Optimization of two-dimensional thin-layer chromatographic separation often relies on selection of the two most orthogonal chromatographic systems which best co-operate in the separation. This is mainly achieved by investigating the correlation between RF values or scoring the distances between the spots. This paper presents and discusses another approach, based on the distances to the closest spot and to the top or bottom of the plate. The theory arises from a well-known geometrical problem about equal-spreading of the points inside a unit square. Two coefficients are proposed — sensitive and insensitive to complete separation, which are the two-dimensional version of previously proposed RU and RD criteria (retention uniformity and retention distance, describing the equal-spreading of the spots in one-dimensional chromatography). They are included in the range 0–1 and their distribution as a random variable is well defined and not affected by the number of separated compounds.
A general equation is presented for modeling retention, using the organic modifier content of the mobile phase. The equation is based on the Box-Cox transform of modifier concentration. Both the semilogarithmic relationship (Soczewiński-Wachtmeister equation) and logarithmic relationship (Snyder-Soczewiński equation) are special cases of the proposed equation. The equation can be fitted easily with free software and an additional coefficient can be interpreted as closeness to the previous models. Even with strong closeness to log-log dependence, the equation still enables extrapolation to zero modifier content. A case study on nine drug-like substances is also discussed, with comparison of 14 previously proposed retention equations found in the literature.
Charged-coupled device (CCD) noise can be a serious problem during videoscanning, especially when scanning dark plates with weakly fluorescent spots. The proper denoising of videoscans inside mathematical environments is a critical part of any advanced chemometric processing. The paper reports comparison and optimization of representative videoscan denoising by different techniques. Several kind of filters (averaging, circular, Gaussian, Savitzky-Golay, median, Wiener, FIR) and wavelet shrinkage (twelve mother wavelets from the Daubechies, Symmlet, and Coiflet family, five decomposition levels, and soft/hard thresholding) were optimized against noise autocorrelation or mean-squared error to the reference image. The reference image was obtained by grabbing and averaging 256 CCD frames. The median filter is the winner of the competition; other filters except Gaussian and wavelet shrinkage at high decomposition level are also sufficient and good ways of videoscan denoising. The Gaussian filter and wavelet shrinkage at low decomposition level performed worst and could not be recommended.
The current guidelines of quantitative method development establish a requirement to validate a set of method parameters and the selectivity is one of the most important parameters to be proven. Currently, one of the most frequently used approaches is to measure the spectrum of a spot together with some reference spots and to correlate them. Additionally, several places of the spot (start, middle, end) can be measured and also correlated. In the current paper we perform a simulation done on 170 real spectra with different levels of contamination and noise, establishing the distribution of correlation coefficient. It can be concluded that contamination up to 10% almost cannot be detected by simple correlation measurement, regardless of the noise level. Moreover, at a high noise level there is a very low margin between correlation of different compounds and the correlation of the same (but noisy) spectra. These observations suggest that the spectral correlation cannot be a reliable measure of method specificity and some another chemometric methods need to be searched in further research.
The paper compares linear, quadratic, and cubical regression together with several weighted and robust approaches in the context of lipophilicity determination. The comparison is done on 35 model compounds on data from different modifiers used on RP18, CN, and silica plates. It can be concluded that the use of weighted and moderately robust regression technique increases correlation between extrapolated retention and real lipophilicity, whereas polynomial and very robust techniques give visibly worse results due to their excessive flexibility and higher extrapolation uncertainty. Additionally, we have compared averaging retention from different modifiers by RF, k, and RM values. The results are similar; however, surprisingly, RF averaging performs slightly better to the other approaches.
A new objective chromatographic response function, RK, based on the kernel density estimate, is introduced for estimation of the fingerprinting performance of a particular TLC system (uniformity of retention) for which a large set of experimental RF values of possible components of the mixture is available. The RK criterion is insensitive to large numbers (hundreds or thousands) of RF values, when the previously proposed criteria cease. It can be applied to one and two-dimensional TLC and is easily computed. As an example of its application, the performance of twelve general screening systems was evaluated in the context of herbal extract fingerprinting (88 phytochemical standards) by both one and two-dimensional TLC.
Authors:T. W. Inglot, A. Gumieniczek, Ł. Komsta, and R. Związek
Comparison of classical densitometry, video-scanning, and capillary electrophoresis was performed for determination of angiotensin II receptor antagonist, valsartan, and calcium channels blocker, amlodipine, in a combined dosage form. Thin layer chromatography was performed on RP8F254 TLC plates with a mobile phase consisting of acetonitrile-phosphate buffer at pH 9.0 (5:5, v/v) and temperature 20 °C. Densitometry was done in the reflectance mode at 217 nm for valsartan and in the absorbance mode at 370 nm for amlodipine. Video-scanning was elaborated at 254 and 366 nm for valsartan and amlodipine, respectively. For chromatographic analysis, calibration plots were constructed in the range of 0.4–2.8 μg per spot for valsartan and 0.02–0.14 μg per spot for amlodipine. Capillary electrophoresis (CE) was performed using a 75 μm × 94 cm fused silica capillary (72 cm effective length), 0.01 mol L−1 borate buffer at pH 8.0, 20 kV voltage, 30 °C temperature, hydrodynamic injection (10 mbar, 6 s) and UV detection at 237 nm. Calibration plots were constructed in the range of 0.1–0.6 mg mL−1 for valsartan and 0.005–0.03 mg mL−1 for amlodipine. All methods were validated in respect to robustness, specificity, stability, linearity, precision, and accuracy. Generally, statistical comparison between the methods did not show significant differences so all procedures are suitable for pharmaceutical analysis.
Authors:M. Gawlik, R. Skibiński, J. Trawiński, and Ł. Komsta
The number of new drugs launched to the market is constantly increasing; however, the metabolism of many of them is still not fully established. The knowledge of drug metabolism pathways is crucial for the efficacy and safety of therapies and, in classical approach, requires the use of animals as well as human volunteers, but this kind of research is expensive and time-consuming. Therefore, nowadays, more and more biological and chemical in vitro methods are developed for the drug metabolism study. This review is focused on the photocatalytic degradation of chemicals and the application of this process in chromatographic methods of drug metabolism research. A theoretical background of photocatalysis and all its applications in a drug metabolism study were reviewed, and other in vitro methods that are actually used were summarized and discussed. Other analytical methods used in this area were also discussed and compared.
Authors:Ł. Komsta, R. Skibiński, A. Bojarczuk, and M. Radoń
This review focuses on the salting-out effect and its use in chromatographic analysis, mainly thin-layer chromatography. First, a theoretical background is given, focusing on the general salting-out theory and a comparison with chaotropic salt addition, practised recently in reversed-phase chromatography. All the salting-out chromatographic applications in literature (from the 1960s to the present moment) are then reviewed.
Authors:M. Dąbrowska, Ł. Komsta, W. Opoka, and M. Starek
The specific aim of this investigation was to study the kinetics of the degradation of cefazolin, cefaclor, cefuroxime axetil, and cefepime in aqueous solution, in the presence (or absence) of various redox agents (iodine solution, potassium permanganate, hydrogen peroxide, sodium thiosulfate, and ascorbic acid) as a function of temperature. Various factors, such as concentration of the analyzed compounds and redox agents, storage time, and temperature, were analyzed. The degradation process of chosen antibiotics was observed chromatographically and fitted to the kinetic models, obtaining model parameters (k, t0.1, t0.5). Principal component analysis (PCA), parallel factor analysis (PARAFAC), and hierarchical cluster analysis (HCA) methods were carried out to interpret the dependencies between these factors on the drug stability.