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Chromatograhy

Select a suitable glass column and plug the outlet with cotton and sand. Make a slurry of 60-75 g of silica gel and pour it into the column. Equilibrate the column with chloroform mobile phase. Dissolve the 15 g sample in methanol and silica gel and apply it to the column. Elute the sample using a step gradient of chloroform and methanol mixtures of varying ratios, collecting 10-15 ml fractions. Analyze the fractions by TLC to determine separation.

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12 views1 page

Chromatograhy

Select a suitable glass column and plug the outlet with cotton and sand. Make a slurry of 60-75 g of silica gel and pour it into the column. Equilibrate the column with chloroform mobile phase. Dissolve the 15 g sample in methanol and silica gel and apply it to the column. Elute the sample using a step gradient of chloroform and methanol mixtures of varying ratios, collecting 10-15 ml fractions. Analyze the fractions by TLC to determine separation.

Uploaded by

rohankarawita
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Procedure for Silica gel Column Chromatography

Select a suitable glass column [Since our sample amount is 15 g, 60x4.5 cm column will be
appropriated].

Generally 100-500 g of silica gel is used for 1 g of crude sample. However our sample (no
18) is fractionated sample with dichloromethane less amount of silica gel can be used (about
60-75 g/sample).

Plug the column outlet with cotton and sand.

Make a slurry in a beaker and settle down (avoid from air) then pour in to the column step by
step

Equilibrium with initial mobile phase (chloroform) until it gets a uniform appearance.

After equilibration keep the solvent level just above the stationary phase.

Dissolve the sample (15 g) with minimum amount of methanol and some silica gel and make
absorb sample in to it, then apply to the column not disturbing stationary phase.

Open the outlet valve and allow the sample to flow in to the bed. Then wash side of the
column with little amount of mobile phase and allow to flow. Repeat the process until sample
absorb in to the bed.

Apply initial mobile phase thereafter different mobile phases (step gradient sequences
( Mobile Phase; Chloroform: Methanol ratio as follows, 100:0, 100:1, 90:1, 80:1, 70:1, 60:1,
50:1) ) and collect 10-15 ml by automated sample fraction collector.

After collection of fractions develop TLC for determination of separation for each.

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