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Summary

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 R F 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 R U and R D 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.

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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.

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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.

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A new objective chromatographic response function, R K, 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 R F values of possible components of the mixture is available. The R K criterion is insensitive to large numbers (hundreds or thousands) of R F 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.

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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 R F, k, and R M values. The results are similar; however, surprisingly, R F averaging performs slightly better to the other approaches.

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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.

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The thin-layer chromatographic (TLC) retention of 35 model compounds has been investigated with ten screening mobile phases on six normal-phase and seven reversed-phase adsorbents. The retention factors formed two cubes with dimensions 35 × 10 × 6 and 35 × 10 × 7, respectively, which enabled three-way analysis by PARAFAC. A one-component PARAFAC model was optimum in both cases and two-component models performed worse. The one-component model explained 78.8% of the variance in normal-phase chromatography and 94.2% of the variance in reversed-phase chromatography. These results showed that the major variability of the retention factor (R F) can be modelled as the product of three factors related to the substance itself, the mobile phase, and the adsorbent. R F modelling was substantially better than using k or R M (rate mobility) values.

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Acta Chromatographica
Authors: Ł. Komsta, R. Skibiński, A. Bojarczuk, and M. Radoń

Summary

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.

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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.

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Ultra high-performance liquid chromatography (UHPLC) coupled with high-resolution quadrupole time-of-flight (Q-TOF) mass spectrometry was used for the preliminary photodegradation study of nine new generation psychotropic drugs. Based on the above method, two ionization sample modes — electrospray and atmospheric pressure chemical ionization were used for the registration of photodegradation profiles of the selected drugs. Multivariate chemometric analysis showed that electrospray ionization (ESI) method is more specific than atmospheric pressure chemical ionization (APCI) in high-resolution mass spectrometry (HR-MS) analysis of the analyzed psychotropic drugs. It was noticed that, with the use of ESI method, more potential photodegradation products can be identified and lower limits of its detection can be obtained.

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