Name:_____________ Date Submitted:_____

Course and Year:____ Date Performed:_____

Lab Activity # 5
Chromatographic Separation

Discussion:

Chromatography is an established technique for the separation of mixture and purification of

compounds . In absorption chromatography, a solution of a material is ran unto a column od
adsorbent and the solvent is then allowed to percolate slowly through the solution. The materials
which are the least firmly adsorbed on the column are eluted first.

Partition chromatography is, in a sense , like extraction, but one of the solvents is held adsorbed
in a solid (silica gel) with adsorbed water is an example). The mixture to be purified dissolved in a
second solvent, is placed in the column, and this latter solvent is then used for elution. The
compounds of the eluting solvent according their partition coefficient , much as in extraction , but the
solutes are, in effect , subjected to an infinite number of partitions as they pass down the column. The
procedure is therefore highly efficient.

Paper chromatography is a form of partition chromatography especially adapted to separating

ultra quantities of materials. The cellulose of the filter paper containing adsorbed water which is the
stationary phase. The sample is applied as a spot to the filter paper and the second solvent is allowed to
pass through the filter paper by capillary ; as it does so, the components of the sample will move in
rates through the filter paper. The rates being proportional to the partition coefficients between the
adsorbed water and the particles used for elution

Reagents and Materials:

Distilled water 3 – petri dishes

Isopropyl alcohol filter paper
3- capillary tubes

Procedure:

Hold a piece of Whatman no 1, 12.5 cm filter paper I half and then another half , in order to find
the center. Using this sheet as guide , punch a whole through two other sheets of similar filter paper
using a pin. The center hole should be bout 1/6 inch in diameter. Prepare a filter paper wick by tearing
s sheet of filter paper in strip about ½ by ¼ inches then roll tightly to form a wick ½ inch long and of
such diameter to fit snugly into the center holes prepared above. Partially fill the large half of the petri
dish with normal propyl alcohol and distilled water 2:1 by volume (about 30 ml per dish).
 Using melting point tubes open at both ends as pipets, place adrop of yellow food coloring at the
center of one sheet and a drop of blue food coloring at the center of another sheet. The dye spot
should be about the size of the a dime. When the spots are dry, insert the wicks so that they extend
about 1/8 inch on one side, and 3/8 inch on the other side, and the rest of the sheets of filter paper on
the rims of the petri dishes with the wicks extending into the solvent. Cover the paper with the petri
dish. Cover and allow the chromatograms to develop for 10 minutes. Carefully remove the wick and
allow the chromatograms to dry. Repeat the same procedure using the green dye instead.

Compounds separated by paper chromatography maybe eluted from the paper and used I
separate experiments. Separate the compounds on chromatogram of the green food coloring and
crumple each color into small ball and place in separate tubes. Add 2 ml of water to each and shake.
Note the yellow and blue dyes have separated have separated and eluted from the paper. Mix two
liquids and note what is obtained.

Questions:

1. Did the solvent moves approximately the same distance in each case?

2. Did the dyes move equal distances?

3. Did the yellow dyes appear to be homogeneous? What about the blue dye?

4. If either was not homogeneous mixture , how many separate dyes were present and what were
their colors?

5. Which dye move the farthest?

6. Is the green food coloring a single dye?

7. Is it made by mixing other dyes? How do you prove it?

8. Is it the same as the yellow and blue dye?