Number Of Theoretical Plates Research Articles

UHPLC assisted simultaneous separation of apigenin and prednisolone and its application in the pharmacokinetics of apigenin

A new ultra-high performance liquid chromatography-mass spectrometry-mass spectrometry (UHPLC-MS/MS) system has been formulated for the resolution of closely related drugs apigenin (API, a bioflavinoid) and prednisolone (PRD) from their mixture. This developed method comprised of a “BEH™ C18 column (50 mm × 2.1 mm, 1.7 μm)” using acetonitrile and 0.1% formic acid (35:65 v/v) at a supply rate of 0.25 mL·min−1 as eluent. It was found that selected eluent provided short run time (≤2.5 min) as well as better peak symmetry. Satisfactory values of chromatographic parameters such as resolution (Rs = 2.5), capacity factor (k; 13.6 and 23.4 for API and PRD respectively, selectivity (α = 1.72) and number of theoretical plates (N; 3789 and 42,435 for API and PRD respectively) indicate the efficiency of the developed method. The obtained separation was then exploited for the detection and measurement of API in rat plasma sample by means of PRD as an “internal standard” (IS). The eluted compounds in plasma were identified by tandem mass spectrometry by means of tandem quadrupole (TQ) detector (“Waters Corp., Milford, MA”) fortified with an “electrospray ionisation (ESI)” source functioning in positive ionization mode. The determination of API in plasma was accomplished by means of “multiple reactions monitoring (MRM)” mode. Assortment of “ionization pairs” (m/z) was displayed in the following manner: API: 270.99 → 152.9 (“cone voltage” 57 V, “collision energy” 34 V), PRD: 403.172 → 385.224 (“cone voltage” 42 V, “collision energy” 13 V). The calibration curves followed linearity in concentration range of 05–1000 ng mL−1 with limit of detection “LOD” and limit of quantification “LOQ” of 7.30 and 22.77 ng mL−1, respectively. The developed method was validated taking into consideration various test conditions and satisfactory values of various parameters such as linearity (r2 ± SD = 0.9995 ± 0.0005), interday accuracy (88–120%), interday precision % RSD = 3.30–13.65% whereas intraday accuracy (91–118%) intraday precision % RSD = 1.18–5.83) indicated its validity. The validation outcomes fulfilled the standards of united states food and drug administration “USFDA” in addition Scientific Working Group for Forensic Toxicology “SWGTOX” guiding principles and were not beyond the tolerable constraint. The process developed in plasma was efficaciously harnessed in the pharmacokinetic investigation of various formulations of API after oral administration in rats.

High separation efficiency of gold nanomaterials of different aspect ratio and size using capillary transient isotachophoresis

Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP), were compared for the detection and separation of spherical gold nanoparticles (AuNPs) and gold nanorods (AuNRs). The development of ctITP using two different leading ions is described. Overall, when compared to traditional capillary zone electrophoresis (CZE), ctITP resulted in improved peak shape and peak efficiency. Specifically, the number of theoretical plates for AuNR samples increased by a factor of 2–2.5 depending on the choice of leading ion. Further, using ctITP two AuNRs differing by aspect ratio were baseline resolved, whereas the same AuNRs could not be separated using CZE or other techniques like single particle inductively coupled plasma mass spectrometry (spICP-MS) and asymmetric flow field-flow fractionation (AF4). The results of this study demonstrate that ctITP is an efficient on-line technique for the improved detection and separation of gold nanomaterials in CE.

Design of Reversed-Phase Chromatography Column Packing Material for Protein Separation: Part 2- Chromatographic Performance

In the second part of the two-part study, monodisperse and porous poly(vinyl acetate-co-divinylbenzene) [poly(VAc-co-DVB)] and poly(divinylbenzene) [poly(DVB)] column packing materials synthesized in the first part of the study were tested in Reverse Phase Chromatography (RPC) under different conditions. The investigation of column performances concluded that columns packed with poly(VAc-co-DVB) (2500 plate/m) and poly(DVB) (1500 plate/m), showed high and stable theoretical plate number and high peak resolution values compared to literature values. Protein recovery data in RPC column, poly(VAc-co-DVB) (93-99%), were determined as similar as quantitative values for all proteins. Reproducibility tests of analytes of poly(VAc-co-DVB) column were assessed "run to run" and "day to day" Relative Standard Deviation (RSD) values were determined under 2.8%.

Detection and analysis of 17 steroid hormones by ultra-high-performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-MS) in different sex and maturity stages of Antarctic krill (Euphausia superba Dana).

A sensitive and accurate method for determination of 17 endogenous and exogenous steroid hormones in Antarctic krill was developed. The method utilized UHPLC-MS in electrospray ionization mode (ESI). Samples were prepared by alkaline hydrolysis; sequential vortex extraction with ethyl acetate, methanol and acetonitrile; followed by a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) clean-up method. The system suitability tests including theoretical plate number, resolution, repeatability, tailing factor proved the system’s resolution and reproducibility that can meet the requirements of sample analysis. The developed method resulted in satisfactory recoveries that varied from 75.4%-110.6% and relative standard deviations (RSDs) that ranged from 3.1%-10.5%. The ranges of the limits of detection (LODs) and the limits of quantitation (LOQs) were 2–30 ng kg-1 and 10–100 ng kg-1, respectively. 14 hormones including cortisone, aldosterone, testosterone propionate, estriol, megestrol acetate, cortisone acetate, dexamethasone, testosterone, hydroxyprogesterone, nandrolone, prednisolone, cortisol, progesterone and estradiol were found in Antarctic krill. Other 3 hormones (Diethylstilbestrol, norethisterone and androsterone) were not detected. The levels of exogenous steroid hormones were much greater than those of endogenous steroid hormones, and the levels of exogenous glucocorticoids were much greater than those of exogenous sex hormones. The changes of hormones in different sex and maturity stages were also explored. Endogenous hormones might regulate the reproductive and development of Antarctic krill. The detected exogenous hormones suggests the potential for hormonal contamination in Antarctic waters that can affect organisms even affect human beings by food chain.

Comparison of five different HPLC columns with different particle sizes, lengths and make for the optimization of seven polycyclic aromatic hydrocarbons (PAH) analysis

The aim of this study is to evaluate the performance of five different analytical columns for the analysis of seven carcinogenic polycyclic aromatic hydrocarbons namely Benz[a]anthracene, Chrysene, Benzo[j]fluoranthene, Benzo[e]Pyrene, Benzo[k]fluoranthene, Benzo[a]Pyrene, Dibenzo[a,h]anthracene and select the best column in terms of separation, peak shape and analysis time and to use the selected column under optimized analytical condition for the detection and estimation of polycyclic aromatic hydrocarbons (PAHs) in light cycle oil; one of aromatic rich petroleum stream. The performance of five different analytical columns with different lengths, particle sizes and make were compared for analysis of above PAHs using HPLC with PDA detector and monitoring the HPLC–UV signals at 254 nm wavelength. Chromatographic parameters including retention time (tR), resolution (R), limit of detection, limit of quantification, number of theoretical plates (N), height equivalent to theoretical plate (HETP) and reduced plate height (h) were evaluated and compared on mentioned columns for analysis of PAHs under study. The mobile phase acetonitrile (95%):water (5%) was used at flow rate of 1.5 mL/min and the injection volume in all the case was 20.0 µL. The best results w.r.t time and acceptable resolution were obtained when these PAHs were analyzed using the shorter Agilent Eclipse PAH column having 1.8 µm, 100 mm × 4.6 mm id.

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE ESTIMATION OF LEDIPASVIR IN BULK AND TABLET DOSAGE FORM

Objective: The objective of this study was to develop a stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the estimation of ledipasvir (LDP) in bulk and tablet formulation.
 Methods: Stability-indicating RP-HPLC method was developed and validated for the estimation of LDP in bulk and tablet formulation. RP-HPLC was carried out on HiQ SiL C18 columns (250 mm × 4.6 mm, 5 μ particle size) using mobile phase acetonitrile:1 mM ammonium acetate buffer in the ratio of 90:10 v/v at a flow rate of 1 ml/min. The analytes were monitored using MD 2010 PDA detector at 333 nm.
 Results: The retention time was found to be 3.843 min. The proposed method was found to be having linearity in the concentration range of 5–30 μg/ml. The number of theoretical plates obtained was 4236.50 which indicate the efficient performance of the column. The limit of detection was 0.305 μg/ml and limit of quantification was 0.923 μg/ml, which indicate the sensitivity of the method; the high percentage recovery indicates that the proposed method is highly accurate. The developed method has been validated according to the ICH guidelines and found to be simple, specific, precise, and accurate.
 Conclusion: The proposed method is precise, accurate, and stability indicating. Therefore, the proposed method can be used for routine quality control and analysis of LDP during stability studies in bulk samples and tablet dosage forms.

Semi-Packed Gas Chromatography Columns With Density Modulated Pillars

The separation column is the most important component which influences the performance of gas chromatography systems. Among a number of column designs, the microfabricated semi-packed column has attracted particular attention in micro gas chromatography because it enhances the separation efficiency and provides higher sample capacity compared to the open-channel counterparts. However, these added advantages come at the cost of higher pressure drops per unit length of the separation column. This paper reports new semi-packed column designs through density modulation of the pillars by changing the spacing between rows or the number of pillars per row from. Various geometries were fabricated and tested against regular semi-packed columns having either one pillar or four pillars per row. Different performance metrics were measured including column pressure drop, optimal flow velocity, number of theoretical plates, and separation efficiency. The density-modulated semi-packed columns provide efficiencies comparable with that of semi-packed columns embedding more pillars per row while requiring a lower inlet pressure which is more comparable with the columns embedding one pillar per row. These density-modulated columns were able to successfully separate diesel and kerosene compounds and to identify the major hydrocarbon constituents of these two widely used samples.

Poly(glycidyl methacrylate-co-1,4-dimethacryloyloxybenzene) monodisperse microspheres - synthesis, characterization and application as chromatographic packings in RP-HPLC

The study describes the synthesis of porous microspheres with glycidyl methacrylate (GMA) and 1,4-dimethacryloyloxybenzene (DMB) using the seed swelling polymerization. As a shape template the polystyrene (PS) seed obtained via the dispersion polymerization was used. Oxirane, esters and aromatic groups are present in the structure of copolymers. Changing the reaction mixture composition – the amounts of crosslinking monomer and PS seed, almost monosized poly(GMA-co-DMB) microspheres of the diameters in the range of 8.20 μm to 11.61 μm and highly developed porous structure with specific surface area (SBET) up to 353 m2/g were obtained. The pore size distribution and the volume of pores measured for the copolymer in the dry and the swollen states differ. In the dry state in the internal copolymer structure mesopores were observed whereas in the swollen state additionally micro- and macropores appeared. The copolymer obtained with the equimolar mixture of monomers was applied as the column packing material and tested in the reverse-phase HPLC. The isocratic separation of alkylbenzenes was achieved with satisfactory resolution using the 100 × 4.6 mm i.d. column with the acetonitrile/phosphate buffer mobile phase. The theoretical plate numbers up to 12,000 were obtained for benzene as the analyte. Using the retention indices for the test compounds it was found that the retention mechanism on the studied copolymer is the sum of π-π hydrophobic interactions, dipole interactions and to a smaller extent of hydrogen acceptance interactions rather than hydrogen donation interactions.

Isopropyl Lactate Obtaining by Esterification in a Reactive Distillation System

The reactive distillation is a way for the intensification of chemical processes by thermal integration into a single process of chemical reactions with products separation. Isopropyl lactate is an important intermediate for drug synthesis what can be synthesized through reactive distillation. Based on scientific studies in literature, a preliminary study was performed in a reactive distillation laboratory system, in order to obtain isopropyl lactate by esterification of lactic acid with isopropyl alcohol. The independent variables studied are: the alcohol/lactic acid molar ratio (R), the catalyst concentration (CA) and the number of theoretical trays in the fractionation zone (NTT). The experimental matrix was established for a 23 factorial experiment, by considering two levels for each variable: R=1 and 3 mole/mole; CA= 3 %wt and 5 %wt; NTT=1.9 and 2.9. A linear mathematical model was proposed to correlate the lactic acid conversion with the molar ratio of reactants, catalyst concentration and the number of theoretical plates in the column. The model shows that R and NTT are the important variables of the process while the acid concentration is less important, in the studied range. The correlation coefficient is R2=0.9978 and the statistical analysis ANOVA of the model�s coefficients proved they are significant at a confidence level of 95%, so the model is reliable.

A Short-Cut Design Technique to Batch Distillation Column

The effects of equipment parameters of batch distillation column on the yield proportion are discussed and analyzed, the relations between maximal yield proportion and the column equipment parameters are correlated, which not only can be used to appraise rationality of the design parameters of the columns being employed and which but also can be used to new batch distillation column design. Under the assistance of the separation difficulty defined in this paper, the minimum number of theoretical plates is determined by the limit loss proportion method given, and further the actual number of theoretical plates and the height for the batch distillation are calculated by using the redundancy coefficient found to complete the whole design of the batch distillation as shown in the computational sample. Research showed that the actual number of theoretical plates and the height of batch distillation column with the column diameter 0.6 m are 17 and 5.1 m in alcohol mixture separation system of the sample proposed. Moreover, the approach can be extended to the design of batch distillation column with a separation system of multi-component liquid mixture after those adjacent components are treated as numerous binary component systems.

Polyethylene Glycols for the Dispersion Development of Graphene in an Aqueous Surfactant Solution Studied by Affinity Capillary Electrophoresis.

Water-soluble nonionic polymers of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), and polyvinylpyrrolidone (PVP) were examined to develop the dispersion of graphene in an aqueous surfactant solution. Sodium dodecylbenzenesulfonate was used as an anionic surfactant to disperse graphene in an aqueous solution and to give negative charge on it. The dispersion of graphene was monitored through the electropherograms in affinity capillary electrophoresis; a broad peak for the dispersed graphene and shot signals for the aggregated one. When PEG was added in the separation buffer as an affinity reagent, the number of the shot signals in the electropherogram was reduced; PEG can develop the dispersion of graphene in an aqueous surfactant solution. The dispersion was also developed with PVP or PVA. The effective electrophoretic mobility of the dispersed graphene was reduced by using the polymer as an affinity reagent. The result suggested that the anionic surfactant on the graphene surface was competitively substituted with the nonionic polymer. The degree of the decrease in the effective electrophoretic mobility was larger with PEG with a high-molecular mass. The broad peak of the dispersed graphene got narrower by the addition of PEG, and the number of theoretical plates was improved.

Design of experiments-based RP – HPLC bioanalytical method development for estimation of Rufinamide in rat plasma and brain and its application in pharmacokinetic study

This study reports a fully validated HPLC-UV (High Performance Liquid Chromatography- Ultra Violet) method for quantitative estimation of Rufinamide (RUFI), an antiepileptic drug, in rat plasma and brain matrices. A response surface methodology based Box Behnken experimental design, using the principles of Design of Experiments (DoE), was employed to optimize critical chromatographic conditions viz. pH and proportion of the buffer and wavelength of detection, for achieving good sensitivity (peak area) and specificity (number of theoretical plates). A desirability function was employed to identify the optimized conditions, which gave a highest value of 0.971. The optimized chromatographic conditions were: pH of the buffer: 4.7, wavelength of detection: 215 nm and proportion of buffer in mobile phase: 84.7% v/v for responses: 124839.6 mV ∗ min as the peak area (1 μg/mL) and 20,000 as the theoretical plate number for the same. A simple protein precipitation method, using methanol, was employed to extract RUFI from the biological matrices. Piribedil was used as the internal standard (IS). At the optimized conditions, the LOQ values of RUFI in plasma and brain were found to be 13.84 ng/mL and 105.24 ng/g respectively. The developed method was validated as per ICH guidelines and its applicability in analysing RUFI in rat plasma and brain matrices was demonstrated by i.v. administration in rats. The AUC0-t in plasma was found to be 91.9% of AUC0−∞ indicating that the method is very sensitive and can capture almost the entire plasma time course of RUFI, at a dose of 0.5 mg/kg.

Six Sigma quality approach for HPLC-UV method optimization

We report a robust HPLC-UV method for ascorbic acid (AS), paracetamol (PA) and guaifenesin (GU) based on response surface/tolerance analysis approach to meet Six Sigma quality of the performance of a chromatographic method. Our goals were the bringing specifications into the optimizations and achieving robustness during the development phase. The critical method attributes (CMAs) were resolution between the peak pairs (Rs), the analysis time(T), tailing factor (Tf), number of theoretical plates (N), and back pressure(P).The critical method parameters (CMPs) included a percentage of organic modifier, a flow rate of the mobile phase, and a temperature of column oven. The separation was performed on a CN column, using an isocratic mode with a mixture of 50 mM potassium dihydrogen phosphate buffer adjusted with ortho-phosphoric acid to pH 2.2 as the aqueous component of the mobile phase and methanol in the ratio 85.9:14.1 (v/v) at 1 ml/min flow rate, with UV detector at 275 nm and a column temperature of 45 °C. The method was validated using the accuracy profiles. Finally, the method was applied to pharmaceutical dosage form.

Effect of the Length-to-Width Aspect Ratio of a Cuboid Packed-Bed Device on Efficiency of Chromatographic Separation

In recent papers we have discussed the use of cuboid packed-bed devices as alternative to columns for chromatographic separations. These devices address some of the major flow distribution challenges faced by preparative columns used for process-scale purification of biologicals. Our previous studies showed that significant improvements in separation metrics such as the number of theoretical plates, peak shape, and peak resolution in multi-protein separation could be achieved. However, the length-to-width aspect ratio of a cuboid packed-bed device could potentially affect its performance. A systematic comparison of six cuboid packed-bed devices having different length-to-width aspect ratios showed that it had a significant effect on separation performance. The number of theoretical plates per meter in the best-performing cuboid packed-bed device was about 4.5 times higher than that in its equivalent commercial column. On the other hand, the corresponding number in the worst-performing cuboid-packed bed was lower than that in the column. A head-to-head comparison of the best-performing cuboid packed bed and its equivalent column was carried out. Performance metrics compared included the widths and dispersion indices of flow-through and eluted protein peaks. The optimized cuboid packed-bed device significantly outperformed its equivalent column with regards to all these attributes.

Study of Monolithic Integrated Micro Gas Chromatography Chip

Abstract A monolithic micro gas chromatography (μGC) chip which integrated the micro separation column (μSC) and the micro thermal conductivity detector (μTCD) based on MEMS (Micro-electro-mechanical systems) technique was fabricated. Compared to the state of the art, the μSC with high depth-to-width ratio channels that was coated with mesoporous silica nanoparticles as stationary phase could effectively improve the column capacity and separation performance. Besides, the stable suspending μTCD, which was designed and fabricated in two ports of the μGC chip, could availably enhance the thermal isolation and reliability of the device. The mixture of light hydrocarbons (methane, ethane, propane and butane) could be separated from each other and detected by this monolithic integrated μGC chip, in which the overall analysis and detection time was only 33 seconds, the separation resolution of ethane and propane was 8.34, and the number of theoretical plate was as high as 11420. The monolithic integrated μGC chip has many advantages such as good separation resolution, high column efficiency and short analysis time, and is suitable for portable gas chromatographic field and onsite detection.

Chiral separation and determination of amino acid enantiomers in fruit juice by open-tubular nano liquid chromatography.

A novel chiral porous-layer stationary phase was developed for use in open-tubular nano liquid chromatography. The stationary phase was prepared by an in-situ polymerization of 3-chloro-2-hydroxypropylmethacrylate (HPMA-Cl) and ethylene dimethacrylate (EDMA). The reactive chloro groups at the surface of the porous stationary phase were reacted with β-Cyclodextrin (β-CD). The resulting morphology was characterized by using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FT-IR). The chromatographic performance of the column was evaluated by hydrophilic interaction chromatography (HILIC). Amino acids were used as test solutes. The running buffer conditions for the enantioseparation were found to be 85% acetonitrile (ACN):10%MeOH: 5% H2 O at 0.1% v/v trifluoro acetic acid (TFA) (flow rate: 800nL/min). The enantioseparation provided high theoretical plate numbers up to 26000 platesm-1 . A good retention capacity within satisfactory retention times was also achieved. Real sample applicability of this column to the separation of amino acid enantiomers in fruit juice sample was demonstrated.

A novel graphical approach for mass exchange networks using composition driving force

Raw materials are considered as a corner stone for both chemical and process industries. Raw materials efficient utilization and usage within the process saves a lot. Mass integration is the most competent path to minimize the economic and environmental losses, by minimizing both the amounts of external mass separating agents and waste disposal. Mass exchange networks or MENs is the tool used to apply this integration. This article presents a new graphical approach, based on the composition driving force, to analyse the mass integration and exchange networks. The new approach is illustrated graphically, and can be applied on existing and new mass networks. The new graphical approach is constructed based on the Pinch Analysis Principle where the lean stream compositions are plotted on the X-axis, while the composition driving forces for each exchanger are plotted on the Y-axis. The performance of MENs is evaluated and analysed in terms of the composition driving force (CDF) inside the mass exchanger. In addition to be the main motive to transfer the composition from rich stream to lean stream, composition driving force is also involved in the calculation of the number of theoretical plates, consequently affecting the cost. Each mass exchanging unit is represented in this graph as a straight line whose slope is related to the mass flow rates of both rich and lean streams and its length is related to the mass load transferred within the exchanger. The CDF is divided into five regions, based on the pinch analysis principles to analyse and perfectly locate the mass exchangers. The new graphical approach is applied to existing MENs to enhance their actual performances by minimizing wastes disposal and external separating agents and accomplish the mass targets defined by the Pinch Analysis Principles.

Treatment of Pharmaceutical Process Wastewater with Hybrid Separation Method: Distillation and Hydrophilic Pervaporation

Abstract The work is motivated by an industrial problem, which is alcohol removal from pharmaceutical process wastewater. The aim of the study was to develop a complete hybrid operation is investigated. Ethanol dehydration, in combination with distillation and hydrophilic pervaporation, is used to investigate about the extent of separation of the ethanol-water mixture. The aim of this research is to rigorously model and optimize this hybrid operation in professional flowsheet simulator environment. The number of minimal theoretical plates of distillation column and minimal effective membrane transfer area are determined. Cost estimation is also examined according to Douglas methodology. Considering our results it can be concluded that, the distillation and hydrophilic pervaporation processes are suitable for separation ethanol and water in 99.5 weight percent purity

SYNTHESIS OF FLOWSHEETS FOR SEPARATION OF MULTIPHASE MIXTURES: STATE OF THE ART

The paper presents an analysis of the current state of research on separation flowsheets based on the combination of distillation and phase separation processes, as well as heteroazeotropic distillation. It is shown that the works of foreign researchers devoted to the study of flowhseets with decanters are more focused on finding ways to reduce energy consumption by introducing additional separators, a combination of several methods (extractive and heteroazeotropic complex columns with external decanters. The task of synthesizing all possible separation flowsheets is not considered in these works. In this paper, a complete set of flowsheets of different structures based on the combination of distillation and phase separation processes, including the use of columns with an external decanter, is proposed for water - butyl acetate - methanol and methanol - heptane-water ternary mixtures separation. Aspen Plus and NRTL model were used for mathematical modelling of phase equilibrium (the relative error of describing liquid-vapor and liquid-liquid equilibrium is less than 5%) was chosen as a method of research. Operating parameters for distillation columns (the number of theoretical plates, feed plate, reflux ratio) and the total energy consumption were obtained for each case. The necessity of using a double feed-plate column for separating of propanol-1 - water - butanol-1 and ethyl acetate - water - butyl acetate ternary systems was explained by the presence of extractive effect. Analytical review of modern publications and results of own research allowed to formulate a number of recommendations for the synthesis of energy effective flowsheets based on a combination of distillation and phase separation processes.

Optimization of L-tryptophan and thiotriazoline compound analysis by high-performance liquid chromatography

Introduction. An actual task of modern medicine is the development of treatment methods of the central nervous system diseases. The solution of this problem was the creation of new, more effective neuropsychotropic drug that shows pronounced antidepressant, nootropic, neuroprotective and antioxidant effects based on the fixed combination of L-tryptophan with thiotriazoline. One of the steps in creating a new drug is the standardization of active ingredients.Today, much attention is being paid to new physical-chemical methods of drugs study, including high-performance liquid chromatography (HPLC). Increasingly, the HPLC method is used for the determination of active ingredients of organic nature, both in mono-preparations and in combined dosage forms.Therefore, we have proposed the HPLC method for new combined L-tryptophan and thiotriazoline drug in order to determine active substances based on their effects.The purpose of our study is to develop a method for the simultaneous standardization of L-tryptophan and thiotriazoline in an artificial compound by high-performance liquid chromatography, in particular the selection of the mobile and stationary phase.Materials and methods. In the first stage of our work, a selection of phase and eluents was carried out for L-tryptophan and thiotriazoline model compound in a ratio of 1:1. The analysis method of thiotriazoline in the reverse phase (C 18) using the eluents, which are water-methanol compounds, was taken as a basis.Results. In the course of study, it was found that the retention volume of both tryptophan and thiotriazoline decreased, and the peak distribution coefficient increased with the number of theoretical plates with increasing content of methanol in phosphate buffer compound. Therefore, as an eluent, attention was drawn to a solution of 20% methanol with 80 % phosphate buffer. Under these conditions, the tryptophan retention volume was 15.11 ml, thiotriazoline – about 6.05 ml, the efficacy of the chromatographic column, calculated on the tryptophan peak of about 3300 theoretical plates.Findings: A highly sensitive technic was developed as part of the study for the simultaneous determination of L-tryptophan and thiotriazoline in a model mixture by HPLC (high performance liquid chromatography method, that is planned to be used in further study, was developed as a part of the study. The method is developed not only for L-tryptophan and thiotriazoline standardization in the combined drug, but also for their determination in biological fluids.