Results

Part A Fractionation of amino acids

Table 1

Fraction No. Response to Ninhydrin test Response to Sakaguchi test

1 Clear solution Pale orange

2 Dark Purple solution Pale pink

3 Purple solution Clear solution

4 Purple solution Pale yellow

5 Light purple Pale yellow

6 Light purple Pink

7 Light purple Light pink

Questions and Discussion

Part A

1. Which of the 2 amino acids in part A is eluted first and which last? Comment on the
reason for the observed order of elution.

This experiment is about the separation of amino acids on a cation exchange resin. The
isoelectric point (pI) of arginine is approximately 11.15 and it is polar compared to the isoelectric
point (pI) of glycine which is approximately 5.97 and non polar. (Amino Acid Properties)

In fact, when pH of the buffer is equal to the proteins pI (pH=pI), the protein carries no net
charge. At pH below the proteins pI (pH<pI), the protein carries a net positive charge. But the
protein will carry a net negative charge if buffer pH is above the proteins pI (pH>pI).
(Cation Exchange Chromatography, 2017)

In this case, the cation exchanger is able to attract positively charged particles. The positive
charged protein ion will bind to the negative charged resin because the buffer pH (5.25) is below
the proteins pI. Thus, proteins will be eluted in an order depends on their net surface charge and
increasing polarity. Because of that, non polar glycine eluted first due to its isoelectric point
which closer to the buffer pH. While arginine eluted last because it is polar and binds strongly to
the negative charged resin.

2. What are the specific structures on the amino acids with which the Sakaguchi test is
associated?

Arginine can be considered to be a basic amino acid as the part of the side chain nearest to the
backbone is long, carbon-containing and hydrophobic, whereas the end of the side chain is a
complex guanidinium group. With a pKa of 12.48, the guanidinium group is positively charged
in neutral, acidic and even most basic environments. (PubChem) Sakaguchi test is specific for
arginine depends on the results of a pink or red colour when guanidine derivatives are treated
with -Naphthol and alkaline hypobromite or hypochlorite.The darker the red colour, the higher
the concentration of arginine. (Baker)
3. Write down structures which show the predominant ionic form of lysine at pH 1.0, 6.0,
9.5 and 11.0.

pH 1.0 pH 6.0 pH 9.5 pH 11.0

Conclusion

In response to the Ninhydrin test, the solution turns a blue or purple color if there is presence of
amino acid. The darker the colour of solution, the higher the concentration of amino acid.

In response to the Sakaguchi test, the solution turns pink or red colour if arginine present. The
darker the colour of solution, the higher the concentration of arginine.

References

Amino Acid Properties. (n.d.). Retrieved February 7, 2017, from Think peptides:
http://www.thinkpeptides.com/aminoacidproperties.html

Baker, J. R. (n.d.). The Histochemical Recognition of Certain Guanidine. Retrieved February 7, 2017, from
http://jcs.biologists.org/content/joces/s3-88/1/115.full.pdf

Cation Exchange Chromatography. ( 2017). Retrieved February 7, 2017, from Bio Rad: http://www.bio-
rad.com/en-my/applications-technologies/cation-exchange-chromatography

PubChem. (n.d.). L-arginine. Retrieved February 7, 2017, from Open Chemistry Database:
https://pubchem.ncbi.nlm.nih.gov/compound/L-arginine#section=Top