Single- and multi-channel detection for generalized quantitative analysis in cases of unresolved chromatographic peaks

Computerized quantification of components under overlapping chromatographic peaks is done by calibration of chromatograms against component mixtures. For conventional (single-channel) detectors, the limitations of earlier methods based on ordinary multiple regression, can be circumvented by data reduction with the aid of principal component analysis with the partial least-squares approach. Simulation studies show that the method can be applied even when there is severe peak overlap, unstable baseline, noisy chromatograms or non-linear detector response. Advantages in the quantification of fused peaks by means of multichannel detectors are outlined. Present limitations on the quantitative evaluation of several overlapping component peaks from a single spectro-chromatogram by means of the partial least-squares method combined with multiple regression on the pure component spectra, are discussed with respect to practical high-performance liquid chromatography.     

Chemometric methods for evaluation of chromatographic separation quality from two-way data--a review

Most traditional chromatographic separation criteria or response functions are defined on chromatograms recorded by single-channel detectors, e.g. a spectrometer measuring the absorbance at a single wavelength or a thermal conductivity detector. When the peaks are seriously overlapped, usually there is a lack of the information concerning the total number of chemical components, overlap degree of the peaks and peak purity. Such information characterizes some crucial aspects of separation process and lack of it will lead to an inaccurate and misleading evaluation of separation quality as well as some computational ambiguity for many traditional response functions. In contrast, hyphenated chromatography-(multi-channel) spectroscopy instruments together with chemometric methods will largely increase the information content available in chromatographic detection. Such information, if properly used, can cast a new light on evaluation of chromatographic separation quality. The main objective of this article is to review chemometric methods devoted to estimation of the number of chemical components, determination of elution sequence and assessment of peak purity. Some newly defined response functions or separation criteria based on extracted information by chemometric methods are also introduced. The methods reviewed are limited to those for treating two-way data obtained by hyphenation of high-performance liquid chromatography with multi-channel detectors. We prefer to provide a comprehensive view of such methods rather than present a full list of all the methods developed. Further details of some important methods are touched upon in favor of employment and understanding of them by researchers not very familiar with chemometrics.     

当归特征组分的识别与定量对比

 将高效液相色谱模式 /二极管阵列检测与自编紫外光谱库管理软件结合 ,用液相色谱 /紫外光谱以及特征参数研究同种当归多个特征组分的识别及快速定量对比。色谱 /光谱及其特征参数共同表达同种当归 2 3个特征组分 ;探讨定量对比的关键技术 ;定量比较两个同种当归相同组分的浓度差异。多指标表达当归特征组分 ,可用于它们的识别 ;定量手段的建立 ,可对比当归特征组分定量结果的差异性。不用化学对照品 ,便能识别和定量比较当归样品中的多组分。方法简便易行、快速 ,结果的代表性强、重现性良好。     

Data handling in HPLC-diode array UV-spectrometry

Summary The mathematical resolution of overlapped chromatographic peaks obtained in HPLC with a diode array UV detector has received considerable attention recently. The purpose of the proposed methods is the quantitation and identification of unknown solutes in complex mixtures by an efficient use of all available data. Basically two approaches have been proposed: the first one resolves the concentration profiles and calculates the pure spectra by applying a minimal number of assumptions, which is denoted self-modeling-curve resolution. The second one is based on a match of pure spectra available in a spectral library with the measured mixture spectra. In this paper both approaches are evaluated with respect to their performance to provide the pure spectra and an accurate quantitation of the concentrations.     

Planar chromatography at the turn of the century.

An overview of the state-of-the-art of modern thin-layer chromatography (planar chromatography) is presented with emphasis on the complementary features of thin-layer and column liquid chromatographic separations. The reasons for selecting thin-layer chromatography for a particular analysis are identified by its attributes: a disposable stationary phase; simultaneous parallel separations; static detection free of time constraints; storage device for chromatographic information; all sample components are observed in the chromatogram. Future prospects for improved separation performance in TLC using zone refocusing, forced flow and electroosmotic flow methods are discussed as well as increasing zone capacity by using two-dimensional development and coupling to column chromatographic methods. Advances in coupling thin-layer chromatography with spectroscopic methods for structural elucidation are also considered. Finally, some predictions are made for how thin-layer chromatography will be practiced in the future.     

Increasing the reliability of the identification by high-performance liquid chromatography by means of selective and/or sensitive detection

An approach for quantitative assessment of the reliability of identification at high-performance liquid chromatography is proposed. The quantitative assessment of identification is useful for determination of selectivity at validation of the analytical methods. Chromatograms and spectra of the analytes are presented as maps in which characteristics as retention times, detector's signals, maxima and minima and another characteristics of spectra are used for identification. A formula for quantitative determination of the contribution of these characteristics on the reliability of identification is given. Using the more selective diode array detector than the convenient UV detector increases the reliability by several orders. A similar result was obtained when the UV detector was replaced with the more sensitive and selective fluorescence detector. Despite of the small contribution of the separation to the reliability its influence is very important for distinguishing of isomers because their spectra are identical.     

Application of Computer-Aided Spectral Deconvolution Technique in Validation of High Performance Liquid Chromatographic Peaks

Abstract

Reliable analysis with high performance liquid chromatography (HPLC) requires purity of the eluting peak. The present work has combined the advantages of the availability of full spectral data from HPLC photodiode array UV detector and computer algorithms to perform chromatographic peak purity check. A deconvolution technique based on multicomponent analysis has been applied to the UV spectra of co-eluting components. This method employs residual error (Relative-fit-error, RFE) between predicted spectrum and analyte's spectrum to detect presence of other component or contaminant. Typical RFE values for uncontaminated chromatographic peaks of norethisterone and ethynyloestradiol range between 1 and 3, while contaminated peaks have RFE values as large as 145. A systematic increase in ‘relative-fit error’ from 1.10 to 145 was observed for peaks of norethisterone when contaminated to varied extent with ethynyloestradiol. Extent of peak overlap in chromatogram was also mapped out with this technique. The co-prescribed oral contraceptive, norethisterone and ethynyloestradiol were used as model in this work. An advantage of the method is its applicability when the contaminant's spectrum is unavailable. The method, unlike several earlier techniques, is also applicable to chromatograms with concidental elution time for the components.     

Isolation and determination of paspalitrem-type tremorgenic mycotoxins using liquid chromatography with diode-array detection

A liquid chromatographic (LC) method is described for the isolation and determination of the tremorgenic mycotoxins paxilline (Penicillium paxilli NRRL 6110), paspaline, paspalinine and paspalicine (Claviceps paspali). Following a Soxhlet extraction of a mould-contaminated matrix using chloroform, the crude extract was partitioned between hexane and 80% aqueous methanol. The latter fraction, containing the desired toxin(s), was evaporated to dryness, the residue dissolved in methylene chloride and the solution analysed by liquid chromatography using a Supelcosil LC-Si column eluted with methylene chloride-diethyl ether (9 + 1, v/v). A mixture containing standards of these compounds was similarly analysed. All toxins were detected using a UV diode-array detector. The generated UV spectra and chromatographic data of the standard toxins were stored in a computer as a library and used to identify these toxins in a crude mixture. The purity of the separated peaks and the amount of toxin in the crude mixture were also determined. The toxins were isolated by selectively collecting the eluted peaks using a programmable fraction collector equipped with a peak level sensor. Further confirmation of compound identity was achieved by mass spectrometry using the direct inlet probe method. In comparison with methods used previously to isolate these toxins, the present technique is fast and allows the acquisition of complete UV spectral information and chromatographic data and the isolation of multiple toxins in a single chromatographic operation.     

Fast Interpretation Of 3D Liquid Chromatography Signals: Application To The Study Of Organic Matter In Wastewaters

Abstract

The aim of this work is to propose a very simple method for the selection and interpretation of the relevant individual spectra of a set of absorbance spectra, as for example in a 3D chromatogram. The method is based on the rank computation of the matrix of experimental values, after or during a chromatographic run. From a classical chromatographic system with a rapid scanning spectrophotometer (diode array detector for example), a computer is used for data acquisition of absorbances of the whole UV spectrum, and later computation. Firstly, acquisition is performed until the end of the elution and then the program gives the number and the corresponding spectra of relevant fractions. If another run is needed, for a different sample for example, the procedure makes it possible to check whether the detection signal is independent of the previous spectra. Different applications are presented for the study of organic matter in wastewaters by low pressure liquid chromatography.     

Evaluation of the Plasma Chromatograph as a Qualitative Detector for Liquid Chromatography

Abstract

The applicability of the plasma chromatograph as a sensitive, qualitative detector for liquid chromatography (LC) is demonstrated. Only a fraction of an LC column effluent directly introduced is needed to produce a qualitative mobility spectrum. Most LC carrier fluids either exhibit no response in plasma chromatographic spectra or do not interfere with LC peak components. Using an indirect technique, components separated by TLC can be identified by plasma chromatography.     

Spectral transformations for deconvolution methods applied on gas chromatography-mass spectrometry data

In hyphenated chromatography, overlapping chromatographic peaks can be resolved into pure spectra and pure chromatographic profiles by several multivariate deconvolution techniques. In general, these methods require bilinearity, which implies that the spectrum of each analyte is constant. The slow scan speeds normally used in gas chromatography-mass spectrometry (GC-MS) will destroy bilinearity and introduce systematic noise in the data because the concentration in the detector changes during the scan. This effect, described as the scan effect, may hinder successful resolution by multivariate deconvolution. In selected ion monitoring (SIM) GC-MS, the scan effect may be removed by simple transformations of the mass spectra. The effects of different transformations are demonstrated both on pure chromatographic peaks and on difficult resolution problems where there are small differences between the spectra of the analytes.     

HPLC analysis of commercial alkyl and aryl quaternary ammonium compounds used in organoclay type rheological additives

Mixtures of aliphatic and aromatic "quats" can be qualitatively identified in a one step chromatographic run. After separation on a strong cation exchange column, the eluted components are detected using a UV and RI detector system in series. The quaternaries present in organoclays (e.g., BENTONE type products) can also be identified by prior destruction of the silicate with hydrofluoric acid followed by chromatography of the residual quat fluorides.     

Standardization of a multi-wavelength UV detector for liquid chromatography-based toxicological analysis.

The performance of a multi-wavelength UV detector for automated drug identification following liquid chromatographic separation was evaluated. The ability of selected wavelength ratios to distinguish two closely related drugs was considered at different concentrations. Calibration of the detector based on wavelength ratios was then utilized to standardize two different detectors and to evaluate instrument-to-instrument variation of a series of detectors. Reproducibility of the second-derivative zero intercept for these drug spectra was also evaluated. Standardization of detector performance by reference to these two parameters permitted the transfer of UV spectral libraries stored on one instrument to another without compromising the reliability of qualitative data.     

HPLC‐DAD in identification and quantification of selected coumarins in crude extracts from plant cultures of Ammi majus and Ruta graveolens

This paper describes a method for the separation and determination of selected coumarins and furanocoumarins in the crude extracts from plant tissue cultures of Ammi majus hairy roots and Ruta graveolens cell suspensions, cultured in vitro, separately or together as co‐cultures. The usefulness of the three main components of the eluent used in reversed‐phase high performance liquid chromatographic analysis, namely: methanol (MeOH), acetonitrile (ACN), and tetrahydrofuran (THF), and different elution programs, was assessed. In the optimal analytical method a Lichrospher® RP‐18e 5‐μm column, a THF‐MeOH elution gradient, and a UV/VIS DAD detector were used. Due to the presence of many different compounds in the investigated plant extracts, the use of a UV/VIS DAD detector was essential. Coumarins were identified by comparison of their UV spectra with those of the analytical standards, and characterization of peak purity.     

Modern approaches for determination and identification of barrenwort (<Emphasis Type="Italic">Epimedium</Emphasis>) flavonoids

The modern methods of analysis of the main active substances barrenwort (Epimedium)—flavonoids—are considered. Various types of extraction are used to isolate these components from plant raw materials. Flavonoids are separated by capillary electrophoresis, thin layer chromatography, and high performance liquid chromatography (HPLC) in combination with UV and mass spectrometric detection.     

Separation and identification of compounds in Rhizoma chuanxiong by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry

A comprehensive two-dimensional liquid chromatographic separation system based on the combination of a CN column and an ODS column is developed for the separation of components in a traditional Chinese medicine (TCM) Rhizoma chuanxiong. Two columns are coupled by a two-position, eight-port valve equipped with two storage loops and controlled by a computer. The effluent is detected by both the diode array detector and atmospheric pressure chemical ionization (APCI) mass spectrometer. More than 52 components in the methanol extract of R. chuanxiong were resolved and 11 of them were preliminary identified according to their UV and mass spectra.     

用紫外-可见光检测器进行色谱峰的光谱分析方法

采用紫外－可见光检测器和自编吸收光谱分析程序对色谱峰在１９５～７００ｎｍ波长范围内进行扫描,得到样品和标准品的吸收光谱图,通过对二者进行比较来鉴定色谱峰纯度,操作简便,具有一定的实用价值。     

Amperometric detection of amines using cobalt electrodes after separation by ion-moderated partition chromatography

A simple and low-cost amperometric sensor for amines has been developed using a cobalt wire electrode working in alkaline solution. The sensor may be used as a detector for high-performance liquid chromatography (HPLC) that avoids the need for derivatization or post-column reaction. Experimental conditions for flow injection analysis (FIA) and HPLC separation, including the applied potential, pH and concentrations of organic modifier and carrier solution, were optimized. A cobalt wire electrode, in the constant potential amperometric mode, gives an excellent response toward amines in ion-exclusion chromatography in unbuffered solution. The sensitivities of the detection and separation of amines on the column are affected by flow rate, the concentration of the mobile phase and the concentration of organic solvent in the mobile phase, whereas the applied potential only affects the sensitivity of the detector. A cobalt electrode is more sensitive than a copper electrode, and comparable in sensitivity to a UV detector for most amines tested. The detection sensitivity is comparable to that obtained with GC methods, but the procedures are far simpler. The detection limits of the order of nanomoles obtained under the chromatographic conditions used offer an alternative for the determination of amines in a variety of matrices, such as in environmental, biomedical and pharmaceutical samples.     

UV-triggered main-component fraction collection method and its application for high-throughput chromatographic purification of combinatorial libraries

A maximum-seeking, algorithm-driven fraction collection method was developed to support high-throughput chromatographic purification, which provides new possibilities for off-line high-performance liquid chromatography mass spectroscopy (HPLC/MS) quality control experiments. The method is based on manipulation of a six-port valve that is installed downstream from the UV detector and equipped with a fraction collector loop. The detector signal is monitored by a programmable microcontroller that controls the state of the fraction collector valve. After detecting a chromatographic peak, the appropriate fraction is stored in the collector loop. The height of the next peak is compared to the previous one (using a maximum-seeking algorithm) and, depending on the result, the collected fraction is or is not exchanged with the new one. At the end of the run, the stored UV main component is pumped into the external fraction vial. This configuration was used for chromatographic purification of large compound libraries (the results of the purification of 5324 compounds are reported here), as well as for high-throughput off-line HPLC quality control experiments, where the collected main component fractions of an analytical-scale separation were subjected to further mass spectrometric molecular weight verification.     

Plasma chromatography of n-alkyl acetates

Plasma chromatography is a technique which permits characterization and analysis of trace constituents in a gaseous mixture at atmospheric pressure. It is especially well suited as a gas chromatographic detector. Operating at atmospheric pressure, the instrument uses either air or nitrogen carrier gas into which the sample is injected directly or through a gas chromatograph. The compounds are identified by their characteristic positive and negative mobility spectra, which consist of simple molecular and dissociative ions. Quantities as low as 10⁻⁶ to 10⁻¹² g are detectable. For use as a gas chromatographic detector, reference mobility spectra of compounds are needed. Those of n-alkanes, alcohols, ketones, halogenated aromatics, substituted nitrobenzenes, polychlorinated biphenyls, alkyl halides, aliphatic N-nitrosamines and isomeric phthalic acids have been previously reported. This study reports the reference mobility spectra produced by n-alkyl acetates. These compounds display strong positive mobility spectra but no negative mobility spectra. The spectra all show characteristic MH⁺, M(H₂O)_nH⁺ and (M₂)H⁺ along with the alkyl fragment ions.