CHEM 3224
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SEPARATION AND PURIFICATION OF PRODUCT BY CHROMATOGRAPHY
CHROMATOGRAPHY IN GENERAL
Chromatography is defined as the separation of a mixture of two or more compounds by
distribution between two phases, one of which is stationary and one of which is moving.
Chromatography is so called because it was originally used to separate substances of different
colors. Various types of chromatography are possible depending on the nature of the two
phases involved: solid-liquid, liquid-liquid, and gas-liquid chromatographic methods are
commonly available.
TLC and column chromatography are often used in conjunction with each other. It is often
convenient to investigate a suitable solvent system for the best mixture separation by TLC,
since TLC is much quicker than running a column. The solvent system may then be used to
elute the column and should give approximately the same types of separation, if the adsorbents
are of similar activity. Both solid-liquid methods have great usefulness to the organic chemist.
Both methods rely on a stationary solid phase and a mobile liquid phase. The solid phase is
usually either silica gel (SiO2 x H2O) or alumina (Al2O3 x H2O).
Some TLC Developing solvents
INCREASING
POLARITY
Alkanes (hexane,
cyclohexane, pet ether)
Carbon tetrachloride
Toluene
Dichloromethane
Chloroform
Ethyl Acetate
Isopropyl alcohol
Acetone
Ethanol
Water
Approximate Polarity of Organic Compounds
INCREASING
POLARITY
Alkanes
Alkenes
Conjugated dienes
Aromatic hydrocarbons
Ethers, Halocarbons
Aldehydes, Ketones, Esters
Amines
Alcohols
Carboxylic acids
DRY COLUMN “FLASH” CHROMATOGRAPHY
The technique as described below combines the advantages of standard flash chromatography
(good separation, speed) with the cheapness of material and simplicity of apparatus and
operation. The distinctive feature of this method is the use of suction as opposed to positive
pressure, simplifying loading of the product mixture and subsequent eluting solvents. A second
feature is that the eluting solvents are added in predetermined volumes, allowing the column to
run dry before adding the next fraction.
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SEPARATION AND PURIFICATION OF PRODUCT BY CHROMATOGRAPHY
The apparatus is set up as for vacuum filtration using a porosity 3 cylindrical sinter. Guidelines
for sinter sizes, weight of silica and ranges of sample weight are shown below:
Sinter Size (mm)
Diameter/Height
30/45
40/50
70/55
Wt. Of
Silica
(g)
15
30
100
Sample
Wt.
15-500mg
500mg-3g
2-15g
Solvent
Fraction
volume (ml)
10-15
15-30
20-50
Use of column lengths appreciably longer than 55mm are not practical, nor are they necessary.
Some lowering of efficiency may be noticeable with the large scale set-up.
Method for performing vacuum flash chromatography
Fill the sinter to the lip with silica and then apply suction, pressing down the silica with extra care
at the circumference. Level the surface, pressing down all the time and finally tap the sides of
the sinter firmly to obtain a totally level surface. The compacting of the silica thus provides a
head space for the addition of product and solvents. Continuing suction, pre-elute the column
with the least polar combination of the required solvents in which the product mixture is readily
soluble. If possible, use pure least polar component for this (e.g., for pentane/ether gradient
elution, pre-elute with pentane). If the silica has been packed correctly, the solvent front will be
seen descending in a horizontal line. If channeling is apparent, suck the column dry and repeat
the packing procedure. Keep the surface of the silica covered with solvent during the pre-elution
until solvent is seen passing into the receiver, and then allow the silica to dry under suction.
Load the mixture, dissolved in the minimum amount of the pre-elution system, evenly onto the
surface of the silica and elute the products by adding successive portions of increasing polarity
solvent mixtures, allowing the column to be sucked dry after each addition (the silica surface is
particularly stable and is only slightly disturbed on addition of solvent). Generally a solvent
gradient whereby the more polar component is increased by 5-10% is the most convenient.
Under these conditions, the product is usually eluted by that solvent mixture which would cause
it to have an Rf = 0.5 on TLC. In addition, for quantities >100 mg elution of product is often
accompanied by frothing on the underside of the sinter.
After a little experience, separations of the same efficiency as TLC are easily possible. There is
minimal material loss and the technique is economical in both time and solvents. During
prolonged chromatography with volatile solvents, condensation of atmospheric moisture may
occur on the apparatus. This does not affect the efficiency of the separation but may be
diminished by substituting less volatile solvents (e.g., hexane for pentane). No solvent is
particularly disfavored for this technique, but combinations of pentane (hexane), ether, ethyl
acetate and methanol are adequate for the vast majority of separation. The low diffusion of
product bands during chromatography usually means that each component is eluted in one or
two fractions with consequently minimal loss of product in cross contaminated fractions.
(Flash chromatography from February, 1982, L.M. Harwood, University of Manchester)
CHEM 3224
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SEPARATION AND PURIFICATION OF PRODUCT BY CHROMATOGRAPHY
PROCEDURE
Practical Information
You will need a pencil, a calculator and a ruler for this experiment, in addition to your
laboratory notebook, pen and safety glasses.
In this experiment, you will be utilizing commercially prepared TLC plates. These thin plates are
in the form of large square sheets, which you will need to carefully cut to the appropriate size.
Visualization of your compounds on these plates can be accomplished via a UV lamp, and is to
be done only under the strict supervision of the instructor or the teaching assistant, due to the
potential for eye injury from improper use of the UV lamp.
Application of the compounds to the TLC plates will be accomplished using commercially
available microcapillary pipettes. The student will not be required to prepare his/her own
pipettes.
Experiment:
THIS IS THE ONLY PART THAT YOU
WILL BE PERFROMING: OBJECTIVE IS TO CHOOSE
THE BEST SOLVENT OR SOLVENT MIXTURE THAT
WOULD BE USED TO DEVELOP A LARGE SAMPLE
OF A MIXTURE OF N,N’-diethl-m-toluamide AND
META- TOLUIC ACID.
1. Prepare enough TLC plates and developing jars to develop N,N’-diethl-m-toluamide and
m-toluic acid in each of the following four solvents (i.e., there will be four slides, each
slide having been spotted with separate spots of the two compounds and those four
slides developed in each of the four solvents):
a.
b.
c.
d.
Petroleum ether (pet ether)
Ethyl acetate
1:3 mixture of pet ether to ethyl acetate
3:1 mixture of pet ether to ethyl acetate
2. Develop the TLC plates
3. Using a pencil, mark the final areas of the compounds on the developed TLC plates
4. From the areas marked in step 3, calculate Rf values for each compound in each of the
solvents
5. Using a mixture of the two compounds, develop the mixture on the following two
solvents (i.e., there will be two slides – one for each compound, each slide having been
spotted with the mixture):
a. 1:3 mixture of pet ether to ethyl acetate
b. 3:1 mixture of pet ether to ethyl acetate
6. Calculate the Rf values for each compound on each of the two slides.
CHEM 3224
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SEPARATION AND PURIFICATION OF PRODUCT BY CHROMATOGRAPHY
7. Based on the results from steps 5 and 6, determine which solvent mixture would
provide the best separation of the two compounds in a larger scale vacuum flash
chromatography experiment.
NOTE: Include reasons for your determination of solvent mixture (step 6) in your
notebook.
NOTE: Include TLC plates and/or sketches of TLC results in your notebook.