Column Characterization and Selection Systems in Reversed-Phase High-Performance Liquid Chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most popular chromatographic mode, accounting for more than 90% of all separations. HPLC itself owes its immense popularity to it being relatively simple and inexpensive, with the equipment being reliable and easy to operate. Due to extensive automation, it can be run virtually unattended with multiple samples at various separation conditions, even by relatively low-skilled personnel. Currently, there are >600 RP-HPLC columns available to end users for purchase, some of which exhibit very large differences in selectivity and production quality. Often, two similar RP-HPLC columns are not equally suitable for the requisite separation, and to date, there is no universal RP-HPLC column covering a variety of analytes. This forces analytical laboratories to keep a multitude of diverse columns. Therefore, column selection is a crucial segment of RP-HPLC method development, especially since sample complexity is constantly increasing. Rationally choosing an appropriate column is complicated. In addition to the differences in the primary intermolecular interactions with analytes of the dispersive (London) type, individual columns can also exhibit a unique character owing to specific polar, hydrogen bond, and electron pair donor-acceptor interactions. They can also vary depending on the type of packing, amount and type of residual silanols, "end-capping", bonding density of ligands, and pore size, among others. Consequently, the chromatographic performance of RP-HPLC systems is often considerably altered depending on the selected column. Although a wide spectrum of knowledge is available on this important subject, there is still a lack of a comprehensive review for an objective comparison and/or selection of chromatographic columns. We aim for this review to be a comprehensive, authoritative, critical, and easily readable monograph of the most relevant publications regarding column selection and characterization in RP-HPLC covering the past four decades. Future perspectives, which involve the integration of state-of-the-art molecular simulations (molecular dynamics or Monte Carlo) with minimal experiments, aimed at nearly "experiment-free" column selection methodology, are proposed.

Analysis of Isocratic-Chromatographic-Retention Data using Bayesian Multilevel Modeling.

The objective of this work was to develop a multilevel (hierarchical) model based on isocratic-reversed-phase-high-performance-chromatographic data collected in methanol and acetonitrile for 58 chemical compounds. Such a multilevel model is a regression model of the analyte-specific chromatographic measurements, in which all the regression parameters are given a probability model. It is a fundamentally different approach from the most common approach, where parameters are separately estimated for each analyte (without sharing information across analytes and different organic modifiers). The statistical analysis was done with Stan software implementing the Bayesian-statistics inference with Markov-chain Monte Carlo sampling. During the model-building process, a series of multilevel models of different complexity were obtained, such as (1) a model with no pooling (separate models were fitted for each analyte), (2) a model with partial pooling (a common distribution was used for analyte-specific parameters), and (3) a model with partial pooling as well as a regression model relating analyte-specific parameters and analyte-specific properties (QSRR equations). All the models were compared with each other using 10-fold cross-validation. The benefits of multilevel models in inference and predictions were shown. In particular the obtained models allowed us to (i) better understand the data and (ii) solve many routine analytical problems, such as obtaining well-calibrated predictions of retention factors for an analyte in acetonitrile-containing mobile phases given zero, one, or several measurements in methanol-containing mobile phases and vice versa.

Quantitative determination of trigonelline in mouse serum by means of hydrophilic interaction liquid chromatography-MS/MS analysis: Application to a pharmacokinetic study.

Trigonelline is a pyridine alkaloid found in fenugreek seeds and coffee beans. Most of the previous studies are concerned with the quantification of trigonelline along with other constituents in coffee herbs or beverages. Only a few have focused on its determination in animal or human tissues by applying different modes of HPLC with UV or MS detection. The aim of the study was to develop and validate a fast and simple method for trigonelline determination in serum by the use of hydrophilic interaction liquid chromatography (HILIC) with ESI-MS/MS detection. Separation of trigonelline was achieved on a Kinetex HILIC column operated at 35°C with acetonitrile-ammonium formate (10 mm, pH = 3) buffer mixture (55:45, v/v) as the mobile phase. The developed method was successfully applied to determine trigonelline concentration in mouse serum after intravenous administration of 10 mg/kg. The developed assay is sensitive (limit of detection = 1.5 ng/mL, limit of quantification = 5.0 ng/mL) and linear in a concentration range from 5.0 to 250.0 ng/mL. Sample preparation is limited to deproteinization, centrifugation and filtration. The application of the HILIC mode of chromatography with MS detection and selection of deuterated trigonelline as internal standard allowed a rapid and precise method of trigonelline quantification to be to developed.

How to model temporal changes in nontargeted metabolomics study? A Bayesian multilevel perspective.

Analysis of time series data addresses the question on mechanisms underlying normal physiology and its alteration under pathological conditions. However, adding time variable to high-dimension, collinear, noisy data is a challenge in terms of mining and analysis. Here, we used Bayesian multilevel modeling for time series metabolomics in vivo study to model different levels of random effects occurring as a consequence of hierarchical data structure. A multilevel linear model assuming different treatment effects with double exponential prior, considering major sources of variability and robustness to outliers was proposed and tested in terms of performance. The treatment effect for each metabolite was close to zero suggesting small if any effect of cancer on metabolomics profile change. The average difference in 964 signals for all metabolites varied by a factor ranging from 0.8 to 1.25. The inter-rat variability (expressed as a coefficient of variation) ranged from 3-30% across all metabolites with median around 10%, whereas the inter-occasion variability ranged from 0-30% with a median around 5%. Approximately 36% of metabolites contained outlying data points. The complex correlation structure between metabolite signals was revealed. We conclude that kinetics of metabolites can be modeled using tools accepted in pharmacokinetics type of studies.

How Much Can We Learn from a Single Chromatographic Experiment? A Bayesian Perspective.

In this work, we proposed and investigated a Bayesian inference procedure to find the desired chromatographic conditions based on known analyte properties (lipophilicity, pKa, and polar surface area) using one preliminary experiment. A previously developed nonlinear mixed effect model was used to specify the prior information about a new analyte with known physicochemical properties. Further, the prior (no preliminary data) and posterior predictive distribution (prior + one experiment) were determined sequentially to search towards the desired separation. The following isocratic high-performance reversed-phase liquid chromatographic conditions were sought: (1) retention time of a single analyte within the range of 4-6 min and (2) baseline separation of two analytes with retention times within the range of 4-10 min. The empirical posterior Bayesian distribution of parameters was estimated using the "slice sampling" Markov Chain Monte Carlo (MCMC) algorithm implemented in Matlab. The simulations with artificial analytes and experimental data of ketoprofen and papaverine were used to test the proposed methodology. The simulation experiment showed that for a single and two randomly selected analytes, there is 97% and 74% probability of obtaining a successful chromatogram using none or one preliminary experiment. The desired separation for ketoprofen and papaverine was established based on a single experiment. It was confirmed that the search for a desired separation rarely requires a large number of chromatographic analyses at least for a simple optimization problem. The proposed Bayesian-based optimization scheme is a powerful method of finding a desired chromatographic separation based on a small number of preliminary experiments.

The pharmacokinetics of dexmedetomidine during long-term infusion in critically ill pediatric patients. A Bayesian approach with informative priors.

The purpose of this study was to assess the pharmacokinetics of dexmedetomidine in the ICU settings during the prolonged infusion and to compare it with the existing literature data using the Bayesian population modeling with literature-based informative priors. Thirty-eight patients were included in the analysis with concentration measurements obtained at two occasions: first from 0 to 24 h after infusion initiation and second from 0 to 8 h after infusion end. Data analysis was conducted using WinBUGS software. The prior information on dexmedetomidine pharmacokinetics was elicited from the literature study pooling results from a relatively large group of 95 children. A two compartment PK model, with allometrically scaled parameters, maturation of clearance and t-student residual distribution on a log-scale was used to describe the data. The incorporation of time-dependent (different between two occasions) PK parameters improved the model. It was observed that volume of distribution is 1.5-fold higher during the second occasion. There was also an evidence of increased (1.3-fold) clearance for the second occasion with posterior probability equal to 62 %. This work demonstrated the usefulness of Bayesian modeling with informative priors in analyzing pharmacokinetic data and comparing it with existing literature knowledge.

Maximum A Posteriori Bayesian Estimation of Chromatographic Parameters by Limited Number of Experiments.

The aim of this work was to develop a nonlinear mixed-effect chromatographic model able to describe the retention times of weak acids and bases in all possible combinations of organic modifier content and mobile-phase pH. Further, we aimed to identify the influence of basic covariates, like lipophilicity (log P), dissociation constant (pK(a)), and polar surface area (PSA), on the intercompound variability of chromatographic parameters. Lastly, we aimed to propose the optimal limited experimental design to the estimation process of parameters through a maximum a posteriori (MAP) Bayesian method to facilitate the method development process. The data set comprised retention times for two series of organic modifier content collected at different pH for a large series of acids and bases. The obtained typical parameters and their distribution were subsequently used as priors to improve the estimation process from reduced design with a variable number of preliminary experiments. The MAP Bayesian estimator was validated using two external-validation data sets. The common literature model was used to relate analyte retention time with mobile-phase pH and organic modifier content. A set of QSRR-based covariate relationships was established. It turned out that four preliminary experiments and prior information that includes analyte pK(a), log P, acid/base type, and PSA are sufficient to accurately predict analyte retention in virtually all combined changes of pH and organic modifier content. The MAP Bayesian estimator of all important chromatographic parameters controlling retention in pH/organic modifier gradient was developed. It can be used to improve parameter estimation using limited experimental design.

Pharmacokinetics and pharmacodynamics of propofol in cancer patients undergoing major lung surgery.

Despite the growing number of cancer cases and cancer surgeries around the world, the pharmacokinetics (PK) and pharmacodynamics (PD) of anesthetics used in this population are poorly understood. Patients operated due to cancer are usually in severe state and often require chemotherapy. It might affect the PK/PD of drugs used in this population. Therefore, in this study we explored the PK/PD of propofol in cancer patients having a major lung surgery. 23 patients that underwent a propofol-fentanyl total intravenous anesthesia were included in the analysis. A large set of demographic, biochemical and hemodynamic parameters was collected for the purpose of covariate analysis. Nonlinear mixed effect modeling in NONMEM was used to analyze the collected data. A three-compartment model was sufficient to describe PK of propofol. The anesthetic effect (AAI index) was linked to the propofol effect site concentrations through a sigmoidal E max model. A slightly higher value of clearance, a lower value of distribution clearance, and a decreased volume of peripheral compartment were observed in our patients, as compared with the literature values reported for healthy volunteers by Schnider et al. and by Eleveld et al. Despite these differences, both models led to a clinically insignificant bias of -8 and -1 % in concentration predictions, as reflected by the median performance error. The C e50 and propofol biophase concentration at the time of postoperative orientation were low and equaled 1.40 and 1.13 mg/L. The population PK/PD model was proposed for cancer patients undergoing a major lung surgery. The large body of studied covariates did not affect PK/PD of propofol significantly. The modification of propofol dosage in the group of patients under study is not necessary when TCI-guided administration of propofol by means of the Schnider model is used.

Determination of ascorbic acid and its degradation products by high-performance liquid chromatography-triple quadrupole mass spectrometry.

This study describes application of liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS) for evaluation of vitamin C stability, the objective being prediction of the degradation products. Detection was performed with an UV detector (UV-Vis) in sequence with a triple-quad mass spectrometer in the multiple reaction mode. The negative ion mode of ESI and MS-MRM transitions of m/z 175→115 (quantifier) and 175→89 (qualifier) for ascorbic acid was used. All the validation parameters were within the range of acceptance proposed by the Food and Drug Administration. The method was fully validated in terms of linearity, LOD, LOQ, accuracy, and interday precision. Validation experiments revealed good linearity with R(2) = 0.999 within the established concentration range, and excellent repeatability (9.3%). The LOD of the method was 0.1524 ng/mL whereas the LOQ was 0.4679 ng/mL. LC-MS methodology proves to be an improved, simple, and fast approach to determining the content of vitamin C and its degradation products with high sensitivity, selectivity, and resolving power within 6 minutes of analysis.

Development and validation of UHPLC method for the determination of cyclosporine A in biological samples.

The aim of the study was to develop and validate a simple and rapid method for the determination of cyclosporine A (CsA) in ocular rabbit tissues using reversed-phase ultra-high-performance liquid chromatography (UHPLC) with UV detection. Previous publications on chromatographic methods of CsA determination in ocular tissues involved only reversed-phase HPLC separation, usually in combination with such detection techniques as radio-immunoassay and mass spectrometry. The application of the UHPLC technique allowed us to significantly decrease the analysis time. Cyclosporine D (CsD) was applied as the internal standard. Satisfactory separation was achieved on an XB-C18 Kinetex column at 60°C with the use of gradient elution mode. The retention times of CsA and CsD were found to be 4.5 and 5.1 min, respectively. The developed assay is specific, sensitive (limit of detection = 6 ng/mL and limit of quantitation = 18 ng/mL) and linear within the analyte concentration range of 0.018-5 µg/mL, with a correlation coefficient of 0.999. High sensitivity, low injection volume (10 µL), short time of analysis (6.5 min) and simplicity make this method useful for the fast analysis of CsA in rabbit ocular tissues and fluids: lacrimal fluid, aqueous humor, cornea, conjunctiva and eye globe.

The quantification of reticulocyte maturation and neocytolysis in normal and erythropoietin stimulated rats.

A technique has recently been proposed for obtaining the reticulocyte (RET) age distribution from the flow cytometric reticulocyte count. It allows for a quantitative characterization of reticulocyte dynamics. In this work this technique was applied to characterize the blood, bone marrow and spleen reticulocytes in homeostatic and erythropoietically stimulated rats in order to determine the reticulocyte maturation times in the bone marrow and blood; and to confirm the presence of ineffective erythropoiesis (neocytolysis). The latter was done by comparing the reticulocyte removal rate from blood with bilirubin formation after erythropoiesis stimulation. A single subcutaneous dose (4050 IU/kg) of recombinant human erythropoietin (rHuEPO) was administered to rats, then their reticulocytes were stained with thiazole orange and the distribution of the fluorescent signal measured using flow cytometry. The obtained signal distribution of the reticulocytes was transformed to the age distribution and a set of basic parameters reflecting reticulocyte dynamics was determined. Bilirubin concentrations were measured to directly assess the presence of reticulocyte irreversible removal. The bilirubin formation was found to be considerably modulated by rHuEPO and corresponded well to the determined reticulocyte removal rate. The initial increase and subsequent decrease of the reticulocyte maturation time in blood was quantitated and directly linked with RET mobilization from the bone marrow. A substantial number (60%) of reticulocytes is sequestrated during homeostasis in rats. This number increases and then decreases after rHuEPO administration, as also reflected by bilirubin formation. Flow cytometry seems to be an excellent method for studying RET dynamics and the presence of young RBC neocytolysis.

Determination of Water-Soluble Components of Abdominal Secretion of Grasshopper (Chorthippus spp.) by GC/MS/MS in Search for Potential Wound Healing Agents.

Wound healing is still a serious medical problem due to process complexity and lack of effective medicaments. This is particularly true in the treatment of wounds arising in the course of such diseases as AIDS or diabetes. Therefore, scientific efforts are focused on the search for new compounds of natural origin, which could be used as medicines or evaluated for subsequent drug design. In folk medicine, grasshopper (Chorthippus spp.) abdominal secretion has been used to accelerate the wound healing process. In this context, the knowledge of the composition of grasshopper abdominal secretion is crucial. The aim of this study was to determine the main water-soluble components of grasshopper abdominal secretion with the use of GC/MS/MS. Liquid-liquid extraction was used as a pretreatment method to clean up, concentrate and fractionate compounds from the complex insect matrix. To obtain more stable and volatile compounds, necessary for GC/MS/MS analysis, a double-step derivatization process was carried out with the use of methoxyamine hydrochloride and a mixture of bis-N,O-trimethylsilyl trifluoroacetamide and chlorotrimethylsilane. As a result, 2,108 compounds were identified, mainly as amino acids, carbohydrates and organic acids. Some of the identified compounds are emphasized due to antimicrobial, antifungal or antioxidant activities reported in the literature. Moreover, a set of compounds characteristic for Chorthippus spp. samples has been selected. In the last part of the study, a statistical analysis was performed to demonstrate differences in composition of aqueous fractions of abdominal secretions from grasshoppers collected at two distant locations: Starogard Gdanski and Lubianka meadows.

Partial least square and hierarchical clustering in ADMET modeling: prediction of blood-brain barrier permeation of α-adrenergic and imidazoline receptor ligands.

PURPOSE. Rate of brain penetration (logPS), brain/plasma equilibration rate (logPS-brain), and extent of blood-brain barrier permeation (logBB) of 29 α-adrenergic and imidazoline-receptors ligands were examined in Quantitative-Structure-Property Relationship (QSPR) study. METHODS. Experimentally determined chromatographic retention data (logKw at pH 4.4, slope (S) at pH 4.4, logKw at pH 7.4, slope (S) at pH 7.4, logKw at pH 9.1, and slope (S) at pH 9.1) and capillary electrophoresis migration parameters (µeff at pH 4.4, µeff at pH 7.4, and µeff at pH 9.1), together with calculated molecular descriptors, were used as independent variables in the QSPR study by use of partial least square (PLS) methodology. RESULTS. Predictive potential of the formed QSPR models, QSPR(logPS), QSPR(logPS-brain), QSPR(logBB), was confirmed by cross- and external validation. Hydrophilicity (Hy) and H-indices (H7m) were selected as significant parameters negatively correlated with both logPS and logPS-brain, while topological polar surface area (TPSA(NO)) was chosen as molecular descriptor negatively correlated with both logPS and logBB. The principal component analysis (PCA) and hierarchical clustering analysis (HCA) were applied to cluster examined drugs based on their chromatographic, electrophoretic and molecular properties. Significant positive correlations were obtained between the slope (S) at pH 7.4 and logBB in A/B cluster and between the logKw at pH 9.1 and logPS in C/D cluster. CONCLUSIONS. Results of the QSPR, clustering and correlation studies could be used as novel tool for evaluation of blood-brain barrier permeation of related α-adrenergic/imidazoline receptor ligands.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.PURPOSE. Rate of brain penetration (logPS), brain/plasma equilibration rate (logPS-brain), and extent of blood-brain barrier permeation (logBB) of 29 α-adrenergic and imidazoline-receptors ligands were examined in Quantitative-Structure-Property Relationship (QSPR) study. METHODS. Experimentally determined chromatographic retention data (logKw at pH 4.4, slope (S) at pH 4.4, logKw at pH 7.4, slope (S) at pH 7.4, logKw at pH 9.1, and slope (S) at pH 9.1) and capillary electrophoresis migration parameters (µeff at pH 4.4, µeff at pH 7.4, and µeff at pH 9.1), together with calculated molecular descriptors, were used as independent variables in the QSPR study by use of partial least square (PLS) methodology. RESULTS. Predictive potential of the formed QSPR models, QSPR(logPS), QSPR(logPS-brain), QSPR(logBB), was confirmed by cross- and external validation. Hydrophilicity (Hy) and H-indices (H7m) were selected as significant parameters negatively correlated with both logPS and logPS-brain, while topological polar surface area (TPSA(NO)) was chosen as molecular descriptor negatively correlated with both logPS and logBB. The principal component analysis (PCA) and hierarchical clustering analysis (HCA) were applied to cluster examined drugs based on their chromatographic, electrophoretic and molecular properties. Significant positive correlations were obtained between the slope (S) at pH 7.4 and logBB in A/B cluster and between the logKw at pH 9.1 and logPS in C/D cluster. CONCLUSIONS. Results of the QSPR, clustering and correlation studies could be used as novel tool for evaluation of blood-brain barrier permeation of related α-adrenergic/imidazoline receptor ligands. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Microscopic and Biopharmaceutical Evaluation of Emulsion and Self-Emulsifying Oil with Cyclosporine.

Among the currently available commercial eye drops with cyclosporine A (Cs) there is a lack of long-acting dosage forms and products with a concentration of the drug substance higher than 0.1%, although Cs is widely used in ophthalmology. The aim of the research was to conduct the microscopic and biopharmaceutical evaluation of two formulations, an emulsion (EM) and a self-emulsifying oil (SEO), both with 0.5% of Cs, proposed for use in eye drops, and the comparison of both. SEO eye drops with Cs or any other drug substance are currently not available as marketed products, and the highest concentration of Cs in the ocular emulsion is only 0.1%. The microscopic evaluation of the emulsion and the SEO after emulsification with water was carried out using a high-resolution digital microscopy. The properties of both preparations were compared using the high dynamic range function or optical shadow effect mode. Images in the 3D composition mode were also recorded. The in vivo study of the Cs formulations was performed on male albino rabbits. The eye tolerance of the preparations was assessed using the ocular irritation test, which is a modified Draize test. Placebo carriers (without the drug substance) were also subjected to irritation testing. The concentration of Cs in the tissues (cornea and conjunctiva) and fluids (tear fluid and aqueous humor) of the rabbit eye was determined after multiple instillations of Cs-EM or Cs-SEO. The tested preparations were compared using the digital microscopy technique, which highlights the features of the formulations and eliminates the risk of unnoticeable properties that are difficult to observe in classical optical microscopy. Both tested Cs-loaded formulations are classified as practically non-irritating. There were also no significant differences when testing the placebo carriers. After a topical administration, Cs was widely distributed in all tissues (e.g., in cornea 1.3 ng/mg and 1.0 ng/mg) and fluids of the eye (e.g., in tear fluid 11.6 µg/mL and 4.3 µg/mL), after the administration of Cs-SEO and Cs-EM, respectively. The obtained results allow us to recognize both tested formulations, the emulsion and the self-emulsifying oil with 0.5% Cs content, as carriers safe for ophthalmic use and effective in delivering the drug substance to the structures of the eye.

Reversed-phase TLC and HPLC retention data in correlation studies with in silico molecular descriptors and druglikeness properties of newly synthesized anticonvulsant succinimide derivatives.

The properties relevant to pharmacokinetics of two series of newly synthesized succinimide derivatives have been studied. The properties under consideration have been either determined empirically, by reversed-phase liquid chromatography (TLC and HPLC technique), or calculated with the use of established theoretical medicinal chemistry/drug design software. Chromatographic techniques allowed determination of the retention constants R(M)° and log k(w), which characterize lipophilicity of compounds. Considering potential pharmaceutical importance of succinimide derivatives, we (i) examined the retention behavior in the reversed-phase liquid chromatographic (RP LC) systems, in both planar and column LC, and (ii) determined the relationships between chromatographic data and selected structural features of analytes that are believed to markedly affect their processes of absorption, distribution, metabolism, excretion and toxicity (ADMETox). Significant relationships were found between the retention constants, R(M)° and log k(w), and the in silico calculated bioactivity descriptors, in particular HIA (human intestinal absorption) and PPB (plasma protein binding) parameters. The R(M)° and log k(w) values of the investigated compounds have been recommended for description of their lipophilicity and evaluating pharmacokinetic properties. In view of results of this study the newly synthesized succinimide agents meet pharmacokinetic criteria of preselection of drug candidates and hence qualify for pharmacodynamic phase of antiepileptic drug development. Best compromising human intestinal absorption and plasma protein binding features appear to be compounds A4, A5, A10 and A11.

The state-of-the-art determination of urinary nucleosides using chromatographic techniques "hyphenated" with advanced bioinformatic methods.

Over the last decade metabolomics has gained increasing popularity and significance in life sciences. Together with genomics, transcriptomics and proteomics, metabolomics provides additional information on specific reactions occurring in humans, allowing us to understand some of the metabolic pathways in pathological processes. Abnormal levels of such metabolites as nucleosides in the urine of cancer patients (abnormal in relation to the levels observed in healthy volunteers) seem to be an original potential diagnostic marker of carcinogenesis. However, the expectations regarding the diagnostic value of nucleosides may only be justified once an appropriate analytical procedure has been applied for their determination. The achievement of good specificity, sensitivity and reproducibility of the analysis depends on the right choice of the phases (e.g. sample pretreatment procedure), the analytical technique and the bioinformatic approach. Improving the techniques and methods applied implies greater interest in exploration of reliable diagnostic markers. This review covers the last 11 years of determination of urinary nucleosides conducted with the use of high-performance liquid chromatography in conjunction with various types of detection, sample pretreatment methods as well as bioinformatic data processing procedures.

Reversed-phase pH gradient thin-layer chromatography of biologically active substances with controlled developing solvent velocity.

For the first time, stepwise pH gradient thin-layer chromatograms of biologically active substances with controlled developing solvent velocity are presented and described in the paper. Change in buffer pH of the mobile phase solution influences retardation, selectivity, and shape of the separated substances' spots. The conducted research has confirmed that the mobile phase's pH gradient could be an essential factor to optimize the conditions of the separation of substances in reversed-phase high-performance thin-layer chromatography. The reproducibility of the gradient retardation factor values of separated substance zones is satisfactory.

pH Gradient as a tool for the separation of ionizable analytes in reversed-phase high-performance chromatography.

The aim of this work was to propose a general scheme of optimizing separation of ionizable analytes and to determine conditions of maximal peak compression in pH-gradient reversed-phase high-performance liquid chromatography (RP HPLC). The approximated explicit equation of the linear pH gradient has been developed. It allows predicting retention times for a given organic modifier content, initial value of pH, and the start and steepness of the pH gradient. Also the formula for calculating maximal peak compression is provided. The developed theory was compared with experimental data on the example of a weak acid (ketoprofen) and a weak base (papaverine). Five parameters characterizing analyte retention (log k(w) and S of the ionized and nonionized forms along with pK(a,chrom)) were determined in a series of isocratic experiments carried out at different pH values and with different methanol contents in the eluent. Next, a series of pH gradients of different pH-gradient steepness and of different pH-gradient starting time has been obtained and used to test the validity of our theoretical approach. The conditions of maximal peak width compression have been found. The derived theory was proved to be in a good agreement with the experimental data. The pH-gradient method led to peak compression of up to 0.2, and minimized peak tailing was obtained for the tested analytes. Since the majority of analytical separations are done in an isocratic mode we proposed a means to transfer an isocratic method to a pH-gradient method.