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Experiment 11: Isolation and Characterization of Casein from Milk
Adapted from Experiment 21, “Isolation of Protein, Carbohydrate and Fat from Milk”, in
Mohr. S.C., Griffin, S.F., and Gensler, W.J. Laboratory Manual for Fundamentals of
Organic and Biological Chemistry by John McMurry and Mary E. Castellion,: nglewood
Cliffs, Prentice-Hall, 1994 and Wayne P. Anderson (4/2002)
You may recallthe Mother Goose nursery rhyme, “Little Miss Muffet sat on a
tuffet, Eating of curds and whey….” When milk is acidified, it is transformed into a solid
component, called curd, and a liquid componentcalled whey. The curds contain the
butterfat and a protein called casein. The carbohydrate, lactose, is present in the whey. In
this experiment you will isolate casein from milk and carry out somequalitative tests for
protein.
The Biuret Test is a general test for protein. When the pale blue Cu2+ ion forms a
complex with adjacent amide nitrogens of the peptide backbone, a very deep violetblue
color forms.
H
2
H
R
C
C
N
N
C
O
O
H
H
R H
Cu
N
N
CH
C O
C O
++
N
N
C
C
C
O
O
H
H
R H
OHCu2
+
The Xantoperoteic Acid Test on the other is a general test for thepresence of the
aromatic amino acids, tryptophan, phenylalanine and tyrosine, in proteins. Aromatic
groups that have an amino group (tryptophan) or a hydroxyl group (tyrosine) are easily
nitrate byconcentrated nitric acid to form yellow (xantho, Greek for yellow) colored
aromatic nitro compounds, thus the term xanthoproteic acids.
Procedure:
Determine the mass of a 125 mL Erlenmeyer flask.Add 50 mL of no-fat milk to
the flask and re-weigh the flask to determine the mass of the milk. Check the label on the
milk container and record the amount of protein per serving in your notebook.Prepare a
water bath by placing 200 mL of water in a 600 mL beaker. Heat the water bath to 40oC;
as the temperature is critical for this experiment monitor the temperature with a
thermometer....
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