1. Compare and explain the gradient column chromatography (liquid chromatography

Question

1. Compare and explain the gradient column chromatography (liquid chromatography) and temperature programming (gas chromatography).

2. Generally, the shape of TLC spot seems like a dot but sometimes it gave draggy spot called 'tailing'. Why 'tailing' takes place and how to avoid or minimize it?

3.When doing the liquid column chromatography, elution should be done in 'proper' rate. Why?

4. In 1978, Still reported flash column chromatography as a efficient chromatographic method. Read this article and answer the questions below. (Journal of Organic Chemistry, 1978, Vol. 43, Page 2923-2925) (a) What is a flash column chromatography? What are the advantages of this method? (b) You have 150 mg of sample composed of two reagents and Rf difference of two reagents is 0.15. Although you used a column diameter of 20 mm, you failed to separate two components. Why? Give all possible reasons of this failure.

Explanation / Answer

1. Gradient column chromatography is a technique wherein the polarity of the mobile phase flown throught he column is varied over certain range of time. This technique is different from single polarity mobile phase used in case of isocratic elution. The technique enables a better separation of components of a mixture based upon the polarity. Say for example you have a mixture with low polarity, medium polarity and high polarity components in it. You start with a mobile phase with low polarity as in 100% hexane a non-polar solvent. The first component comes out with this solvent. We increase the polarity to 10% ethylacetae in hexane and pass it through the column. The second components comes out of column. Finally we pour 50% ethylacetate in hexane solvent as mobile phase which allows the third component to come our of the column.

Similarly a temperature gradient column is utilized in gas chromatography based on the principle that different components of the mixture have different boiling points and their affinity for the staionary phase and mobile phase changes according to the temperature of the column in gas chromatography. We start with a low temperature to separate the first component (lowest boiling point), followed by second component eluted when temperature is slightly increased. Likewise the temperature can be varied depending upon the number and type of component present in the mixture.

2. Tailing is a phenomenon which arises due to higher concentration of the compound we have spotted on the tlc plate. To avoid tailing we must reduce the concentration of substance by diluting in proper solvent and then spotting on the tlc plate.

3. In a column chromatography elution is done at a proper rate, which helps separate the components of the mixture between the stationary phase and the mobile phase. If elution is doen at an increased rate, all the components would flow together and no separation would be obtained.

4. (a) Flash chromatography is a technique wherein. fine particle grade of silica is used as stationary phase in column chromatography and an inert gas is used to push mobile phase and mixture through the column. Due to fine grade silica separation is better on this type of column chromatography.

(b) The separation in this may have failed due to chorter length column employed. The components did not get enough time to separate on the column before getting eluted. Another possibility could be the flow rate for mobile phase may have been used would be too high which again did not allow the somponents to separate on the stationary phase properly.

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