The Measurement of Retention Time

Since multi-wavelength detection has now been mentioned and will feature quite prominently in other apphcations, it is worthwhile discussing the reasons for this and why forensic laboratories have been keen to use and develop new multiwavelength detection techniques. Traditionally, solute characterisation in HPLC is based upon the measurement of retention time. However, for the purpose of sample identification or discrimination retention time is a very poor parameter because of the possibility of this being similar or identical to other compounds. 

Pathways 8, 9 and 10 all involve two-parameter, linear regression equations using the log of each parameter. The utility of pathway 9 is enhanced by an available compilation of various solvent/water partition coefficients (K. ) for thousands of chemicals [28). The utility of pathway 09 is fairly well recognized the regression equations are included in Chapter 2, which covers estimation methods for solubility (S). Pathway 10 is more of a laboratory estimation method than a computational method it derives its main benefit from the fact that the measurement of retention time takes only about 25 minutes [44]. In a test of 18 compounds, the HPLC/RT method estimated values of log Kow with average absolute error of 23% [44]. 

The Measurement of Retention Time.— The accurate measurement of retention time /e [equation (4)] should not be determined from ruler measurements on the chromatogram but should be obtained from good stop-watch measurements. Paper stretching and mains-frequency fluctuations (which alter the rate of drive of synchronous motors) are two important factors that are responsible for the inaccuracies of the ruler method. 

Mass spectrometer equipped with desorption electrospray (DESI) ion source is capable of detection and identification of different substances from the surface. Moreover, the information about spatial distribution of those substances is retained. Connection of this technique with TLC not only allows for the measurement of retention times for separated chemicals (spatial distribution), but also for their unambiguous identification, based on the molecular weight of certain substances and their fragmentation spectra. Additionally, because DESI works under ambient conditions, there is no need to apply a high vacuum system for the sample introduction. Moreover, samples analyzed by DESI practically do not require any kind of preparation (e.g., covering with matrix prior to MALDI analysis), thus connection of those two techniques is relatively easy. Certainly, not all the substances, due to their chemical features, may be detected with this technique. Only compounds, which are able to ionize in this type of ion source, may be analyzed. 

Systematic studies in our laboratory have indicated the presence of a second lift force that acts on the particles. These studies involved the measurement of retention times for a set of PS particle standards under wide ranging conditions of flowrate and field strength. 

Several methods for measuring drug binding to human serum albumin involving the determination of retention times on HPLC columns with bound albumin have been reported [77,78]. Solid-phase microextraction [79,80], capillary electrophoresis [81], and displacement of near-infrared fluorescent labels [82] have all been studied. 

Individual components are identified by their retention time, usually measured along the time axis of the chart paper, but the reproducibility of retention times is significantly affected by alterations in the gas flow and column temperature. It is not adequate to rely on quoted values for retention times but it is necessary to determine the values at frequent intervals using identical experimental conditions for tests and reference compounds. 

The effluent volume is the most convenient parameter to measure in gel permeation chromatography because flow rates are often variable, making the use of retention times unsuitable. The sequence of different solutes emerging from a column will therefore be reported as the total volume of solvent that has emerged from the column when the substance appears in the effluent (Ve). 

The problem of determining V-j in SEC is similar to that of determining zero retention time (tg) in other liquid chromatography columns. Recently, there have been several papers dealing with the determination of retention time of a retained peak in HPLC (12-19). In high-performance reversed-phase chromatography, McCormick and Karger (1 ) and Berendsen, et al., (16) have employed D2O to measure tg. Neidhart et al.,... 

The uncertainty in the measurement of elution time / or elution volume of an unretained tracer is another potential source of error in the evaluation of thermodynamic quantities for the chromatographic process. It can be shown that a small relative error in the determination of r , will give rise to a commensurate relative error in both the retention factor and the related Gibbs free energy. Thus, a 5% error in leads to errors of nearly 5% in both k and AG. An analysis of error propagation showed that if the... 

Individual VOC. The term VOC is commonly used to describe speciated measurements of individual organics. The almost universal approach to the identification and measurement of individual VOC is GC with either FID or mass spectrometry (MS). GC-MS is used to establish the identity of a particular compound through the combination of retention times and mass spectra and, of course, can also be used for quantification. However, for a given type of air mass, GC-FID is commonly used for more extensive quantitative measurements after the individual peaks have been identified. For reviews of various aspects of sampling and measurement of VOC in air, see Westberg and Zimmerman (1993), Apel et al. (1994), Klemp et al. (1994), Sacks and Akard (1994), and Dewulf and Van Langen-hove (1997). 

The capacity factor (Equation 23-16) is a measure of retention time, fp in units of the time tw required for mobile phase or an unretained solute to pass through the column. Reasonable separations demand that the capacity factors for all peaks be in the range 0.5-20. If the capacity factor is too small, the first peak is distorted by the solvent front. If the capacity factor is too great, the run takes too long. In the lowest trace in Figure 25-12, tm is the time when the first baseline disturbance is observed near 3 min. If you do not observe a baseline disturbance, you can estimate... 

Tests of the reproducibility of retention times, retention factors, separation selec-tivities, and column efficiencies for our methacrylate monolithic capillary columns are summarized in Table 6.2. This table shows averaged data obtained for 9 different analytes injected 14 times repeatedly every other day over a period of 6 days, as well as for 7 different capillary columns prepared from the same polymerization mixture. As expected, both injection-to-injection and day-to-day reproducibilities measured for the same column are very good. Slightly larger RSD values were observed for col-umn-to-column reproducibility. While the selectivity effectively did not change, larger differences were found for the efficiencies of the columns. 

The majority of chromatographic separations as well as the theory assume that each component elutes out of the column as a narrow band or a Gaussian peak. Using the position of the maximum of the peak as a measure of retention time, the peak shape conforms closely to the equation C = Cjjjg, exp[-(t -1] ) The modelling of this process, by traditional descriptive models, has been extensively reported in the literature. 

But K and k depend markedly on the operation temperature, and cannot be used to compare data obtained in programmed temperature conditions. A way to increase the reproducibility of measures is to use a reference compound. In the case of retention time, flow rate and column geometry affect all the compounds in the same way the reproducibility is increased by using retention times relative to a reference, instead of absolute values. 

A first prediction attempt of GCxGC retention using this strategy was carried out by Beens et al. [10]. Retention times in the D column were calculated from those of n-alkanes and the analyte retention index. For the analyte retention times in D, k was first obtained by interpolation for the n-alkane series at the elution temperatures, the retention times f of these compounds were calculated using the corresponding holdup times, and then t Ri for the analyte was calculated from its RI at the elution temperature using Equation (3). Differences between calculated and experimental values were observed for D retention times, although predicted elution profiles were similar to the experimental patterns. A similar approach [27] used k values measured at several temperatures to obtain a better interpolation. The accuracy of the prediction of retention times was... 

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