Elution of Column
When properly packed, the silica gel fills the column to just below the indent on the pipette. This leaves a space of 4-5 cm on top of the adsorbent for the addition of solvent. Clamp the filled column securely to any stand using a clamp

59. Pour a good amount of your elution solvent onto the silica gel.
The solvent flows slowly down the column.
Monitor the solvent level, both as it flows through the silica gel and the level at the top.
Make sure it does not go below the top of the silica.

60. The process can be speed up byOR
Pipette bulb
Automated System
This helps in the quick travel of solvent through the column and save the time (it is the main importance of flash column chromatography)

61. When the bottom solvent level is at the bottom of the column, the pre-elution process is completed and the column is ready to load.

62. Step 3: Sample Loading
WET LOADING
DRY LOADING

63. WET LOADING
Dissolve your sample in the minimum possible volume of pre-elution solvent and apply it evenly to the surface of the silica.
Once the sample is in the column, fresh eluting solvent is added to the top and you are ready to begin the elution process.

64. DRY LOADING
Dissolve the sample to be analyzed in the minimum amount of solvent and little amount of silica gel.
Swirl the mixture until the solvent evaporates and only a dry powder remains.

65. Transfer this dried powder onto the top of the prepared column
Add fresh eluting solvent to the top. Now you are ready to begin the elution process

66. Step 4: Eluting the Sample
Step 5: Detection of Sample
Step 6: Isolation of Separated or Desired Compound

67. PHASES USED IN COLUMN CHROMATOGRAPHY

68. STATIONARY PHASE USED IN COLUMN CHROMATOGRAPHY
Most common stationary phase used are:
Alumina may be acidic, neutral or basic
Silica gel is slightly acidic.
SELECTION OF STATIONARY PHASE
removal of impurities
No. of components to be separated
affinity differenced between components
length of the column used
quantity of the adsorbent used

69. PROPERTIES OF STATIONARY PHASE (ADSORBENT)
Most stationary phases influence the flow rate and resolution characteristics
The larger the particle faster the flow rate but smaller the particle the larger the surface area and potentially greater the resolving power.
For greatest effectiveness the particles of adsorbent should be of uniform size and large surface area (for instance, 150 mesh alumina has a surface area of 155 m2/g)
concentration of the mixture

70. The strength of adsorption depends upon the compounds involved
The strength of adsorption depends upon the compounds involved. Since the adsorbents are polar, the more polar compounds are adsorbed more strongly. Thus, non-polar compounds are eluted first.

71. MOBILE PHASE USED IN COLUMN CHROMATOGRAPHY
THE FUNCTIONS OF THE MOBILE PHASE ARE:
to introduce the mixture into the column as a solvent
to develop the zones of separation as  developing agent
to remove pure component out of the column as an eluent.

72. CHARACTERISTICS OF MOBILE PHASE
Selection of solvents requires a balancing act between solvent and compound polarities.
For most separations, the solvent should be less polar than the compounds.
If the solvent is much more polar than the compounds, the compounds will remain in the mobile phase, and separation will not occur.
If the compounds are much more polar than the solvent, no compounds will elute since the solvent is unable to move compounds from the adsorbent sites.
order of polarity for silica gel and alumina is as follows:
hexane < petroleum <ether < carbon tetrachloride < toluene < dichloromethane < chloroform < diethyl ether < ethyl acetate < acetone < propanol < ethanol < methanol < acetic acid < water.

73. In complex separations, a series of increasingly polar solvents is used.
A large increase in polarity might cause all of the components to elute at once, as well as cause other problems with the column packing. Consequently, small polarity changes are accomplished by careful use of mixed solvents. For example, pure hexane may be used as the first solvent.

74. ADVANTAGES AND DISADVANTAGES OF COLUMN CHROMATOGRAPHY

75. ADVANTAGES OF COLUMN CHROMATOGRAPHY
Column chromatography is used to determine the number of components of a mixture and used to separate and purify substantial quantities.
Column chromatography should be used to separate a mixture of liquids or solutes into its components individually. In fact, it is the most frequently used method of purifying mixtures of products in research laboratories.
Wider choice of mobile phase.

76. DISADVANTAGES OF COLUMN CHROMATOGRAPHY
Requires some technical skill and take some time.
Column chromatography is less full proof than paper chromatography and requires constant attention while the experiment is being performed.
This method is time consuming and tedious especially for large samples. If it is unnecessary to separate large quantities of sample, analytical methods such as paper chromatography may be more suitable and easier to perform.
Air bubbles can entrapped between stationary and mobile phase in case of dry packing due to which cracks appear in the adsorbent layer

77. FACTORS AFFECTING COLUMN EFFICIENCY

78. Dimension of the column
Particle size of adsorbent
Nature of solvent
Temperature of the column
Pressure

79. APPLICATIONS OF COLUMN CHROMATOGRAPHY

80. Separation of mixtures of compounds
Removal of impurities or purification process
Isolation of active constituents
Isolation of metabolites from active fluids
In establishing the identity or non-identity of two substances

81. THANK YOU FOR YOUR TIME