Solvents_Reagents_2014_Text - page 36

34
For more information, or to download product instructions,
Developments in Amino Acid Analysis
Improvements in amino acid analysis by ion exchange
chromatography have involved the analytical system, as
well as the instrumentation. Systems have been developed
(by varying buffer pH or ionic strength) that work to displace
the amino acids into discrete bands. The buffer systems are
compatible with single- or two-column analysis of amino
acids found in protein hydrolyzates or physiological fluids.
Buffer systems are determined by the counter ion used
(sodium or lithium) and by the method of buffer changes
introduced to the resin (step changes or gradient elution).
9-15
The most commonly used buffering component, citrate, is
suitable for solutions below pH 7.
16
Buffers are prepared
either with citric acid or an alkali salt, and citrate
concentrations of 0.05 to 0.06 7 M are common.
Unfortunately, for high-sensitivity work, citric acid is a
significant contributor to amino acid contamination.
Therefore, to achieve consistent analyses, it is essential to
use high-purity reagents for buffer preparation.
Alternatives to ion exchange are available for the
separation of amino acids. Because amino acid analysis
is one of the basic protein chemistry tools available, more
rapid and sensitive methods for separation and quantitation
are desirable.
17
Several pre-column derivatization methods
using reverse-phase HPLC have been developed.
Two of the most widely used of these methods involve the
formation of dansyl
18-19
or
o
-phthalaldehyde (OPA)
20-24
derivatives of amino acids prior to HPLC analysis. Both
methods offer greater sensitivity and shorter analysis time
than post-column derivatization techniques. Other methods
include the quantitative derivatization of amino acids with
phenylisothiocyanate (PITC) and the separation and
quantitation of the resulting phenylthiocarbonyl derivatives
via HPLC. These derivatives are stable enough to eliminate
in-line derivatization.
ProPac, DNAPac, ProSwift
columns
Industry leading columns
High efficiency and
resolution
Excellent reproducibility
Ultimate
Bio HPLC performance
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