Product Overview

Chromatography has been developed into a new method of separation of mixture of substances mainly when they are available in small amounts. This method is very useful when the components of a mixture have almost the same physical and chemical properties and hence can’t be separated by other

Product Description

Chromatography has been developed into a new method of separation of mixture of substances mainly when they are available in small amounts. This method is very useful when the components of a mixture have almost the same physical and chemical properties and hence can’t be separated by other usual methods of separations. The term chromatography means writing in colour (in Greek: Khromatos-colour, and graphos- written). It was discovered by Mikhail Tswett in 1906. 
The methods of separation in chromatography are based on the distribution of the components in a mixture between a fixed (stationary) and a moving (mobile) phase. The stationary phase may be a column of adsorbent, a paper, a thin layer of adsorbent on a glass plate, etc., through which the mobile phase moves on. The mobile phase may be a liquid or a gas. When a solid stationary phase is taken as a column it is known as column chromatography. We are the major manufacturers of Column Chromatography Silica Gel in india. To meet the different needs of the clients, we offer. Column Chromatography Silica Gel in various particle sizes for pilot plant or other plant applications.

Column Chromatography

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. The principle of column chromatography is based on differential adsorption of substance by the adsorbent.

The usual adsorbents employed in column chromatography are silica, alumina, calcium carbonate, calcium phosphate, magnesia, starch, etc., selection of solvent is based on the nature of both the solvent and the adsorbent. The rate at which the components of a mixture are separated depends on the activity of the adsorbent and polarity of the solvent. If the activity of the adsorbent is very high and polarity of the solvent is very low, then the separation is very slow but gives a good separation. On the other hand, if the activity of adsorbent is low and polarity of the solvent is high the separation is rapid but gives only a poor separation, i.e., the components separated are not 100% pure.
The adsorbent is made into slurry with a suitable liquid and placed in a cylindrical tube that is plugged at the bottom by a piece of glass wool or porous disc. The mixture to be separated is dissolved in a suitable solvent and introduced at the top of the column and is allowed to pass through the column. As the mixture moves down through the column, the components are adsorbed at different regions depending on their ability for adsorption. The component with greater adsorption power will be adsorbed at the top and the other will be adsorbed at the bottom. The different components can be desorbed and collected separately by adding more solvent at the top and this process is known as elution. That is, the process of dissolving out of the components from the adsorbent is called elution and the solvent is called is called eluent. The weakly adsorbed component will be eluted more rapidly than the other. The different fractions are collected separately. Distillation or evaporation of the solvent from the different fractions gives the pure components.

Intermolecular forces, which vary in strength according to their type, make organic molecules to bind to the stationary phase. The stronger the intermolecular force, the stronger the binding to the stationary phase, therefore the longer the compound takes to go through the column.

Column Chromatography Procedure

COLUMN CHROMATOGRAPHY GRADE SILICA GEL

1. Use a piece of wire to add a plug of cotton to the bottom of the column. There should be just enough cotton that the sand and silica will not fall out of the column.
2. Clamp the column to a ring stand and add enough sand to fill the curved portion of the column.
3. Place a pinch clamp on the tubing, then fill the column 1/4 to 1/3 full with the initial eluent.
4. Prepare a slurry of silica in the initial eluent by pouring dry silica into a beaker of eluent. (Add a volume of silica gel, such as 20 mL, to approximately double the volume of eluent, 40 mL.) CAUTION: keep the dry silica in your hood and be careful not to inhale the lightweight substance. A picture of packed column given below.
5. Quickly but carefully pour the slurry into the column. Stir and pour immediately to maximize the amount of silica that goes into the column instead of remaining behind in the beaker. You may find a clean spatula or glass rod helpful in transferring the silica.
6. Remove the pinch clamp to allow solvent to drip into a clean flask. Tap on the side of the column with a rubber stopper or tubing to help the silica settle uniformly.
7. Use a Pasteur pipet to rinse any silica that is sticking to the sides of the column. Allow the silica to settle while eluent continues to drip into the flask.
8. Once the silica has settled, carefully add sand to the top of the column. Sand is heavier than silica. If the silica has not settled, the sand may sink into the silica instead of forming a layer on top of it. (You may need to rinse down sand that sticks to the side of the column.
   The Loading a sample onto the column:
9. Drain eluent from the column until no solvent remains above the surface of the sand.
10. Using a long Pasteur pipet, carefully add your sample to the column.
11. Drain eluent from the column until no sample remains above the surface of the sand.
12. Use ~ 1 mL of eluent to rinse your container and pipet. Add this milliliter of sample to the sand. Drain eluent from the column until no liquid remains above the surface of the sand.
13. Repeat step 12 two or three times to completely transfer your sample onto the silica gel. If you do not do and repeat step 12, your sample will remain in the sand instead of on the silica. Sample remaining in the sand will dissolve in the eluent that you add in step 14, ruining the possibility of good separation of components.
    Eluting the sample
14. Once you have rinsed your sample onto the silica, carefully add eluent to the top of the column. To avoid disturbing the top of the column, it's a good idea to carefully pipet an inch or two of solvent onto the column instead of pouring solvent directly onto the sand.
15. Add more eluent as necessary. The eluent collected prior to the elution of sample can be recycled. The composition of the eluent can be changed as the column progresses. If the eluent composition is to be changed, ALWAYS start with least polar solvent/mixture and change to the more polar solvent/mixture.
    Analyzing the fractions
16. Analyze the fractions by thin-layer chromatography to determine a) if the fraction contains more than one component and b) if fractions can be combined without affecting the purity of those fractions.

Other Tips

The success of your separation will be dependant on how well you pack and load the column. It is important to have level sand and silica. It is also important to carefully and evenly add your sample to the packed column.

• Do not allow the silica to dry out as the column progresses. Cracks will form within the silica column if it dries, and compounds can fall down the cracks instead of partitioning between mobile and stationary phases.
• Compounds pass through sand quickly and do not stick to it. Sand is used at the bottom of the column to help ensure a level silica gel line. The bottom of the column is typically cone shaped. If no sand were present at the bottom of the column, molecules traveling down the center of the column would encounter less silica gel than molecules traveling down the edge, closer to the glass. As a result, a particular component would elute as a broader band which is undesirable.
• Sand is used at the top of the column to aid even loading of the sample. Sample diffuses evenly through the sand. Once the pinch clamp is removed from the bottom of the column, sample loads evenly onto the silica. Without sand, the sample would be added directly to the silica and would stick wherever it is added, not evenly across the surface of the silica.

Technical Specification