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Ion-Exchange Analysis of Amino Acids
Ion-exchange chromatography followed by post-column
derivatization has been the method of choice for amino acid analysis
since S. Moore and W.H. Stein published it in 1948-- work which
merited a Nobel prize. Now it is possible to apply either sodium- or
lithium-based ion exchange chromatography to obtain consistent
results with sensitivity, stability and speed.
No other techniques, including reversed-phase
chromatography, have been shown to match post-column ion-exchange
methods in quality and reproducibility. This is because the
retention mechanism in ion exchange provides for chromatography that
is almost completely matrix-insensitive. Simplified sample
preparation for native samples is an added benefit of the
ion-exchange method.
The most popular post-column reactions for amino
acid analysis are Ninhydrin and OPA. A detailed discussion is found
in the Chemicals section.
Although pre-column derivatization followed by
reversed-phase chromatography is popular with some HPLC users, its
successful application is limited to protein hydrolysates. Without
extensive pretreatment, the carbohydrates and organic acids in wine,
the fats in tobacco, and the macromolecules in human serum will
cause the amino acids to elute at a different time for each unique
matrix. Moreover, as yet no combination of pre-column chemistry and
reversed-phase chromatography has been able to resolve the full
complement of amino acids found in typical native samples such as
serum or urine.
In the reversed-phase environment all species in
solution compete simultaneously for derivatizing reagent. The yield
of any particular species will be influenced by the concentration of
all other solutes present.
In contrast, ion-exchange chromatography followed by
post-column derivatization is intrinsically more rugged and
repeatable than pre-column analysis. Since chromatography and
derivatization represent two separate and distinct events, each can
be optimized independently.
The retention mechanism in ion-exchange
chromatography is almost completely matrix-insensitive. Since the
sulfonated divinylbenzene polymer comprising the stationary phase
has a high ion-exchange capacity, the positively charged amino acids
exhibit a strong affinity for these fixed negative sites.
Consequently, all amino acids, except for the very acidic, are fixed
in a narrow band in the guard column, while the sample matrix moves
on. Thus the same program used by one laboratory for the analysis of
wine can be employed by another to analyze human urine.
The most important difference between ion-exchange
resins and reversed-phase silicas is in chemical selectivity. Bonded
reversed-phase silicas are manufactured to exhibit monotypical
chromatographic behavior, e.g., partitioning. In contrast
ion-exchange resins are polytypical. Separation is based not only
upon ion-exchange, but also partitioning, adsorption, size ion
exclusion, and more. Because of the involvement of so many retention
mechanisms, a single change in any operational parameter&emdash;cation
concentration, pH, flow rate, column temperature-- can result in
multiple changes in peak position. This flexibility is particularly
advantageous, for example, when developing a method to optimize the
separation of a few amino acids of interest at the expense of the
others, as in the PKU and other rapid-screens.
There are two main mechanisms for effecting
separation in ion-exchange chromatography. One is titration, or
increasing the pH. As pH increases, the amino acids go from a
positive to a neutral charge state, and so are released from the
fixed anionic sites in the resin (stationary phase).
The other mechanism is competitive ion exchange. The
positive amino acids show a higher affinity for the ion-exchange
sites than do the eluting cations (H+, Li+ or Na+) and so are bound
strongly to the anionic sites. They are eluted by the continuous
flow of cation or by increasing cation concentration developed by
gradient formation.
The earliest protocols for ion-exchange of amino
acids were based on step gradients-- isocratic procedures that step
through a set of 3-6 solutions of varying pH and cation
concentration. Both titration and elution adjustments are made with
each eluant step. This technique persists today in the design of
dedicated amino acid analyzers, such as the Beckman System 6300.
The modern liquid chromatographic (HPLC) techniques
for ion exchange use three solutions and continuous gradients.
Gradient capability allows for easier methods development and
results in flatter baselines-- especially important at high
sensitivity.
As with dedicated analyzers, the eluants are either
sodium- or lithium-based. The need for two cation systems derives
from the two fundamental types of samples encountered in amino acid
analysis: hydrolysed hydrolyzed and non-hydrolyzed ("native")
samples. Hydrolyzing the sample results in a smaller array of amines
(typically up to 21) than is present in native samples (typically
40-60). This wider range of amines in native samples requires a more
discriminating or weaker eluant to fully resolve them. A lithium
ion-based eluant provides the necessary resolution. If the sample is
hydrolyzed, a sodium system can be used for a shorter run time.
Both Na+ and Li+ eluants for gradient HPLC are
designed in the same way. The first, low-pH eluant is a buffer with
low cation concentration. The second eluant has no buffering
capacity and a high cation concentration. The third, high-pH eluant
(usually referred to as the column regenerant) is based on the
cation hydroxide and is of comparable cation concentration to the
first eluant. These solutions allow for titration relatively
independent of changes in eluant strength, or allow for increasing
eluant strength.
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