A study of the practical limitations of principal components analysis and the labelling of unresolved HPLC peaks

Summary The method of iterative target transformation factor analysis (ITTFA) used in conjunction with second derivative peak finding has shown to be a practical method for the peak deconvolution and reconstruction of HPLC chromatograms and spectra. The second derivative method of peak finding is acceptable for resolutions above 0.5 for peaks of similar heights. Above 0.5 resolution the labelling gives correct results where the spectra are substantially different and also when reasonably similar. Below this value the peak labelling was still accurate where the spectra were different. Solvent effects on the spectra of the compounds studied are small and do not hinder the peak labelling process. Thus small “local” libraries are feasible. Presented at the 17th International Symposium on Chromatography, September 25–30, 1988. Vienna, Austria.     

Improved particle-packed HPLC/MS microchips for proteomic analysis

The influence of packing process parameters (packing pressure, application of ultrasound) and the stationary phase particle size (3.5 and 5 μm) on the chromatographic performance of HPLC/MS chips was systematically investigated for proteomic samples. First, reproducibility and detection limits of the separation were evaluated with a low‐complexity sample of tryptic BSA peptides. The influence of adsorbent packing quality on protein identification was then tested with a typical proteomics sample of high complexity, a human plasma protein fraction (Cohn fraction IV‐4). All HPLC/MS chips provided highly reproducible separations of these proteomic samples, but improved packing conditions and smaller particle sizes resulted in chromatograms with narrower peaks and correspondingly higher signal intensities. Improved separation performance increased the peak capacity, the number of identified peptides, and thus the sequence coverage in the proteomic samples, particularly for low sample amounts.     

Probability of failure of the watershed algorithm for peak detection in comprehensive two-dimensional chromatography

The watershed algorithm is the most common method used for peak detection and integration in two-dimensional chromatography. However, the retention time variability in the second dimension may render the algorithm to fail. A study calculating the probabilities of failure of the watershed algorithm was performed. The main objective was to calculate the maximum second-dimension retention time variability, Δ²t_R,crit, above which the algorithm fails. Several models to calculate Δ²t_R,crit were developed and evaluated: (a) exact model; (b) simplified model and (c) simple-modified model. Model (c) gave the best performance and allowed to deduce an analytical expression for the probability of failure of the watershed algorithm as a function of experimental Δ²t_R, modulation time and peak width in the first and second dimensions. It could be demonstrated that the probability of failure of the watershed algorithm under normal conditions in GC × GC is around 15–20%. Small changes of Δ²t_R, modulation time and/or peak width in the first and second dimension could induce subtle changes in the probability of failure of the watershed algorithm. Theoretical equations were verified with experimental results from a diesel sample injected in GC × GC and were found to be in good agreement with the experiments.     

Comparative evaluation of software for retention time alignment of gas chromatography/time-of-flight mass spectrometry-based metabonomic data

In chromatography-based metabonomic research, retention time (RT) alignment of chromatographic peaks poses a challenge for the accurate profiling of biomarkers. Although a number of RT alignment software has been reported, the performance of these software packages have not been comprehensively evaluated. This study aimed to evaluate the RT alignment accuracy of publicly available and commercial RT alignment software. Two gas chromatography/mass spectrometry (GC/MS) datasets acquired from a mixture of standard metabolites and human bladder cancer urine samples, were used to assess three publicly available software packages, MetAlign, MZmine and TagFinder, and two commercial applications comprising the Calibration feature and Statistical Compare of ChromaTOF software. The overall RT alignment accuracies in aligning standard compounds mixture were 93, 92, 74, 73 and 42% for Calibration feature, MZmine, MetAlign, Statistical Compare and TagFinder, respectively. Additionally, unique trends were observed for the individual software with regards to the different experimental conditions related to extent and direction of RT shifts. Conflicting performance was observed for human urine samples suggesting that RT misalignments still occurred despite the use of RT alignment software. While RT alignment remains an inevitable step in data preprocessing, metabonomic researchers are recommended to perform manual check on the RT alignment of important biomarkers as part of their validation process.     

A study of the precision and accuracy of peak quantification in comprehensive two-dimensional liquid chromatography in time

Simulated chromatographic data were used to determine the precision and accuracy in the estimation of peak volumes (i.e., peak sizes) in comprehensive two-dimensional liquid chromatography in time (LC × LC). Peak volumes were determined both by summing the areas in the second dimension chromatograms and by fitting the second dimension areas to a Gaussian peak. The Gaussian method is better at predicting the peak volume than the moments method provided there are at least three second dimension injections above the limit of detection (LOD). However, when only two of the second dimension signals are substantially above baseline, the accuracy and precision of the Gaussian fit method become quite poor because the results from the fitting algorithm become indeterminate. Based on simulations in which the modulation ratio (M_R = 4¹σ/t_s) and sampling phase (?) were varied, we conclude for well-resolved peaks that the optimum precision in peak volumes in 2D separations will be obtained when the M_R is between two and five, such that there are typically four to ten second dimension peaks recorded over the eight σ width of the first dimension peak. This sampling rate is similar to that suggested by the Murphy–Schure–Foley criterion. This provides an RSD of approximately 2% for the signal-to-noise ratio used in the present simulations. The precision of the peak volume of experimental data was also assessed, and RSD values were in the range of 4–5%. We conclude that the poorer precision found in the LC × LC experimental data as compared to LC may be due to experimental imprecision in sampling the effluent from the first dimension column.     

Evaluation of 1.0 mm i.d. column performances on ultra high pressure liquid chromatography instrumentation

The present study focuses on the evaluation of 1.0 mm i.d. (internal diameter) columns on a commercial Ultra-High Pressure system. These systems have been developed specifically to operate columns with small volumes, typically 2.1 mm i.d., by reducing extra-column volume dispersion. The use of columns with smaller i.d. results in a reduced solvent consumption and required sample volume. The evaluation of the columns was carried out with samples containing neutral and pharmaceutical compounds. In isocratic mode, the extra-column volume produced additional band broadening leading to poor performances compared to equivalent 2.1 mm i.d. columns. By increasing the length of the column, the influence of the extra-column bandspreading could be reduced and 75,000 plates were obtained when four columns were coupled. In gradient mode, the effect of the extra-column contribution on efficiency was limited and about 80% of the performance of the 2.1 mm i.d. columns was obtained. Optimum conditions in gradient mode were further investigated by changing flow rate, gradient time and column length. A different approach of the calculation of peak capacity was also considered for the comparison of the influence of these different parameters.     

基于二极管阵列检测器的色谱峰纯度的快速直观鉴定

基于二极管阵列检测器（DAD）得到的色谱-光谱图,通过去噪、归一化、计算欧氏距离,聚类分析等方法,建立了一种快速直观的评价色谱峰纯度的有效方法：可直观的判断色谱峰的纯度,并判断存在干扰峰的位置,为色谱工作者评价色谱峰,进行下一步工作提供了依据。     

Nondestructive evaluation for remanent life of modified 9Cr-1Mo steel by reversible magnetic permeability

We present a magnetic and nondestructive method to evaluate the remanent life of modified 9Cr-1Mo steel by measuring the reversible magnetic permeability. Specimens with ten different kinds of aging periods were prepared using an isothermal heat treatment at 690 °C. The Larson-Miller parameter (LMP) was calculated and the peak interval of reversible magnetic permeability (PIRMP) was measured using the surface type probe. PIRMP was inversely proportional to LMP. We can evaluate the remanent life of modified 9Cr-1Mo steel using the relationship between PIRMP and LMP. Also, we present the possibility that the tensile strength and yield strength measured by destructive methods could be estimated by PIRMP measured nondestructively.     

The role of ion-pairing in peak deformations in overloaded reversed-phase chromatography of peptides

The paper reports a study on the role of ion-pairing behind peak deformations, e.g. peak splitting and even peak disappearance, during the elution of a peptide at highly overloaded conditions in reversed-phase chromatography. Deformation of component peaks is not uncommon in chromatography. There are reports which discuss their occurrence, but mostly at analytical scale, while their occurrence is quite common also in the preparative scale, as in the case discussed in this work. This paper first describes the conditions leading to peak splitting and peak disappearance of an industrial peptide, then explains the plausible reasons behind such behaviour, and finally with experimental analysis demonstrates the role of ion-pairing in causing such behaviour.