Amino Acid Analysis Part 1
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.