正交投影用于多波长色谱重叠峰分析

 将正交投影分辨 (OPR)技术用于多波长色谱重叠峰分辨 ,当色谱峰中最大重叠度小于或等于波长数时 ,用这一方法能从多波长色谱重叠峰中获得完全真解。基于双波长色谱分析 ,提出了一种新的色谱重叠峰中背景校正、组分数和纯组分信号区确定以及各组分重叠情况的分析方法 ,即双波长特征信息分析 (DWCI)。该法被成功的用于三组分双峰和双组分单峰重叠色谱的分析。     

三元重叠色谱峰面积的定量方法

本从色谱峰的ＥＭＧ模型出发，通过对重叠色谱峰的模拟和回归分析，提出了一种三元重叠色谱峰的面积的定量方法，三元重叠色谱峰的每一个峰面积可以由峰面积比和总面积求得，此法所需的数据都由实验色谱图上测得，峰面积计算结果的相对误差小于±５％，适用于相对峰谷为５０％－９５％的三元重叠色谱峰面积的定量。     

高效液相色谱多波长定性方法研究

介绍了高效液相色谱法中除常用的保留时间和光谱图定性以外的另一方法──多波长定性分析方法。根据Beer's定律可知,同一化合物在不同波长下测得的峰面积之比为定值。利用高效波相色谱多波长同时检测的方法,比较化合物在不同波长下的峰面积大小,建立了简便的定性方法,并在禁用假氮染料测定时对18种芳香腔的定性分析中得到成功的应用。     

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。     

模差渐进投影法解析重叠色谱峰

提出了一种鉴别重叠色谱峰类型的化学计量学方法——模差渐进投影(MDEP)法。该方法利用二维色谱数据中相邻两个色谱流出点的光谱矢量模的和与该两个矢量和的模之差,建立模差-时间(dm-t)曲线判断非纯色谱峰的纯组分区,同时判定第二组分色谱流出点和倒数第二组分色谱结束点。分别以纯组分区的光谱矢量建立正交阵对该色谱峰进行投影运算,得到投影后转换色谱。该转换色谱消除了纯组分区所对应化合物的色谱和光谱信息。对转换色谱重复上述操作至整个色谱峰的模差-时间曲线均为0时终止,得到重叠色谱峰各组分的色谱流出点和结束点。利用上述结果,采用不包含待测组分光谱信息的该重叠峰二维色谱数据建立正交矩阵对该色谱峰进行投影运算,获得待测组分的相似色谱图。通过比对分析即可得到该组分在重叠色谱峰中的光谱和色谱信息。依次操作,即可获得重叠色谱峰中各组分的光谱和色谱信息。采用模差渐近投影法对4组分模拟重叠二维色谱峰及杜香挥发油GC-MS的重叠色谱峰进行了解析,方法简便、结果准确。     

高效液相色谱切换波长法同时测定葡萄组织中单体酚

建立了切换检测波长同时测定葡萄组织中单体酚含量的高效液相色谱方法,使得全部待测物质能够在其最大吸收波长下得到检测。应用ZorbaxC18柱,以1.3%的乙酸水溶液和乙腈为流动相,梯度洗脱,流速0.8mL/min,柱温30℃。结果显示:各组分均得到很好分离,校准曲线的线性关系良好(r≥0.9990);平均加标回收率≥89.5%;保留时间及峰面积在日内(n=10)和日间(n=10)实验中的RSD分别≤0.3%、2.9%和0.3%、3.1%;与恒定检测波长的方法相比,该方法明显地降低了被测物质的检出限,并且在测定实际样品中得到了基线平稳、分离效果和峰形皆好的色谱图。该方法预处理简单、灵敏、重现性好。     

基于积分渐进展开的气相色谱重叠峰快速解析法

论文提出用积分渐进展开解析气相色谱重叠峰,该方法有3个主要步骤：首先将谷峰或肩峰分成两个积分区域,得到一个子区域的积分方程和一个重叠峰面积的代数方程;然后用数值积分求出这两个方程计算中所需要的峰面积,再用积分渐进公式将积分方程展开成代数方程;最后,将这两个方程与峰高约束方程联立后,得到一个非线性代数方程组,用Gauss-Seidel迭代可以快速求解方程组,方程收敛的最大迭代次数不超过20次。仿真和实验结果表明,解析的峰高和峰面积误差均很小,峰面积最大误差低于6.44%,峰高的最大误差约为6.80%。由于该算法精度高,效率高,所以这个方法可以用于气相色谱重叠峰和一般色谱峰的实时在线解析。     

联用色谱数据的双窗口因子分析

利用组分光谱的特征信息,发展了一种能直接对联用色谱重叠峰中组分进行定性定量分析的新方法──双窗口因子分析（dualwindowfactoranalysis,DWPA）。该法可从多组分重叠峰中定性目标组分,且在未经其它组分的分辨下可直接对目标组分的光谱、色谱进行分辨。因此更适应于联用色谱对复杂体系中待测组分的定性定量分析。用该法成功地对4组分重叠峰进行了分析,实验结果令人满意。     

基于紫外检测器的壳寡糖色谱分析与比较

基于氨基葡萄糖的紫外吸收特性,通过对氨基葡萄糖标准溶液在波长190～400 nm的检测,确定了壳寡糖(氨基葡萄糖分子的低聚物)的紫外检测波长为190～210 nm。比较了常用于寡糖分析的两种商品色谱柱(Shodex氨基柱和TOSOH酰胺柱)分离效果,两种色谱柱分离效果相当,但前者的出峰时间更早。基于Shodex氨基柱,拟合出紫外检测器条件下氨基葡萄糖定量计算的标准曲线,开发并验证了壳寡糖定性定量的分析方法,不同聚合度壳寡糖的分离程度和色谱峰分布规律与示差折光检测器的检测效果相当,并以此确定了壳寡糖混合标准样品的聚合度分布。方法可作为现行壳寡糖行业标准检测方法的有效补充。     

直观推导式演进特征投影法解析多环芳烃的重叠色谱峰

用高效液相色谱法(HPLC)测定多环芳烃时,因芴、苊和菲,茚(1,2,3-cd)芘和苯并(g,h,i)苝的色谱峰严重重叠而影响测定结果。本研究用高效液相色谱-二极管阵列检测器(DAD)和荧光检测器(FLD)测定多环芳烃,在激发波长λex=230nm,发射波长λem=300～500nm范围内采集重叠峰的HPLC-FLD二维色谱数据,再用直观推导式演进特征投影法(HELP)解析它们的重叠色谱峰,分辨结果令人满意。该方法对重叠组分的分辨下限为0.02mg/L。结果表明,用二维色谱荧光数据解析色谱重叠峰,灵敏度更高,可用于环境样品中多环芳烃的测定。     

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。     

Mutual peak matching in a series of HPLC–DAD mixture analyses

One of the largest challenges in high performance liquid chromatography (HPLC) method development is the necessity for tracking the movement of peaks as separation conditions are changed. Peak increments are then used to build a mathematical model capable of minimizing the number of experiments in an optimization circuit. Method optimization for an unknown mixture is, moreover, complicated by the absence of any a priori information on component properties and retention times when direct signal assignment is not possible. On the contrary, achievement of the maximum separation becomes an important factor for successful identification or quantitation. In this case, the optimization may be based on assigning peaks of the same component chosen from different experiments to each other. In other words, mutual peak matching between the HPLC runs is required.

A new method for mutual peak matching in a series of HPLC with diode array detector (HPLC–DAD) analyses of the same unknown mixture acquired at varying separation conditions has been developed. This approach, called mutual automated peak matching (MAP), does not require any prior knowledge of the mixture composition. Applying abstract factor analysis (AFA) and iterative key set factor analysis (IKSFA) on the augmented data matrix, the algorithm detects the number of mixture components and calculates the retention times of every individual compound in each of the input chromatograms. Every candidate component is then validated by target testing for presence in each HPLC run to provide quantitative criteria for the detection of “missing” peaks and non-analyte components as well as confirming successful matches. The matching algorithm by itself does not perform full curve resolution. However, its output may serve as a good initial estimate for further modeling. A common set of UV-Vis spectra of pure components can be obtained, as well as their corresponding concentration profiles in separate runs, by means of alternating least-square multivariate curve resolution (ALS MCR), resulting in reconstruction of overlapped peaks.

The algorithms were programmed in MATLAB^® and tested on a number of sets of simulated data. Possible ways to improve the stability of results, reduce calculation time, and minimize operator interaction are discussed. The technique can be used to optimize HPLC analysis of a complex mixture without preliminary identification of its components.     

Computerized separation of chromatographically unresolved peaks

A computerized peak deconvolution software and mass spectra were successfully applied for the deconvolution of overlapped peak cluster in the chromatogram obtained separating the complex mixture of pesticides by retention time locking gas chromatography-mass spectroscopy. The method based on the unique fragment ions in the spectra can be used for deconvolution of peak clusters if mass spectra of overlapped peaks differ. This method allows determining actual retention times of overlapped peaks. Peak areas found by this method however, cannot be used naturally for the quantitative purposes as the abundance of fragment ions used for this deconvolution procedure can dramatically differ. Computer assisted deconvolution of peaks in the peak clusters gives more realistic peak area ratios as at this method it is supposed equal response for all peaks overlapped in a cluster.     

Characterization of a nematic mixture by reversed-phase HPLC and UV spectroscopy: an application to phase behaviour studies in liquid crystal-CO2 systems

In the present work a simple but selective reversed-phase high performance liquid chromatographic method (HPLC) was developed for the analysis of the nematic liquid crystal mixture E7 to determine precisely the composition of the liquid crystal mixture used in PDLC film preparation. Ultraviolet absorption spectrophotometry experiments were carried out to determine the HPLC detection wavelength and to characterize the absorptivity constants of E7 constituents. The technique developed is applied in the case of equilibrium solubility studies for E7 in supercritical carbon dioxide (scCO₂). The results indicate unambiguously that scCO₂ can fractionate the mixture towards the E7 components. The four single component peaks of the E7 mixture were distinctively separated by this method, which enabled the determination of the solubility of E7 constituents in the scCO₂.     

Kalman filter quantitative resolution of shifted ESCA spectra after optimal alignment by simulated annealing

Summary The quantitative separation of overlapped responses accomplished by the Kalman filter algorithm is deteriorated by the errors in the model due to shifts of the component peaks. This is particularly true for spectral resolution in Electron Spectroscopy for Chemical Analysis (ESCA). A computational approach for the quantitative resolution of overlapping ESCA spectra when there is loss of collinearity between the pure component peaks and the mixture peak, has been developed. The procedure makes itera tive use of the Kalman filter for resolving the mixture spectrum with the component spectra aligned according to some values of the position parameters, and of the simulated annealing algorithm to find the optimal alignment. The performance of simulated annealing in pursuing this task, has been compared with those of simplex and steepest descent, by analyzing a set of overlapped synthetic spectra and some ESCA spectra of powder mixtures of lead compounds.     

Quantitative impurity profiling by principal component analysis of high-performance liquid chromatography-diode array detection data

Related organic impurities generally have approximately similar molar absorption coefficients (epsilon) due to their structural similarities. On the assumption that all peaks in an impurity profiling chromatogram have approximately the same maximum molar absorption coefficients (epsilon(max)) and the chromatogram contains one major peak and several much smaller ones, all of which are completely separated, integration of the summed score vectors from the principal component analysis (PCA) decomposition of high-performance liquid chromatography-diode array detection (HPLC-DAD) data will give areas that are quantitatively proportional to the actual content of the compounds. Due to the sequential nature of PCA, the first principal component (PC) will primarily be related to the main compound and all peaks showing a similar spectrum, while the second PC will be related to the impurities with a spectrum different from the main peak. Summing the two score vectors thus makes it possible to take account of different spectra in the score chromatogram, which make the method proposed give better quantitative estimates of the impurities than any single wavelength chromatogram. Multivariate curve resolution alternating least squares (MCR-ALS) is used for comparison. The results are presented for two examples of simulated HPLC-DAD data as well as for three examples of real HPLC-DAD data from impurity profiling. The results show that integration of the score chromatograms can handle differences in the unknown epsilon(max) of the peaks and take account of the different spectra of the impurity peaks, giving quantitative estimates of the content of the impurities that closely correspond to the reference values. The results obtained are also better than integration with the best possible separate wavelength. The method could be a straightforward approach to impurity profiling in order to obtain a good estimate of the content or relative response factors of small chromatographic impurity peaks without knowledge of their molar absorption coefficients and without any precalibration.     

Parallel column liquid chromatography with a single multi-wavelength absorbance detector for enhanced selectivity using chemometric analysis

The selectivity of high performance liquid chromatography (HPLC) separations is increased using a parallel column configuration. In this system, an injected sample is first split between two HPLC columns that provide complementary separations. The effluent from the two columns is recombined prior to detection with a single multiwavelength absorbance detector. Complementary stationary phases are used so that each chemical component produces a detected concentration profile consisting of two peaks. A parallel column configuration, when coupled with multivariate detection, provides increased chemical selectivity relative to a single column configuration with the same multivariate detection. This enhanced selectivity is achieved by doubling the number of peaks in the chromatographic dimension while keeping the run time constant. Unlike traditional single column separation methodology, the parallel column system sacrifices chromatographic resolution while actually increasing the chemical selectivity, thus allowing chemometric data analysis methods to mathematically resolve the multivariate chromatographic data. The parallel column system can be used to reduce analysis times for partially resolved peaks and simplify initial method development as well as provide a more robust methodology if and when subsequent changes in the sample matrix occur (such as when new interferences show up in subsequent samples). Here, a mixture of common aromatic compounds were separated with this system and analyzed using the generalized rank annihilation method (GRAM). Analytes that were significantly overlapped on both stationary phases applied, ZirChrom PBD and CARB phases, when used in traditional single column format, were successfully quantified with a R.S.D.% of typically 2% when the same stationary phases were used in the parallel column format. These results indicate that a parallel column system should substantially improve the chemical selectivity and quantitative precision of the analysis relative to a single-column instrument.     

Quantitation in multianalyte overlapping peaks from capillary electrophoresis runs using artificial neural networks

The potentiality of artificial neural networks for multicomponent analysis in unresolved peaks from capillary electrophoresis (CE) is evaluated. The system chosen consists of mixtures of three ebrotidine metabolites, which cannot be successfully separated by CE. Data selected for analysis consist of UV spectra taken at the maximum of the CE peak. The most dissimilar analyte, in terms of spectral differences, is accurately quantitated in any type of mixture with an overall prediction error of 5%. Because of the strong interference of the two most overlapped compounds, a preliminary procedure for spectral data filtering based on principal component analysis is performed to improve their quantitation.