Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Amino Acid Column Chromatography
Columns and Buffers

Sulfonated polystyrene cross-linked with divinylbenzene was first used by Moore and Stein for the Ion-exchange Chromatography of Amino Acids [263–267, 348] and remains widely applied in laboratory practice today [347].

At low pH, amino acids exist in a cationic form and bind to the negatively charged sulfo groups of the stationary phase. As the pH of the eluting buffer (typically sodium citrate) increases, the positive charges on the amino acids decrease, weakening their interaction with the resin and leading to their sequential elution from the Column. The elution order of amino acids is dictated by the pH of the elution buffer. However, Na⁺ cations in the buffer compete for binding sites at the sulfo groups; although Amino acids have a higher affinity for the resin, they are progressively displaced by sodium ions due to the much higher concentration of the latter in the medium. The Nature of the side chain also affects the elution order—for example, Tyrosine (pKa = 9.1), which contains an aromatic ring, elutes after leucine (pKa = 9.7). In the two-column method, the first column (0.9 × 120 cm) packed with Amberlite IR-120 is used to separate acidic and neutral amino acids using two eluents: 1) 0.2 M Na⁺ + 0.1 M citrate buffer (pH 3.25); 2) 0.2 M Na⁺ + 0.1 M citrate buffer (pH 2.25). The second column (0.9 × 15 cm) is employed for the analysis of basic amino acids, with an eluent composition of 0.35 M Na⁺ + 0.12 M citrate (pH 5.28). These results are critically discussed in [357].

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FIG. 8.3. Two-dimensional Thin-Layer Chromatography of Amino Acids on Cellulose plates. Solvent compositions are given in the text.

1 — Ala, 2 — Arg, 3 — Asp, 4 — Asn, 5 — Cys, 6 — Cys-Cys, 7 — Cya, 8 — Glu, 9 — Gln, 10 — Gly, 11 — His, 12 — Hyp, 13 — Ile, 14 — Leu, 15 — Lys, 16 — Met, 17 — Nle, 18 — Phe, 19 — Pro, 20 — Ser, 21 — Thr, 22 — Trp, 23 — Tyr, 24 — Val [147] (reprinted with permission of the authors).

Implementing a two-column system is technically complex; moreover, it fails to meet modern requirements because it doubles material consumption and correspondingly increases the probability of errors.

Certain difficulties in quantitative evaluation arise when there are large differences in the concentrations of mixture components, causing some amino acid peaks to fall within the measurement scale while others exceed it and cannot be accurately integrated. In such cases, the analysis is repeated with a smaller Sample size, and the final results are derived from the sum of both determinations. This problem can be resolved by recording the elution curve simultaneously at high and low detector sensitivities, or by applying a linear-logarithmic transformation of the electrical signal to a chart recorder or integrator, which allows the areas of all peaks to be determined.

Numerous buffer systems have been developed for the complete Separation of all amino acids in a single run on a single column. These programs typically incorporate three or even four discrete buffer system changes. Although straightforward to set up and control, switching buffers invariably causes baseline drift resulting from the chromatographic shift of impurity peaks and minor Changes in the refractive indices of the solutions [10].

Continuous buffer gradients [297] are less popular due to the difficulty of achieving exact reproducibility; however, with the advent of modern HPLC technology, the challenges associated with forming continuous buffer gradients and precisely delivering small volumes of solutions to the column have been largely overcome. A system utilizing three Buffer solutions at pH 3.25, 3.50, and 3.65 with increasing sodium ion concentrations (0.02, 0.70, and 1.60 mol/L) was described in 1972 [18]. Under these conditions, complications may only arise from the Swelling or shrinking of the column bed when the eluent molarity changes. Concurrently, a four-buffer system with a constant Na⁺ content (0.20 mol/L) at pH 3.25, 4.15, 5.25, and 10.10 was reported [141].

Operating at the highest possible sensitivity requires the absence of Background noise on the chart recorder, which is achieved by using ultra-pure Solvents and Reagents (Section 8.3).

Buffer purity test. To prepare the sodium citrate buffer, dissolve 20 mg of Na₃C₆H₅O₇·2H₂O in 1 mL of Water (Na⁺ concentration ~0.2 M) and add 40 µL of "ultrapure" 1 M HCl to adjust the pH to 2.0. Inject 100 µL of this solution into the ion-exchange column and compare the resulting chromatogram with control runs of water and Hydrochloric acid [142].

To optimize separation conditions, one can adjust the pH, molarity, buffer flow rate, and column Temperature. Buffers must be prepared using high-purity reagents and water. pH values should be set with an accuracy of 0.001 units, and buffer solutions ought to be stored under nitrogen. Typically, ~2% (w/w) isopropyl or ethyl alcohol is added to the first buffer (pH 3.25) to improve the separation of Thr and Ser. Commercial instruments are supplied with comprehensive operating manuals, but every analyst should, of course, fine-tune the analytical program through trial and error.

Figures 8.4 and 8.5 show amino acid elution curves obtained using a Beckman analyzer.

Note to Fig. 8.4: baseline elevation occurs upon introducing a new buffer onto the column after 42 and 56 min. Figure 8.5 illustrates the high-sensitivity separation of a glycoprotein hydrolysate containing glucosamine and galactosamine (indicated). Reviews on Amino acid analysis can be found in [19, 20, 28, 138, 139, 142, 300].

To improve the resolution of complex multi-component mixtures of ninhydrin-positive compounds in plasma and other biological fluids, The Use of lithium citrate buffers in ion-exchange chromatography has been proposed [264]. Single-column chromatography has been applied to separate 65 compounds in 20 hours [296]. A two-column system successfully resolved 41 compounds in 7.5 hours [18] and 55 compounds in 9.5 hours [270].