Smirnova O. Separation and determination of 1,4-naphthoquinone derivatives by liquid chromatography

The manuscript is devoted to the research of separation and determination of 29 derivatives of 1,4-naphthoquinone reversed phase high-performance liquid chromatography (RP HPLC) and normal phase thin layer chromatography (NP TLC). The effect was investigated for the organic modifier amount in the mobile phase (MP) on the retention of 1,4-naphthoquinone derivatives under conditions of RP HPLC. The most complete separation for the investigated compounds in isocratic mode using simple binary MP «water-organic modifier» was achieved for MP containing 60 % vol. acetonitrile (MeCN) and 75 % vol. methanol (MeOH). For amino acid derivatives of 1,4-naphthoquinone, the effect of pH for MP on retention was investigated and it was found that at values less than pH ≈ 5 the bifurcation of disappears, so these analytes turn into a molecular form, for which chromatographic peaks have longer retention times under HPLC. The dependences of the capacity factor of amino acid derivatives on the pH value of the MP (pH = 3.5-7) have the form of part of the S-shaped curve, which is characteristical in the conditions of RP HPLC for analytes that are weak acids. The possibilities of computer modeling assisted by the ChromDream software for predicting the retention of 1,4-naphthoquinone derivatives in RP HPLC were tested. The values of V and ΔG were calculated for the investigated 1,4-naphthoquinone derivatives. When calibrating the chromatographic column for reference substances (n = 5), which were selected from the investigated derivatives of 1,4-naphthoquinone, it has been shown that the values of a, b and c (model Galushko S.V. and coworkers) significantly depend on both the amount of substances and which substances were selected for their calculation, as evidenced by the different calculation options. The number of satisfactory results from all prediction data (n = 5/24) in the case of MP with MeCN is approximately 2 times less than for MP with MeOH. The generalisation was made for the MP composition effect on the retention and separation of 1,4-naphthoquinone derivatives under NP TLC according to the Snyder‒Soczewinsky model. An increase in the TLC efficiency in terms of the number of theoretical plates from N = 700-1000 to N = 1400-1500, that is, within 1.5-2 times, is observed when the polar component of the mobile phase is replaced in the series: acetonitrile <acetone <methanol <propane-2-ol <chloroform. Methods were developed for the quantitative determination of 1,4-naphthoquinone derivatives using RP HPLC and NP TLC based on the found optimal compositions of binary MP.