Controlling the correctness of retention parameters variations in reversed phase HPLC using recurrent relations

Abstract

Recurrent approximation of analytes’ net retention times (tR) in reversed phase high performance liquid chromatography (RP HPLC) at different contents of organic constitu­ent in the eluent (C) is recommended as a method of revealing the reversible hydrate for­ma­tion. The criterion of that are the deviations of the dependences tR(С + DС) = atR(С) + b (*) from linearity, where DС is the constant increment of concentration variations; in our case DС = 5%. However, such deviations are rather small and, hence their measuring requires high robustness of the equipment involved. Besides hydrate formation, there are additional reasons for deviations, namely discrepancies between the real and the selected flows of the eluent. Compa­ring tR values obtained for the same analytes using the same chromatogra­phic column at the same conditions, but with different HPLC instruments using the systems methanol – water as the eluent confirms that tR values of one data set are equal only to approx. 76-98% tR values of another data set. Therefore, the eluent flow in the second case exceeds that in the first regime at the same pro­por­tion. The simple method for revealing such flow deviations is proposed. It is based on the recurrent approximation of tR = f(C) data sets for any compounds forming no hydrates in RP HPLC conditions (chlorobenzene was selected). The absence of the influ­en­ce of any distorted factors is confirmed with values of correlation coefficients for re­cur­rent depen­dencies (*) exceeding 0.999.

Evidence for the Hydration of Some Organic Compounds during Reverse-Phase HPLC Analysis

“…If the HPLC pump(s) does not provide the fixed eluent flow rate under increasing eluent viscosity, this may lead to unpredictable distortions of the t R -values. This is why the use of one of the available HPLC instruments to us was rejected [ 14 ].…”