Peak alignment using wavelet pattern matching and differential evolution

Retention time shifts badly impair qualitative or quantitative results of chemometric analyses when entire chromatographic data are used. Hence, chromatograms should be aligned to perform further analysis. Being inspired and motivated by this purpose, a practical and handy peak alignment method (alignDE) is proposed, implemented in this research for one-way chromatograms, which basically consists of five steps: (1) chromatogram lengths equalization using linear interpolation; (2) accurate peak pattern matching by continuous wavelet transform (CWT) with the Mexican Hat and Haar wavelets as its mother wavelets; (3) flexible baseline fitting utilizing penalized least squares; (4) peak clustering when gap of two peaks is smaller than a certain threshold; (5) peak alignment using differential evolution (DE) to maximize linear correlation coefficient between reference signal and signal to be aligned. This method is demonstrated with both simulated chromatograms and real chromatograms, for example, chromatograms of fungal extracts and Red Peony Root obtained by HPLC-DAD. It is implemented in R language and available as open source software to a broad range of chromatograph users (http://code.google.com/p/alignde).     

Determination of vancomycin related substances by gradient high-performance liquid chromatography

A gradient high-performance liquid chromatographic method for the determination of vancomycin related substances is described. This method was developed to profile vancomycin, co-fermentation products, and degradation products. The resultant chromatograms confirm the multifactored nature of vancomycin, separating a number of peaks from the main component. The development of acceptable chromatographic performance is described, with the final method intended for use as a control assay. Quantitation of total related substances is made by comparing the relative area of the main peak to total peak area in a pair of chromatograms from a stock solution and a twenty-five fold dilution. A rapid-scan UV detector was used to demonstrate the similar spectral characteristics of the vancomycin related substances, confirming the validity of the relative area approach. Statistical method validation data are included, evaluating the use of this method for quantitative applications. Example applications demonstrate the effectiveness of this method.     

Monolithic silica columns for high-efficiency chromatographic separations

A deconvolution methodology for overlapped chromatographic signals is proposed. Several single-wavelength chromatograms of binary mixtures, obtained in different runs at diverse concentration ratios of the individual components, were simultaneously processed (multi-batch approach), after being arranged as two-way data. The chromatograms were modelled as linear combinations of forced peak profiles according to a polynomially modified Gaussian equation. The fitting was performed with a previously reported hybrid genetic algorithm with local search, leaving all model parameters free. The approach yielded more accurate solutions than those found when each experimental chromatogram was fitted independently to the peak model (single-batch approach). The improvement was especially significant for those chromatograms where the peaks were severely affected by the tails of the preceding compounds. Peak shifts among chromatograms, which are a usual source of non-bilinearity, were modelled in a continuous domain instead of in a discrete way, which avoided some drawbacks associated with latent variable methods. An experimental design involving simulated chromatograms was applied to check the method performance. Five main factors affecting the deconvolution were examined: concentration pattern, chromatographic resolution, number of batches and replicates, and noise level, which were evaluated using first- and second-order figures of merit. The method was also tested on three real samples containing compounds showing different overlap. Four multi-batch deconvolution methods were considered differing in the nature of the processed information and kind of peak matching among chromatograms. In all cases, the multi-batch deconvolution yielded better performance than the single-batch approach.     

Colachrom,a command language for chromatographic data processing

A command language for the interactive computer processing of chromatograms is described. Report data files, resulting from a peak evaluation program, are processed with single-line commands. Reference peaks for quantitative evaluation or for retention index calculations can be specified, compound names or response factors be assigned, etc. Customer-specific reports can be generated. Several commands operate on the unreduced digitized chromatograms, e.g., repeat peak evaluation with modified peak search parameters or with a user-corrected baseline. Commands can be combined into procedures for semi-automatic processing of series of chromatograms. Various forms of interactive graphical presentations supplement the command language.     

Chromatogram surgery

A computer program is developed on a Hewlett-Packard 3354 B Laboratory Automation System to eliminate background in gas and liquid chromatograms. Moreover undesired peaks, such as solvent peaks and spikes, can be excluded by selecting appropriate method parameters or by application of the integrator-timed events directly affecting peak integration. Injudicious application of these codes deforms the chromatograms. Peaks can be labelled with retention times, areas or amounts.     

Chromatogram simulation by area reproduction

A modified Poisson function has been developed for the simulation of chromatographic peaks. The proposed model is shown to have the property of exactly recreating the experimentally determined peak area. Model parameters are obtained directly from the experimental peak, and overlapping peaks are deconvoluted such that the area sum of overlapping peaks is kept unchanged. The method was applied to real, complex chromatograms.     

Automatic program for peak detection and deconvolution of multi-overlapped chromatographic signals part II: peak model and deconvolution algorithms

Several interlinked algorithms for peak deconvolution by non-linear regression are presented. These procedures, together with the peak detection methods outlined in Part I, have allowed the implementation of an automatic method able to process multi-overlapped signals, requiring little user interaction. A criterion based on the evaluation of the multivariate selectivity of the chromatographic signal is used to auto-select the most efficient deconvolution procedure for each chromatographic situation. In this way, non-optimal local solutions are avoided in cases of high overlap, and short computation times are obtained in situations of high resolution. A new algorithm, fitting both the original signal and the second derivatives is proved to avoid local optima in intermediate coelution situations. This allows achieving the global optimum without the need of background knowledge by the user. A previously reported peak model, a Gaussian with a polynomial standard deviation whose complexity can be modulated to enhance the fitting quality, was applied. However, the original formulation was modified to account baseline outside the peak region. Also, the optimal model complexity was auto-selected via error propagation theory. The method is able to process simultaneously several related chromatograms. The software was tested with both simulated and experimental chromatograms obtained with monolithic silica columns.     

A novel determination of peak areas for application on large computers

Summary A computer program with some new algorithms for the determination of peak areas of gas chromatograms has been developed which has been used for several years with a satellite computer system. In contrast to most gc-programs the first and second derivatives of the curve are not used for peak detection. The maximum of a peak is defined by ordinates of the sample points alone; the base line is constructed by drawing curves of higher order through those parts of the chromatogram which are defined to be base line by special criteria. Consequently, the peak areas on the tailing of a solvent are determined more correctly than with skimming and, furthermore, the calculated base line of chromatograms with temperature program and subsequent isothermal run can be approximated to the real base line. The base line divides the chromatogram into several peak groups which are further separated by the democratic distribution method. This program is best suited for nonroutine analysis in research laboratories, because only a few input parameters are necessary for peak area determination with unknown chromatograms.     

Inner chromatogram projection (ICP) for resolution of GC-MS data with embedded chromatographic peaks

The chromatographic peak located inside another peak in the time direction is called an embedded or inner peak in contradistinction with the embedding peak, which is called an outer peak. The chemical components corresponding to inner and outer peaks are called inner and outer components, respectively. This special case of co-eluting chromatograms was investigated using chemometric approaches taking GC-MS as an example. A novel method, named inner chromatogram projection (ICP), for resolution of GC-MS data with embedded chromatographic peaks is derived. Orthogonal projection resolution is first utilized to obtain the chromatographic profile of the inner component. Projection of the two-way data matrix columnwise-normalized along the time direction to the normalized profile of the inner component found is subsequently performed to find the selective m/z points, if they exist, which represent the chromatogram of the outer component by itself. With the profiles obtained, the mass spectra can easily be found by means of a least-squares procedure. The results for both simulated data and real samples demonstrate that the proposed method is capable of achieving satisfactory resolution performance not affected by the shapes of chromatograms and the relative positions of the components involved.     

离子色谱线性梯度淋洗分离阴离子的计算机模拟

建立了使用氢氧化钠淋洗液时用计算机模拟离子色谱梯度淋洗分离阴离子的新方法,研究了在线性梯度淋洗过程中不同时间阴离子在色谱柱中的位置及所在位置的淋洗液浓度,得出阴离子在不同时间的容量因子、迁移速度,通过积分得到保留时间。再根据离子色谱峰峰形变化的规律,得到色谱峰峰形的参数。模拟色谱图保留值与实验值的相对误差小于5%,模拟色谱图的色谱峰峰形与实验所得到的色谱图的色谱峰峰形也非常接近。     

The usefulness of the deconvolution of chromatograms into orthogonal polynomials for characterizing the quality of separation

Simulated chromatograms are deconvoluted into series of orthogonal polynomials. The number of terms necessary to achieve a preset goodness of fit, the reproducibility of coefficients in the presence of noise and the effects of signal-to-noise ratio, peak-height ratio and peak asymmetry on the value of coefficients from the deconvolution are investigated. A convenient method for the deconvolution of real chromatograms is described in order to characterize the quality of separation in the chromatogram. A known quality criterion is modified by using coefficients from the deconvolution in order to enhance its performance. Performance is tested on a simulated separation of five sulfa drugs on a reversed- phase column.     

Application of the TLC image analysis technique for the simultaneous quantitative determination of L-proline and L-lysine in dietary supplement

A simple and sensitive thin-layer chromatography (TLC) method coupled with an image analysis technique was developed for the simultaneous quantitative determination of L-proline and L-lysine in dietary supplement with good precision and accuracy. Separation was performed on silica gel plates using ethanol‒toluene (2:3, V/V) as the mobile phase. The visualization of chromatograms was based on iodine–azide reaction; therefore, pre-chromatographic derivatization reaction of amino acids with phenyl isothiocyanate was performed. Digital images of TLC plate chromatograms were converted into peak chromatograms, and quantitative analysis was conducted using TLSee software.

     

An On-Line Wavelet Transform for De-Noising of High Performance Liquid Chromatograms

ABSTRACT

A novel algorithm of wavelet transform, called the on-line wavelet transform, which can be used in on-line processing of signals of an instrumental analysis, is proposed. And application of the on-line wavelet transform in denoising of high performance liquid chromatograms was investigated. Results showed that the noise in chromatograms was cleanly removed by the method, peak position after the on-line de-noising dose not change, and the linearity of calibration curves of concentration versus peak area remained and even was improved. Quantitative determination of three mixed samples were investigated with five standard samples; the recoveries were between 94.0-105.0%.     

A new mathematical procedure to evaluate peaks in complex chromatograms

Automatic peak evaluation in chromatograms and subsequent quantification of compound concentrations is still a challenge in the analysis of complex samples containing hundreds or thousands of compounds. Although a number of software packages for peak evaluation exist, baseline definition and overlapping peaks of different shapes are the main reasons which prevent reliable automatic analysis of complex chromatograms. A new mathematical procedure is presented which uses peak shapes extracted from the chromatogram itself and modified by nonlinear (in fact, hyperbolic) stretching of the peak head and tail. With this approach, the peak parameters are position, height, scale of front, scale of tail, and smoothness of transition from front to tail scaling. This approach is found to give a substantially better fit than traditional analytically defined peak shapes. Together with a good peak finding heuristic and nonlinear optimization of parameters this allows a reliable automatic analysis of chromatograms with a large number of peaks, even with large groups of overlapping peaks. The analysis matches the quality of standard interactive methods, but still permits interactive refinement. This approach has been implemented and tested on a large set of data from chromatography of hydrocarbons in ambient air samples.     

Package for on-line data acquisition and processing with microcomputers

Professor Ducauze and his co-workers from the Institut National Agronomique Paris-Grignon interfaced their CBM 8032 microcomputer with several chromatographs and developed a software package for the data acquisition and processing of the chromatograms. A specific option of the package is the correction of peak positions for long term drift by a comparison with reference chromatograms stored on disk.     

Quantitative Auswertung von Gas-Chromatogrammen partiell getrennter Stoffgemisch

A simple method is described for the qualitative and quantitative analysis of gas chromatograms with considerably overlapping peaks, based on the assumption of known retention indices and peak shapes. Its application to mixtures of deuteriated hydrocarbons is indicated. The extent to which the results are reproducible is shown to be about the same as that of high-resolution gas chromatography or mass spectrometry.     

沉香的高效液相色谱-四极杆-飞行时间质谱数字化指纹图谱研究

采用高效液相色谱-四极杆-飞行时间质谱联用(HPLC-Q-TOF-MS)技术,研究构建了一种沉香数字化色谱-质谱指纹图谱的新方法。沉香药材经乙醇提取后,采用HPLC-Q-TOF-MS测定,并同时采集HPLC-Q-TOF-MS及液相色谱-紫外数据,得到液相色谱-紫外检测(HPLC-UV)色谱图和高分辨飞行时间质谱(TOF-MS)总离子流色谱图。对色谱图中的各个色谱峰进行精确质量数识别,建立数字化指纹图谱,以精确质量数结合保留时间表征沉香中的化学成分,即为每个色谱峰给出具有唯一性的数字信息,以数字化的形式反映其化学成分,并根据精确质量及同位素推算出分子式,结合二级质谱及文献资料共鉴定出30个化学成分。该方法对沉香的每种化学成分给出了类似于身份认定的数字化信息,具有唯一性,能全面反映沉香的物质成分,可为沉香的药理、药效及质量标准研究提供科学的数据。     

小波变换用于重叠色谱峰组分信息的提取

提出了一个小波变换用于提取重叠色谱中组分信息的理论公式，并将它应用于三组分和五组分重叠色谱实验数据的解析。结果表明：在定定分离度范围内，当相邻色谱峰峰宽相近时，根据此公式的计算结果，可以将各组分的信息从重叠色谱峰信号中提取出来。洒工作对小波变换用于重叠峰的解析具有重要意义。     

Fingerprint chromatogram analysis of extracts from the leaves of Tripterygium wilfordii Hook. F. by high performance liquid chromatography

A simple and reliable high performance liquid chromatographic (HPLC) method has been developed and validated for the study of fingerprint chromatograms of extracts from the leaves of Tripterygium wilfordii Hook. F. (TWHF) and for controlling the quality of the herb. HPLC separation of the extracts was performed on a Lichrospher RP-18 column and detected by ultraviolet absorbance at 210 nm. The column temperature was maintained at 35 degrees C. A mobile phase composed of acetonitrile:H2O in the ratio of 39:61 (v/v) was found to be most suitable for this separation at a flow rate of 0.8 mL/min with isocratic elution. Under the chromatographic conditions described, the peak profile of the 10 components collected within 35 min made up the fingerprint of the extracts from leaves of TWHF with universal features. The fingerprint chromatograms had a good stability, precision, and reproducibility. The similarity of the extracts from leaves of TWHF collected in summer and winter was studied with triptolide as a reference peak. The method is suitable for differentiation of extracts from the leaves of TWHF, and can be used as a quality control method for this herb.     

Analysis of plant oils by subcritical fluid chromatography using pattern fitting

A new method for analyzing plant oils was developed, which allows determination of fatty acid (FA) and triglyceride (TG) compositions of oils directly from their chromatograms without complete separation and peak identification. TGs of oils were separated by subcritical fluid chromatography using an octadecyl-silica column, carbon dioxide mobile phase and flame ionization detector. Observed chromatograms were compared with simulated ones, which were generated using FA compositions and predicted retentions of TGs on the assumption that FAs are combined with glycerol at random. FA compositions were determined by minimizing the differences between observed and simulated chromatograms through trial and error. Compared with GC analysis, relative errors of calculated FA compositions were less than 10% for main components (mol fraction>0.2). However, cocoa butter presented large errors even for main components because of the selective bonding of FAs to glycerol. Application of this method to the analysis of blended oils was also demonstrated, where FA compositions and mixing ratio were determined.