Coeluting

A mixture of methyl paraben, ethyl paraben, propyl paraben, diethyl phthalate, and butyl paraben is separated by HPLC. This experiment emphasizes the development of a mobile-phase composition capable of separating the mixture. A photodiode array detector demonstrates the coelution of the two compounds. 

The limits of lifetime detection and resolution in on-the-flight fluorescence lifetime detection in hplc were evaluated for simple, binary systems of polycycHc hydrocarbons (70). Peak homogeneity owing to coelution was clearly indicated for two compounds having fluorescence lifetime ratios as small as 1.2 and the individual peaks could be recovered using predeterrnined lifetimes of the compounds. Limits of lifetime detection were deterrnined to be 6 and 0.3 pmol for benzo[b]fluoranthene and benzo[k]fluoranthene, respectively. 

An interesting and practical example of the use of thermodynamic analysis is to explain and predict certain features that arise in the application of chromatography to chiral separations. The separation of enantiomers is achieved by making one or both phases chirally active so that different enantiomers will interact slightly differently with the one or both phases. In practice, it is usual to make the stationary phase comprise one specific isomer so that it offers specific selectivity to one enantiomer of the chiral solute pair. The basis of the selectivity is thought to be spatial, in that one enantiomer can approach the stationary phase closer than the other. If there is no chiral selectivity in the stationary phase, both enantiomers (being chemically identical) will coelute and will provide identical log(Vr ) against 1/T curve. If, however, one... 

The use of hexafluoroisopropanol (HFIP) as an SEC eluent has become popular for the analysis of polyesters and polyamides. Conventional PS/DVB-based SEC columns have been widely used for HFIP applications, although the relatively high polarity of HFIP has led to some practical difficulties (1) the SEC calibration curve can exhibit excessive curvature, (2) polydisperse samples can exhibit dislocations or shoulders on the peaks, and (3) low molecular weight resolution can be lost, causing additive/system peaks to coelute with the low molecular weight tail of the polymer distribution... 

Flow markers are often chosen to be chemically pure small molecules that can fully permeate the GPC packing and elute as a sharp peak at the total permeation volume (Vp) of the column. Examples of a few common flow markers reported in the literature for nonaqueous GPC include xylene, dioctyl phthalate, ethylbenzene, and sulfur. The flow marker must in no way perturb the chromatography of the analyte, either by coeluting with the analyte peak of interest or by influencing the retention of the analyte. In all cases it is essential that the flow marker experience no adsorption on the stationary phase of the column. The variability that occurs in a flow marker when it experiences differences in how it adsorbs to a column is more than sufficient to obscure the flow rate deviations that one is trying to monitor and correct for. 

A more difficult criterion to meet with flow markers is that the polymer samples not contain interferents that coelute with or very near the flow marker and either affect its retention time or the ability of the analyst to reproducibly identify the retention time of the peak. Water is a ubiquitous problem in nonaqueous GPC and, when using a refractive index detector, it can cause a variable magnitude, negative area peak that may coelute with certain choices of totally permeated flow markers. This variable area negative peak may alter the apparent position of the flow marker when the flow rate has actually been invariant, thereby causing the user to falsely adjust data to compensate for the flow error. Similar problems can occur with the elution of positive peaks that are not exactly identical in elution to the totally permeated flow marker. Species that often contribute to these problems are residual monomer, reactants, surfactants, by-products, or buffers from the synthesis of the polymer. 

Some GPC analysts use totally excluded, rather than totally permeated, flow markers to make flow rate corrections. Most of the previously mentioned requirements for totally permeated flow marker selection still are requirements for a totally excluded flow marker. Coelution effects can often be avoided in this approach. It must be pointed out that species eluting at the excluded volume of a column set are not immune to adsorption problems and may even have variability issues arising from viscosity effects of these necessarily higher molecular weight species from the column. 

G. M. Frame, A collaborative study of 209 PCB congeners and 6 Aroclor s on 20 different HRGC columns. Part 1. Retention and coelution database , Fresenius 7. Anal. Chem. 357 701-713 (1997). 

An application of an LC-SFC system has been demonstrated by the separation of non-ionic surfactants consisting of mono- and di-laurates of poly (ethyleneglycol) (23). Without fractionation in the precolumn by normal phase HPLC (Figure 12.18 (a)) and transfer of the whole sample into the SFC system, the different homologues coeluted with each other. (Figure 12.18(b)). In contrast with prior fractionation by HPLC into two fractions and consequent analysis by SFC, the homologues in the two fractions were well resolved (Figures 12.18(c) and 12.18(d)). 

A liquid chromatographic experiment resulted in the same retention time for the electroactive compounds A and B. Which electrochemical detection scheme would offer a selective detection of the two coeluting analytes Explain your selection. (E = +0.43 V Eg = +0.77 V.)... 

TLC analysis of the crude product (elution with 50 1 pentane ether, visualization with iodine) showed three non-baseline spots Rf 0.65 (cis isomer), Rf 0.52 (unknown impurity), and Rf 0.32 (trans isomer). The unknown impurity is intensely sensitive to iodine and largely coelutes with the cw-isomer in the subsequent column chromatography. However, the ll NMR spectrum of this isomer shows excellent purity despite the presence of this spot on TLC. In 100 1 pentane ether, Rf values of the cis and trans isomers are about 0.50 and 0.15, respectively. 

There are two notable features of the quantitative performance of this type of interface. It has been found that non-linear responses are often obtained at low analyte concentrations. This has been attributed to the formation of smaller particles than at higher concentrations and their more easy removal by the jet separator. Signal enhancement has been observed due to the presence of (a) coeluting compounds (including any isotopically labelled internal standard that may be used), and (b) mobile-phase additives such as ammonium acetate. It has been suggested that ion-molecule aggregates are formed and these cause larger particles to be produced in the desolvation chamber. Such particles are transferred to the mass spectrometer more efficiently. It was found, however, that the particle size distribution after addition of ammonium acetate, when enhancement was observed, was little different to that in the absence of ammonium acetate when no enhancement was observed. 

It is clear that neither NMEA nor NDPA is appropriate for an internal standard in NDMA determination if criteria are interpreted strictly, but both compounds have been used for this purpose. Addition of a nitrosamine, not normally present in the sample, is helpful in detecting any gross errors in the procedure, but the addition should not be considered to be internal standardization. Utilization of NMEA or NDPA to indicate recovery of NDMA can lead to significant errors. In most reports of the application of these "internal standards", recovery of all nitrosamines was close to 100%. Under these conditions, any added compound would appear to be a good internal standard, but none is necessary. NDMA is a particularly difficult compound for use of internal standardization because of its anomalous distribution behavior. I mass j ectrometry is employed for quantitative determination, H- or N-labeled NDMA could be added as internal standard. Because the labeled material would coelute from GC columns with the unlabeled NDMA, this approach is unworkable when GC-TEA is employed or when high resolution MS selected ion monitoring is used with the equipment described above. 

The experiments showing the influence of lateral interaction on coelution of the two species were discussed in Subsection 2.4.2. Figure 2.18 and Figure 2.19 give a comparison of single profiles of acid and ketone or of alcohol and ketone with those attained for the binary mixture. Very similar peak profiles can be obtained upon solving Equation 2.21 separately for the alcohol, acid, and ketone with isotherms (Equation 2.4 and Equation 2.7a), and for the binary mixture with the isotherms (Equation 2.9 and Equation 2.10). 

Two-dimensional development is commonly used in analytical TLC for the separation, identification, and sometimes, quantification of complex lipid species especially when one-dimensional development does not provide satisfactory separation where some individual hpids coelute in a number of common solvent systems. 

To obtain reliable chromatograms in the final step of the determination of the analytes by LC or GC, it is important to remove interfering signals resulting from coelution of other compounds. To this end, a variety of techniques are applied for cleanup of the sample extract. The most effective procedures for sample cleanup for PAH measurements are partitioning between M, N-dimethylformamide/water/cyclo-hexane and LC on silica and on Sephadex LH 20. Other cleanup procedures include LC on alumina or XAD-2 and preparative thin-layer chromatography. 

Alternatively, LC is used for the separation and quantification of PAHs using both UV and fluorescence detection. The analytes are identified based on their relative retention times and UV and/or fluorescence emission spectra. For UV detection an efficient cleanup is a prerequisite since this detection method is not very selective (almost universal for PAHs), and hence it also responds to many coeluting compounds. Due to the high specificity of fluorescence detection for most PAHs, this LC detection method is less susceptible to potential interferences. As in the case of GC the apphcation of internal standard(s) is mandatory since solvents have to be evaporated during the cleanup, which may result in partial losses of some of the more volatile analytes. 

Pigiure 5.10 An exaaple of the use of spectral overlays to detect coeluting inpurities. (Reproduced with pemission from ref. 73. Copyright Preston Publications, Inc.)... 

Figure 2 shows the elution profiles of several neuropeptides and the PCP-like material. When the PCP-active fractions were rechromatographed over a flatter gradient and in a different solvent system, it was clear that bombesin- and substance P-immunoreactiv-ity no longer coeluted with the PCP-like material. 

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