Derivative Chromatogram Research Articles

Protein profile study of breast‐tissue homogenates by HPLC‐LIF

Proteomics is a promising approach for molecular understanding of neoplastic processes including response to treatment. Widely used 2D-gel electrophoresis/Liquid chromatography coupled with mass spectrometry (LC-MS) are time consuming and not cost effective. We have developed a high-sensitivity (femto/subfemtomoles of protein/20 mul) High Performance Liquid Chromatography-Laser Induced Fluorescence HPLC-LIF instrument for studying protein profiles of biological samples. In this study, we have explored the feasibility of classifying breast tissues by multivariate analysis of chromatographic data. We have analyzed 13 normal, 17 malignant, 5 benign and 4 post-treatment breast-tissue homogenates. Data was analyzed by Principal Component Analysis PCA in both unsupervised and supervised modes on derivative and baseline-corrected chromatograms. Our findings suggest that PCA of derivative chromatograms gives better classification. Thus, the HPLC-LIF instrument is not only suitable for generation of chromatographic data using femto/subfemto moles of proteins but the data can also be used for objective diagnosis via multivariate analysis. Prospectively, identified fractions can be collected and analyzed by biochemical and/or MS methods.

Validation of an analytical method to determine sulfamides in kidney by HPLC-DAD and PARAFAC2 with first-order derivative chromatograms

Six sulfamides were extracted from kidney and analysed by high-performance liquid chromatography with diode array detection (HPLC-DAD): sulfadiazine, sulfamethazine, sulfamethoxypyridazine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline. Two main difficulties arose in identifying and quantifying the analytes. Firstly, the chromatographic peaks of the matrix interferences overlapped with those of the analytes. The uniqueness property of PARAFAC2 solved this problem. Secondly, the gradient elution caused a baseline drift. The first-order derivative of the chromatograms minimized its effect. The analytical method was validated. As the performance criteria detailed in the European Decision 2002/657/EC are based on specific signals, this paper generalizes those criteria for higher-order and non-specific signals. In this sense the proposed methodology is general and can be applied to any chromatographic method (HPLC or GC) with a detector that provide a multivariate signal (MS, DAD, EC, etc.).

Separation and determination of podophyllum lignans by micellar electrokinetic chromatography

A micellar electrokinetic chromatography method was established for the quantitative analysis of seven podophyllum lignans in Podophyllum emodi Wall. var. chinesis sprague. The optimum buffer system was 10 m M NaH 2PO 4–5 m M borate–100 m M sodium dodecylsulfate–30% isopropanol (pH 7.20). Voltage was 18 kV and detection at 214 nm. The second derivative chromatogram was used to determine a low-content component and those not fully separated from adjacent ones. The RSD values of migration times and peak areas were <2.2 and <5.5%, respectively. The effects of several CE parameters on the resolutions were studied systematically.

Stepwise Self-Association of Penicillin V As Deduced by Frontal Derivative Chromatography

The aggregation pattern of penicillin V (PCV) in a 0.15 M potassium chloride solution at 298.2 K is investigated by frontal derivative chromatography on Sephadex G-10 columns and is quantitatively analyzed on the basis of a stepwise aggregation model and a trimer−dodecamer model. The aggregation parameters for these models are best fitted to the observed centroid volume data. The derivative chromatogram of PCV is simulated on the basis of plate theory with the aggregation models. The height and position of the peak simulated on the basis of the stepwise aggregation model are in excellent agreement with the observed ones, whereas those simulated on the basis of the trimer−dodecamer model remarkably differ from the observed ones, particularly in dilute solutions. Thus, PCV forms dimer and self-associates stepwise.

Self-Association Patterns of Sodium Taurocholate and Taurodeoxycholate As Studied by Frontal Derivative Chromatography

The aggregation patterns of sodium taurocholate (TC) and taurodeoxycholate (TDC) in an isotonic sodium chloride solution at 298.2 K are investigated by frontal derivative chromatography on Sephadex G-10 columns and are quantitatively analyzed on the basis of several aggregation models. The aggregation parameters are best fitted to the observed centroid volume data. The derivative chromatogram of TC at 6.006 mM simulated on the basis of a stepwise aggregation model is in excellent agreement with the observed one, whereas the simulation on the basis of the pentamerization model remarkably differs in shape from the observed one. The derivative chromatograms of TDC at 1.500 and 2.003 mM simulated on the basis of another stepwise aggregation model are closer to the observed ones than those of the Bottari−Festa model. Thus, TC and TDC form their dimers and polydisperse micelles. These conclusions are supported by the fitness to the centroid volume data. In light of the present results and current reports, the structure and counterion binding of bile salt micelles are discussed.

Micellar liquid chromatography of plant growth regulators detected by derivative fluorometry

A micellar liquid chromatographic method with fluorimetric detection was developed for the determination of the plant growth regulators indol 3-yl acetic acid (IAA), 2-(1-naphthyl) acetic acid (1-NAA), indol 3-yl propionic acid (IPA), 2-(2-naphthyl) acetic acid (2-NAA), indol 3-yl butyric acid (IBA), 2-(1-naphthyl) acetamide (1-Namide) and indol 3-yl acetic acid ethyl ester (IAA ethyl ester). Extraction of plant hormones was performed with methanol, followed by purification with ethyl acetate, solid-phase extraction and again partitioning against diethyl ether. Sodium dodecylsulphate (10 m M) is used as mobile phase to elute the compounds in a maximum run-time of 23 min. Partially or unresolved peaks are separated by calculation of the 1st derivative chromatogram. Detection limits are between 0.30 μg g −1 and 1.10 μg g −1.

Nonmicellar Self-Association of Propantheline Bromide: Effect of the Column Length on the Chromatogram

The self-association behavior of propantheline bromide in a 154 mmol dm-3 sodium bromide solution has been investigated by frontal chromatography on two Sephadex columns and by the surface tension method. Analysis of centroid volume data by asymptotic theory yields the monomer concentration of propantheline bromide as a function of its total concentration. Further analysis of the concentration dependence of the monomer concentration by a multiple equilibrium model provides weight-average and number-average aggregation numbers. The aggregates of propantheline bromide are small and are formed in a stepwise manner without a cmc. Stepwise aggregation constants for propantheline bromide are estimated and are used to predict the aggregate size distribution. Derivative chromatograms support the above results based on the centroid volume data. For instance, the shape of the derivative chromatogram of propantheline bromide at the trailing boundary is closer to that of penicillin V than to that of the surfactant, suggesting that propantheline bromide self-associates in a nonmicellar mode. The dimerization constant of propantheline bromide estimated from the trailing peak volume is close to that obtained from the centroid volume. The shape of the frontal chromatogram is almost independent of the size of the column. This fact indicates that our chromatographic method for investigating the self-association behavior is reliable. The surface tension of the propantheline bromide solution has a transition, albeit diffuse, when plotted against the logarithm of the total concentration and decreases gradually with increasing concentration above this transition. A reasonable molecular area of propantheline bromide on the aqueous surface is obtained by using the monomer concentration, determined by the chromatographic data, in the Gibbs adsorption equation.

Evaluation of different techniques for peak purity assessment on a diode-array detector in liquid chromatography

The efficiencies of nine different techniques implemented on a diode-array detector in liquid chromatography for peak homogeneity determination were compared at a concentration level of a drug that can be found in biological samples. Solutions of cefotaxime sodium (analyte) spiked with various amounts of a model impurity (theophylline) were injected under chromatographic conditions giving severe overlap of the peaks ( R S = 0.14). Under the conditions used, the most efficient techniques implemented in the instrument were spectral suppression (ca. 0.5%), derivative spectrum (<1%), followed by spectral overlay, absorbance ratio plot (<5%), multiple-wavelength chromatograms overlay and purity parameter (ca. 5%). The numerical value of the absorbance ratio could also be exploited statistically to detect 0.5% of impurity. Theophylline at levels up to 10% could not be detected using three-dimensional plots, contour diagrams and derivative chromatograms.

Frontal Chromatography for Nonassociable and Micellar Systems

Abstract The continuous flow model of chromatography for computer simulations is developed and applied for nonassociable, dimerizing, and micellar systems. This model is more realistic than the discontinuous flow model, since the former takes into account the infinitesimal flow of the mobile phase. The frontal and zonal chromatograms of sucrose, a nonassociable solute, are determined on a Sephadex G-10 column as a function of the total sucrose concentration. The derivative of the frontal chromatogram at the leading boundary completely overlaps with that at the trailing boundary and has the same shape with the zonal chromatogram. Therefore the number of plate can be calculated from the observed derivative chromatogram. For some dimerizing systems the dimerization constant can be estimated from the concentration dependence of the derivative at the trailing boundary, if an appropriate correction is applied. Some characteristic parameters obtained from the derivative chromatogram of the micellar system provide information on the aggregation pattern. The observed results outlined above can be semiquantitatively predicted by the continuous flow model and by the discontinuous flow model, if the void volume and the number of plate are regarded as adjustable parameters in the latter model.

Self-Association of Penicillins in Aqueous Solution as Revealed by Gel Filtration Chromatography.

The self-association of penicillin G (PCG) and penicillin V (PCV) in 0.15 M potassium chloride solution has been investigated by frontal gel filtration chromatography (GFC) on Sephadex G-10 columns at 25°C. Analysis of the centroid volume, Vc, of a GFC profile yields the monomer concentration of a penicillin and the weight-average aggregation number, nw. No critical micelle concentration is observed in the Vc vs. concentration profile for PCV. The nw value for PCV is 2 at low concentrations and gradually increases with increasing concentration. PCG forms dimers only below 0.06 M, and provides useful GFC data for the investigation of dimerization. The dimerization constants of PCG and PCV are also evaluated from the concentration dependence of the derivative peak position at the trailing boundary. Stepwise aggregation constants, obtained from non-linear least-squares fitting of the centroid volume data, satisfactorily reproduce observed derivative chromatograms, and are used for calculations of the aggregate size distribution of PCV. The structure-association relationship and the effect of self-association on the bactericidal activity and chemical stability of the penicillins are discussed.

Chromatographic study on stepwise aggregation patterns of zwitterionic derivatives of cholic acid

The frontal chromatogram of the 2-hydroxy derivative (CHAPSO) of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) has been obtained on a Sephadex G-10 column at 298 K. From the centroid volume, Vc, of the chromatogram, the monomer concentration, the weight- and number-average aggregation numbers (nw and nn), the critical micelle concentration (c.m.c.) and the dimerization constant, k2, are evaluated, and furthermore, stepwise aggregation constants, ki, are estimated on the basis of several self-association models. At small aggregation numbers, an odd–even alternation of the aggregation number dependence of ki is found and CHAPSO self-associates cooperatively, whereas it does so anti-cooperatively at high aggregation numbers. The c.m.c. depends on the treatment of the Vc data, and this result suggests that CHAPSO forms small micelles. The derivative chromatogram of CHAPSO and some characteristic values obtained therefrom are markedly different from those of a dye and typical surfactants, and reflect their aggregation patterns. The derivative chromatogram simulated on the basis of the above aggregation models is very similar to the observed one. The differences in limiting aggregation constant k and nw/nn between CHAPSO and CHAPS are explained on the basis of the fact that the head group of CHAPSO is more bulky than that of CHAPS. The micelle size distribution of CHAPSO is calculated on the basis of the above aggregation models. The concentrations of the monomer and each aggregate species will be useful for applications of CHAPSO and CHAPS to the solubilization of membrane proteins as well as for the investigation of the aggregation patterns of bile salts.

Gel filtration chromatographic study on the self-association of surfactants and related compounds

After a brief survey of gel filtration chromatography (GFC) and the self-association equilibrium, it is shown that GFC is a unique tool for investigating the self-association of many substances, such as surfactants, chlorpromazine hydrochloride (CPZ), Methylene Blue (MB) and a sulfobetaine derivative (CHAPS) of cholic acid. The hydrodynamic radius of an aggregate can be estimated with the gel of large pore size, such as Sephadex G-200. This method is utilized for the study of the sphere-rod transition of surfactant micelles. A precise value of monomer concentration can be determined from the centroid elution volume of a frontal chromatogram on Sephadex G-10. This important parameter in self-associating systems allows us to determine the micellar aggregation number as a function of the total surfactant concentration. The derivative chromatogram can be used to detect slight changes in self-association. From these results, it is found that nonionic surfactants form premicelles including dimer and that multiple equilibrium model for micelle formation is more appropriate than mass-action model. The Tanford theory with some modifications appears to fit the concentration dependence of the aggregation properties of surfactants forming small globular micelles and allows us to estimate the stepwise aggregation constant and the micelle size distribution function. The pronounced cooperativity in the self-association of surfactants forms the basis of mass action model and the concept of the cmc, though they are actually approximations even for surfactants. They would be more inadequate, though often used, for the self-association of drugs and bile salts. The stepwise aggregation constant of MB is almost independent of aggregation numbers, which may be expected from the stacking mode of aggregation. The main aggregates of CPZ in 154 mM sodium chloride solution are dimer and 38-mer and its dimerization constant is smaller than that of MB and larger than those of surfactants. The stepwise aggregation constant of CHAPS has the maximum at hexamer and oscillates, depending on odd or even aggregation numbers. Thus, the aggregation pattern of a substance is closely related with the chemical structure of the hydrophobic group of its molecule. The definition of cmc is discussed together with its significance.

Improved algorithm for resolution of overlapped asymmetric chromatographic peaks

A simple and improved procedure to resolve an overlapped asymmetric chromatogram into its component peaks is proposed. The overlapped asymmetric peak profile was assumed to be a convolution of its component peaks, which were characterized by an exponentially modified Gaussian, and further simplified by the use of its derivative chromatogram. A new technique is suggested for initial guessing of peak parameters. The simulation study showed that peak parameters were able to be recovered within 5% deviations when the reduced resolution ( RR), the ratio of the resolution to the critical resolution, was larger than 1.0 and were sufficient for recovery process. For RR values from 1.0 to 0.6, however, the recovery was not so efficient with the initial guessed values alone and was achieved with a non-linear least-squares routine within 3% deviations in most instances utilizing these values as initial guesses of iterations. The lower limit of RR for this technique was found to be 0.6. The validity of this algorithm for recovered parameters was confirmed by comparison with experimental observations and its recovery ability was found to show no more than a 1.6% deviation from true values and a 2.2% standard deviation throughout the study.

Efficiency of four different techniques in coupled HPLC-UV/VIS to quantify overlapping peaks with known spectral features

Four different techniques to quantify unresolved chromatographic peaks with known spectral features combined with photodiode array detection, are investigated as regards their efficiency for the accurate and precise determination of drugs in the low μg-range. The comparison includes peak suppression utilising difference chromatograms, first-order derivative chromatograms, selective chromatograms, generated by the calculation of orthogonal polynomial shares, and the powerful least-squares multicomponent analysis approach. Each of these methods uses UV-spectra taken throughout, the peak. The results presented and conclusions reached should enable the chromatographer to come to a decision about the reasonable use of these options now provided by multichannel detection in HPLC.

Characterization of peak shape parameters with normal and derivative chromatograms

Characterization of peak shape parameters with normal and derivative chromatograms