No abstract

Some polar analytes (X) can reversibly form hydrates in water-containing eluents under the conditions of reversed-phase HPLC analysis, X + H2O ⇄ X × H2O. One of the methods to detect their formation is the recurrent approximation of the net retention times of such analytes, tR(C + ΔC) = atR(C) + b, where ΔC = const is the constant step in the variation of the organic modifier content of an eluent. These dependencies are linear if hydrates are not formed, but in the case of hydrate formation, they deviate from linearity under high water content. It has been shown that UV spectroscopic parameters, namely, relative optical densities: Arel = A(λ1)/A(λ2), depend on eluent composition for some organic compounds, but their variations cannot be used as indicators for hydrate formation. The coefficients that characterize the dependence of the analyte retention indices on the organic component concentration of an eluent, dRI/dC, appeared to be the most informative additional criterion for hydration. The values of these coefficients for most polar analytes are largely negative (dRI/dC < 0), whereas, for nonpolar compounds, they are largely positive (dRI/dC > 0).

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.

The confirmation of the correspondence of the detected forms of analytes to their molecular structures seems to be one of the principal problems of analytical chemistry. The previous analysis of the series of drugs by reversed-phase HPLC allowed revealing that the retention regularities of the complex polyfunctional organic compounds containing sulfonamide groups -SO2-N< differ from those of other compounds. It was explained by reversible formation of their hydrate forms. To verify this proposition, three model N-substituted arylsulfon­ami­des were synthesized, namely N,N-diethyl- (I), N-allyl- (II), and N-phenyl (III). The consideration of the dependencies of these analytes net retention times (tR) in reverse-phase HPLC on the content of orga­nic solvent (C, methanol) in an eluent did not permit us to reveal any regularities or anomalies due to the non-linearity of such depen­den­cies. However, to achieve this, the recurrent appro­ximation was re­com­mended, tR(C + DC) = atR(C ) + b (*), where DC = const – con­stant varia­tions of concentration of methanol (5% in our case), а and b – coefficients calculated by Least Squares Method. If the chemical origin of the analytes at the outlet of the chromatographic column remained the same within concentration range Сmin < С < Сmax, dependencies (*) were linear with correlation coefficients R > 0.999. Yet, if additional chemical transformations of analytes took place, na­me­ly rever­sible formation of hydrates (or variations of their composition) due to the presence of water in an eluent, it led to the deviations of recurrent dependencies (*) from linea­rity. Three sulfonamides under characterization corresponded to different kinds of such deviations: non-linearity within the whole range Сmin < С < Сmax (amide I), presence of two linear dependencies instead of one (amide II), and linearity within the whole range Сmin < С < Сmax (amide III). First two cases corres­ponded to the interconver­si­on of anhydrous forms and hydrates, or (less probable) different hydrates, while the latter meant the existence of analyte in the single form (pro­ba­b­ly hydrate). Thus, the ana­ly­sis of mo­del compounds confirmed that the presence of the polar functionality -SO2-N< was just the reason of hydrate formation in water soluti­ons.