Effect of the mobile phase on antibody-based enantiomer separations of amino acids in high-performance liquid chromatography

Enantiomer separation of amino acids in immunoaffinity micro LC‐MS

“…The temperature dependence is based on the fact that the interaction between the antibody and D-amino acids is enthalpy driven and, therefore, is stronger at lower temperatures. 33 The interaction of the unretained enantiomer with the antibody, however, is not affected by temperature changes. Thus, the setup shown in Figure 1 may be used to improve separation of poorly resolved peaks by lowering the temperature or, conversely, to shorten the run times of well-resolved peaks by increasing the temperature.…”

“…32,33,40 As phosphate buffers are not compatible with MS-detection, we decided to use a low-ionic strength ammonium bicarbonate buffer as solvent for LC-MS hyphenation; this buffer appeared to be particularly suitable for this purpose since it is not only volatile, but also buffers in the neutral pH range. Using Chirality DOI 10.1002/chir anti-D-AA 67.36 immobilized onto a synthetic high-flowthrough support material, it was recently demonstrated that the affinity between this antibody and D-amino acids is highest at around neutral pH values and that it slightly increases with a decrease in ionic strength.…”

“…Using Chirality DOI 10.1002/chir anti-D-AA 67.36 immobilized onto a synthetic high-flowthrough support material, it was recently demonstrated that the affinity between this antibody and D-amino acids is highest at around neutral pH values and that it slightly increases with a decrease in ionic strength. 33 Furthermore, since organic modifiers were found to cause denaturation of the antibody, 33 no organic solvents were added to the buffer. As seen in Figure 2, the use of a 1 mM ammonium bicarbonate buffer, pH 7.8, as mobile phase enabled enantiomer separation of tryptophan on column A under isocratic conditions at RT.…”

“…41 Binding characteristics of this and other stereoselective anti-amino acid antibodies, and applications in chromatography, immunoassays, and immunosensors have been reported in detail elsewhere. 4,32,33,[43][44][45][46][47][48] The affinity of the antibody is highest for D-amino acids with aromatic or bulky side chains; however, also D-amino acids with aliphatic side chains are stereoselectively recognized.…”

“…32,33 Using antibodies directed against the stereogenic center of a-amino acids as a model system, it was shown that the strength of interaction between the immobilized chiral selector and analytes can be modulated conveniently by varying chromatographic parameters such as flow rate and temperature. Thus, the whole chromatographic separation process can be carried out in an aqueous, physiological buffer that does not impair the structural integrity of the antibody-CSP.…”