MODULE 8

ISOLATION OF CASEIN AND SEPARATION OF

AMINO ACIDS USING PAPER
CHROMATOGRAPHY

Name: Jayson P. Jalbuna Rating: _________________

Date Performed: November 9, 2021 Date Submitted: November 9, 2021

Data and Report Sheet

A. Isolation of Casein
Mass of dry casein plus filter paper
Mass of filter paper
Mass of Casein isolated 25g
% Casein Isolated 25%
Mass of Lactose 10g
% of Lactose Isolated 10%

B. Isolation of Casein Separation of Amino Acids Using Paper Chromatography

Mobile Phase Distance
Samples Rf Value
0.47
Glycine
0.83
Valine
0.24
Aspartic Acid

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 C. Ink Separation Using Paper Chromatography (Add images of your
actual experiment)
Mobile Phase Distance
Samples Rf Value
0.74
HBW 9801 (BLACK)
0.30
PILOT BP-S FINE (RED)
0.85
PANDA CLASSIQUE (BLUE)

Documentation:

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3 Isolation of Casein and Separation of Amino Acids Using Paper Chromatography – Module
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POST LABORATORY QUESTIONS:
1. Why does ninhydrin stain the skin blue? What is the purpose of ninhydrin?
- Our skin contains a Keratin, a type of Protein that can be found on the skin, and
it contains Amino Acids since it is the building blocks of proteins, the specific
amino acids are the Glycine and Phenylalanine. Therefore, Ninhydrin stain the
skin blue because of the presence of Glycine and Phenylalanine which are amino
acids on the skin. The purpose of ninhydrin reagent is to detect the presence of
Amino acids or Amines in many reactions, it can also use to detect fingerprints.

2. What are the certain technical aspects which are important in performing paper
chromatography?
- Partition chromatography or adsorption chromatography are two methods or
technical aspects that can be used. Because the compounds are partitioned or
spread between liquid phases, partition chromatography is used. Water is retained
in the filter paper's pores, and the other phase is a mobile phase that passes through
the paper. The separation of the mixture occurs when the mobile phase moves. The
compounds in the mixture separate themselves based on the differences in their
affinity towards stationary and mobile phase solvents under the capillary action of
pores in the paper. Adsorption chromatography between solid and liquid phases,
wherein the solid surface of the paper is the stationary phase and the liquid phase
is the mobile phase.
3. Why would it be unwise to use an ink pen to mark the reference lines and points
on the chromatography paper?
- Because ink pen reacts to the solvent, if we use it for the reference lines and
points, it will react with the alcohol together with the test samples which can make
the test failed. That is why we use pencil, because it is made of graphite and it
does not react with the alcohol.

4. Suppose while performing Part A, you weigh the casein and filter paper before
they are completely dry. How will this error influence the reported percentage of
casein?
-

5. How can one use the Rf value in predicting the unknown solute? Suppose we have
a known Rf value of 0.17mm and 3unknown values: a) 0.56 mm b) 0.16 mm c)
0.100 mm. Which among the unknown can be compared with the actual identity of
the compound?
- If it is comparable to the level or range of the reference substances, we can use
Rf value for predicting unknown solute. For the three unknown values, b) 0.16 is
the one that can be compared with the actual identity of the compound, due to the
fact that its value is the closest to the Rf value of 0.17mm.

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6. What are the guidelines in making a good and reliable paper chromatogram?
- It is quite important that the paper strip is then placed in a container that contains
an appropriate solvent. The paper strip should be positioned so that only the
bottom edge is covered so that the air inside will be saturated with solvent vapor.
As the solvent moves up the paper strip, the compounds(s) deposited at the origin
dissolves in it to some extent and thus is carried along up on the strip. Lastly,
cover the container so that the solvent will not easily evaporate. To sum up all of
these general rules, it will create a good and reliable paper chromatogram.

7. What are the purpose of Calcium carbonate, charcoal and ethanol in the Isolation
of Casein?
- The purpose of the Calcium carbonate in the isolation of the Casein is to
decompose the lactose or excess acetic acid in the next step of heating the solution,
the purpose of charcoal on the other hand is to decolorized the solution. Lastly, the
purpose of ethanol in isolation of the Casein is to isolate the lactose, because
lactose is soluble in ethanol, and to make the lactose forcefully crystallize out.

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