Application of UV spectrophotometry and reversed-phase HPLC to determine the ionization constant of a new biologically active compound

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Abstract

Aim – to determine the ionization constant (pKa) of the biologically active compound 2-ABPPC by UV spectrophotometry and high performance liquid chromatography (HPLC).

Material and methods. The object of the study was the substance 2-ABPPC (2-amino-1-(4-bromophenyl)-5-(3,3-dimethyl-2-oxobutylidene)-4-oxo-4,5-dihydro-1H-pyrrole-3- carboxamide). The methods of UV spectrophotometry and high performance liquid chromatography were used to determine the ionization constant. The spectrophotometric analysis was carried out on a Shimadzu UV-1800 spectrophotometer. The chromatographic determination of the pKa value was carried out on an LC-20 Prominence liquid chromatograph (Shimadzu) using a Zorbax Extend-C18 reversed-phase column.

Results. In the course of study, it was found that 2-ABPPC has one pKa value. When using two different methods to estimate the ionization constant, the comparable results were obtained: 7.64 (UV spectrophotometry method) and 7.40 (reversed phase HPLC method).

About the authors

Arthur T. Tsecheev

Perm State Pharmaceutical Academy

Author for correspondence.
Email: arthurtse@yandex.ru
ORCID iD: 0000-0001-5774-9504

a postgraduate student of the Department of Toxicological chemistry

Russian Federation, Perm

Yurii N. Karpenko

Perm State Pharmaceutical Academy; Parma Clinical LLC

Email: arthurtse@yandex.ru
ORCID iD: 0000-0003-3174-3678

PhD, Associate professor of the Department of Toxicological chemistry

Russian Federation, Perm; Perm

References

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Supplementary files

Supplementary Files
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2. Figure 1. Structure of 2-ABPPC.

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3. Figure 2. UV absorption spectra of 2-ABPPC at different buffers’ pH values.

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4. Figure 3. Dependence of the optical density of 2-ABPPC on the pH at 250 nm.

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5. Figure 4. UV absorption spectra of 2-ABPPC in buffer solutions (pH 7.2–8.23).

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6. Figure 5. Absorbance diagram of 2-ABPPC in different buffer solutions (pH 1–12).

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7. Figure 6. Chromatograms of 2-ABPPC at different pH level of the eluent (а – 2.35; б – 6.90; в – 11.10).

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8. Figure 7. The first derivative curve for pKa determination.

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Copyright (c) 2023 Tsecheev A.T., Karpenko Y.N.

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