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33
The History
The development of amino acid analysis began in 1820
when Braconnot isolated glycine from a hydrolyzate of
gelatin.
1
Later, in 1848, the Dutch chemist Mulder showed
that glycine contains nitrogen, a major component of
amino acids.
2
It was not until 1883, however, that Kjeldahl
introduced a method that accurately determined the
amount of nitrogen in a protein/amino acid sample.
3
By 1910, most of the amino acids had been isolated and
their structures discovered. As the number of known
amino acids accumulated, it became possible to group
them on the basis of common chemical features. At that
time, it was discovered that all amino acids have the same
general formula and differ only by the chemical structure
of the side chains.
H
C
NH
2
R
COOH
From 1910-1940, amino acid research was characterized by
the work of quantitative analysts, as opposed to the organic
chemists of the 1800s. Amino acid analyses conducted
during the 1800s and early 1900s were laborious, often
extending over weeks and months. While the amino acid
content of a number of proteins was discovered, exact
information was not always obtainable using the
equipment available during that time period.
4
The introduction of chromatography opened new doors for
amino acid analysis. The first breakthrough came when
Martin and Synge introduced partition chromatography,
which separates the acetyl derivatives of certain amino
acids.
5
In this method, an equilibrium is established between
two liquid phases. Silica gel is mixed with a solution of
water and an indicator. The resulting slurry is packed into
a column, forming the stationary phase. Next, the acetyl
amino acids are dissolved in solvent, forming the mobile
phase. The acids then are placed in the same column.
The acetyl amino acids flow through the column at
different rates. Separation is made visible by the bands of
color change in the indicator.
While this system successfully separated mono-amino and
mono-carboxylic acids, it was impractical for other types of
amino acids.
Later, Martin and his associates used filter paper as an
alternative to silica gel, developing a paper chromatography
method that is still in use today. The amino acids were
dissolved in butanol and allowed to seep onto the filter
paper for a set amount of time. The paper then was dried
and sprayed with a dilute solution of ninhydrin (2,2-dihy-
droxy-1,3-indandione) in butanol. The colored spots were
measured and compared with the set values for those
experimental conditions.
The separations achieved with paper chromatography were
only semi-quantitative. Column chromatography, on the
other hand, had potential for quantitation, but the
separations were imperfect. The introduction of ion
exchange chromatography solved these problems,
allowing column separation of amino acids without any
prior derivatization.
6,7
Initially used to remove carbohydrate
contamination from starch columns, ion exchange resins
were quickly found to have great potential for separating
amino acids. While many types of polymeric exchange
resins were tested, polysulfonic resins (such as Dowex
®
50)
provided the best separations.
8
Modifications to these procedures have improved amino
acid separations. Resin characteristics, column size,
column temperature, buffer pH and ionic strength all have
been modified to improve resolution of amino acid mixtures
and achieve specific separations. Also, quantitation was
greatly improved by the use of post-column reactions with
ninhydrin. At one time ninhydrin was the most widely used
detection system; however, more sensitive indicators, such
as
o
-phthalaldehyde were developed to increase analytical
sensitivity.
Introduction to
Amino Acid Analysis
