The Selection of “Green” Conditions for Components Identification in a Combined Medicine by TLC/HPTLC Methods




“green” chemistry, thin-layer chromatography, high-performance thin-layer chromatography, glycine, glutamic acid, ascorbic acid


Aim. To select “green conditions” for identifying components in a combined medicine for the treatment of alcohol intoxication.
Materials and methods. Thin-layer chromatography and high performance thin-layer chromatography methods were used. An analytical GREEnness calculator was applied to assess the environmental friendliness of the analytical procedure.
Results and discussion. The choice of mobile and stationary phases that comply with the principles of “green chemistry” and can be used to detect glutamic acid and glycine in the composition of a combined medicine has been substantiated. It has been determined that by the indicators Rf, Rs, ΔRf, α, N, H the most effective for the division is the ethanol (96 per cent) – water (70:30) mobile phase (the length of the solvent front is 10 cm, the application volume is 5 μL), which allows, in addition to amino acids, to determine another prescription component – ascorbic acid. The conditions for identification of substances by the high-performance thin-layer chromatography method (the length of the solvent front is 7 cm, the application volume is 2 μL) have been selected. It has been found that to detect chromatographic zones, it is optimal to use ninhydrin solution R1 with further heating of the plate at a temperature of 100-105°С for 5 min. The specificity of determination of glutamic acid, glycine and ascorbic acid in comparison with solutions of standard substances has been proven. While studying the robustness of the method the influence of chromatographic conditions on the final result (the impact of the stationary phase type, chamber saturation, application volume, distance from the “start line” to the “finish line”, the effect of the detection solution, stability of solutions for application) has been researched. The precision of the method on one and three plates of the same type has been studied; the intermediate precision has been researched. The calculated assessment of greenness of the analytical procedure is 0.66.
Conclusions. As a result of the studies conducted, “green conditions” for identifying amino acids (glutamic acid, glycine), as well as ascorbic acid in a combined medicine by thin-layer chromatography and high-performance thin-layer chromatography methods have been selected. The validation characteristics of the method (specificity, robustness and precision) have been studied.


Byrne, F. P.; Jin, S.; Paggiola, G.; Petchey, T. H. M.; Clark, J. H.; Farmer, T. J.; Hunt, A. J.; Robert McElroy, C.; Sherwood, J. Tools and techniques for solvent selection: green solvent selection guides. Sustainable Chem. Processes 2016, 4 (1), art. No. 7.

Curyło, J.; Wardencki, W.; Namieśnik, J. Green Aspects of Sample Preparation – a Need for Solvent Reduction. J. Environ. Stud. 2007, 16 (1), 5-16.

Tobiszewski, M.; Namieśnik, J. Greener organic solvents in analytical chemistry. Opin. Green Sustainable Chem. 2017, 5, 1-4.

Prat, D.; Hayler, J.; Wells, A. A survey of solvent selection guides. Green Chem. 2014, 16 (10), 4546-4551.

Clarke, C. J.; Tu, W.-C.; Levers, O.; Bröhl, A.; Hallett, J. P. Green and Sustainable Solvents in Chemical Processes. Rev. 2018, 118 (2), 747-800.

Vorozheikin, S. B.; Shtykov, S. N.; Bashko, E. S. Thin-layer Chromatography of Amino Acids in Micelle Mobile Phases on Silicagel [in Russian]. Sorbtsionnye Khromatogr. Protsessy 2011, 11 (6), 840-847.

Bhawani, S. A.; Mohamad Ibrahim, M. N.; Sulaiman, O.; Hashim, R.; Mohammad, A.; Hena, S. Thin-layer chromatography of amino acids: a review. Liq. Chromatogr. Relat. Technol. 2012, 35 (11), 1497-1516.

Trineeva, O. V.; Safonova, E. F.; Slivkin, I. Development of method for the quantitative determination of ascorbic acid by high-efficiency thin-layer chromatography [in Russian]. Khim.-Farm. Zh. 2017, 51 (10), 58-64.

Thanh Van, Ng.; Mironenko, N. V.; Brezhneva, T. A.; Selemenev, V. F.; Berezhnova, T. A.; Preobrazhenskaya, S. Development of technique for qualitative identification of individual Quillaja saponins by TLC [in Russian]. Proceedings of Voronezh State University. Series: Chemistry. Biology. Pharmacy 2018, 1, 15-21.

Qiu, T.; Li, H.; Cao, Y. Pre-staining thin layer chromatography method for amino acid detection. J. Biotechnol. 2010, 9 (50), 8679-8681.

Khokhlova, E. A., Vishnevska, L. I., Garna, S. V., Kotov, A. G. Development and validation of the method of identification of isoflavonoids and triterpenoid saponins in the tincture “Аterofit-norma” by TLC [in Ukrainian]. Pharmacom 2013, 1, 38-52.

Reich, E.; Schibli, A. High-performance thin-layer chromatography for the analysis of medicinal plants; Thieme: Stuttgart, 2007.

Mohammad, A.; Moheman, A.; El-Desoky, G. Amino acid and vitamin determinations by TLC/HPTLC: review of the current state. Open Chem. 2012, 10 (3), 731-750.

Mohammad, A.; Inamuddin; Siddiq, A.; Naushad, M.; El-Desoky, G. E. Green solvents in thin-layer chromatography. In Green Solvents I; Mohammad, A., Inamuddin, Eds.; Springer: Dordrecht; 2012, 331-361.

Pena-Pereira, F.; Kloskowski, A.; Namieśnik, J. Perspectives on the replacement of harmful organic solvents in analytical methodologies: a framework toward the implementation of a generation of eco-friendly alternatives. Green Chem. 2015, 17 (7), 3687-3705.

Pena-Pereira, F.; Wojnowski, W.; Tobiszewski, M. AGREE – Analytical GREEnness Metric Approach and Software. Chem. 2020, 92 (14), 10076-10082.

Rudakova, O. V.; Gubar, S. M.; Bezchasnyuk, O. M.; Bevz, N. Y.; Smielova, N. M.; Georgiyants, V. A. Development of composition and technology of combined medicine for pharmacotherapy of alcohol intoxication [in Ukrainian]. Pharmaceutical review 2022, 1, 39-48.

Rudakova, O.; Gubar, S.; Smielova, N.; Lytkin, D.; Briukhanova, T.; Bezchasnyuk, E.; Bevz, N.; Georgiyants, V. Study of compatibility of components of a new combined drug for treatment of alcoholic intoxication and its hepatoprotective effect on a model of alcoholic liver injury. ScienceRise: Pharm. Sci. 2021, 6, 91-100.

Derzhavna farmakopeia Ukrainy: v 3 tomakh, 2 vydannia [The State Pharmacopoeia of Ukraine: in 3 volumes, 2nd, in Ukrainian]; State Enterprise “Ukrainian Scientific Pharmacopoeial Center for Quality of Medicines”: Kharkiv, 2015; Vol. 1.

European Pharmacopoeia, 10th; Euroрean Department for the Quality of Medicines: Strasbourg, 2019; Vol. 1.

Maltseva, A. A.; Trineyeva, O. V.; Chistyakova, A. S.; Brezhneva, T. A.; Slivkin, A. I.; Sorokina, A. A. Thin layer chromatography in the analysis of flavonoids of plant objects [in Russian]. Farmatsiya (Moscow, Russ. Fed.) 2013, 1, 13-16.

Sumina, E. G.; Shtykov, S. N.; Uglanova, V. Z.; Kulakova, N. V. Tonkoslojnaya hromatografiya. Teoreticheskie osnovy i prakticheskoe primenenie, 3rd [Thin-layer chromatography. Theoretical foundations and practical application, in Russian]; Saratov State University: Saratov, 2012.

Khokhlova, K.O.; Zdoryk, O. A.; Georgiyants, V. A.; Development and validation of identification method of flavonoids in the Marigold tincture. Message 1 [in Russian]. Rastit. Syr'ya 2015, 1, 133-139.

Derzhavna farmakopeia Ukrainy: v 3 tomakh, 2 vydannia [The State Pharmacopoeia of Ukraine: in 3 volumes, 2nd, in Ukrainian]; State Enterprise “Ukrainian Scientific Pharmacopoeial Center for Quality of Medicines”: Kharkiv, 2014; Vol. 2.

Zakrzewski, R.; Ciesielski, W.; Kaźmierczak, D. Application of the iodine–azide procedure for the detection of glycine, alanine, and aspartic acid in planar chromatography. Liq. Chromatogr. Relat. Technol. 2002, 25 (10-11), 1599-1614.

Sorokina, O. N.; Sumina, E. G.; Shtykov, S. N.; Atayan, V. Z.; Barysheva, S. V. TLC Separation of D-, L- of Amino Acids in the 2-Hydroxypropyl-β-Cyclodextrin Aqueous Mobile Phase [in Russian]. Sorbtsionnye Khromatogr. Protsessy 2010, 10 (1). 135-141.

Simonyan, A. V.; Salamatov, A. A.; Pokrovskaya, Yu. S.; Avanesyan, A. A. Ispolzovanie ningidrinovoj reakcii dlya kolichestvennogo opredeleniya a-aminokislot v razlichnyh obektah: Metodicheskie rekomendacii [The use of the ninhydrin reaction for the quantitative determination of α-amino acids in various objects: Guidelines, in Russian]; Volgograd State Medical University: Volgograd, 2007.

Alikina, E. N. Analiticheskaya himiya. Kachestvennyj analiz [Analytical chemistry. Qualitative Analysis, in Russian]; Perm State National Research University: Perm, 2019.

Smielova, N. M.; Gubar, S. M.; Yevtifieieva, O. A.; Bezchasnyuk, E. M. Selection of the optimal conditions for analysis of plant active pharmaceutical inulin ingredients by thin-layer chromatography to develop the draft for the national monograph “Inulin”. Res. J. Pharm. Technol. 2019, 12 (6), 2862-2870.




How to Cite

Rudakova, O. V.; Gubar, S. M.; Smielova, N. M.; Kriukova, A. I.; Bevz, N. Y.; Georgiyants, V. A. The Selection of “Green” Conditions for Components Identification in a Combined Medicine by TLC/HPTLC Methods. J. Org. Pharm. Chem. 2022, 20, 52-63.



Original Researches