Altered Retention Times

This is usually caused by changes in the carrier gas flow rate or column temperature. Flow rate changes can be caused by leaks in the system upstream from the column inlet, such as in the injection port (e.g., the septum) by low pressure in the system due to an empty or nearly empty carrier supply or by faulty hardware, 

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A parameter that alters retention time (and hence resolution) is altered in the middle of the run in each case. 

Metal complexation — One of the most insidious and widely occurrent sources of analytical variation in IEC is product complexation with metal ions. Most proteins can form complexes with metals, regardless of whether or not they are metalloproteins.1 Participant metal ions can derive from the cell culture production process, purification process buffers, or even stainless steel chromatography systems. Complexation can alter retention times, create aberrant peaks, and substantially increase peak width. To the extent that metal contamination of your sample is uncontrolled, so too will be the performance of your assay. 

Doubled peaks or altered retention times (Figure 25-16) sometimes occur if the solvent in which sample is dissolved has much greater eluent strength than the mobile phase. Try to dissolve the sample in a solvent of lower eluent strength or in the mobile phase itself. 

Deviations from classical Van t Hoff behaviour in reversed phase chromatography have been described (Snyder, 1979 Melander and Horvath, 1979) in which an increased retention of specific chemical types occurs at higher temperatures this phenomenon may be observed when molecules adopt a compact, near spherical configuration compared with their molecular conformers and are retained for longer times. Also, where the extent of ionisation of either the buffer or the solute molecules is affected by changes in column temperature an altered retention time may be obtained and may result in improved peak symmetry (Knox and Vasvari, 1973 Melander et al., 1979). 

Unfortunately retention times are not on their own sufficiently definitive to characterize compounds. Slight differences in carrier gas flow rate, column temperature and column condition can alter retention times. Such differences are usually more pronounced when the same column is used in different gas chromatographs or with different columns (same packing) in the same gas chromatograph, and for this reason retention data are most frequently reported as relative retention times or retention indices. The relative retention time of a solute is the ratio of the adjusted retention time of the solute to that of a selected reference compound and in many of the chromatograms shown elsewhere in the book (see also the Appendix), n-tetracosane is used as the... 

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. 

Three isomeric tetrachlorodibenzo-p-dioxins were studied. All were insoluble in TFMS acid. To dissolve these compounds and form cation radicals, UV irradiation was necessary. The 1,2,3,4-tetrachloro compound was particularly sensitive to UV irradiation, and as a solid, even turned pink when exposed to ordinary fluorescent light. When subjected to constant UV irradiation, radical ions were induced rapidly. The change in the cation radical concentration was monitored by the ESR signal as illustrated in Figure 10. To determine whether the tetrachloro isomer had been converted to lower chlorinated derivatives after UV irradiation, the dissolved dioxin was then poured into ice water and recovered. The GLC retention time of the recovered dioxin was unchanged in addition, no new GLC peaks were observed. Moreover, the ESR spectrum see Figure 11) for the recovered material was not altered between widely... 

Treatment of the extract with UV light (4) for 16 hours completely destroyed TCDD in the amended soil. The extract from the unamended soil was not changed by irradiation. Peaks close to the TCDD peak in retention time ( 0.2 min) were not altered. Recovery of ca. 100% was obtained when 5 ppb TCDD was added to the soil. A peak should be discernible at a concentration of 1 ppb as seen on the control core. However, background interference, even in the cleaned up residue, increased tremendously at the very low levels, and confirmation of a peak s identity was very difficult. 

One of the first applications of the HPLC method was the investigation of differences in toxin profiles between shellfish species from various localities ( ). It became apparent immediately that there were vast differences in these toxin profiles even among shellfish from the same beach. There were subtle differences between the various shellfish species, and butter clams had a completely different suite of toxins than the other clams and mussels. It was presumed that all of the shellfish fed on the same dinoflagellate population, so there must have been other factors influencing toxin profiles such as differences in toxin uptake, release, or metabolism. These presumptions were strengthened when toxin profiles in the littleneck clam (Prototheca Staminea) were examined. It was found that, in this species, none of the toxin peaks in the HPLC chromatogram had retention times that matched the normal PSP toxins. It was evident that some alteration in toxin structure had occurred that was unique in this particular shellfish species. 

Microparticulate silica can be used in a number of modes for hplc of these, reverse phase chromatography using bonded phases is the most widely used. In normal and reverse phase chromatography the retention times and selectivities of solutes can be altered by adjustment of the nature and composition of the mobile phase. 

Hi) The retention times of solutes in exclusion chromatography can be altered by changing the polarity Of the mobile phase. 

When retention times of mixture components decrease, there may be problems with either the mobile or stationary phase. It may be that the mobile phase composition was not restored after a gradient elution, or it may be that the stationary phase was altered due to irreversed adsorption of mixture components, or simply chemical decomposition. Use of guard columns may avoid stationary phase problems. 

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. 

Internal standardization circumvents the effects of time-variant instrument response, but does not compensate for different ionization efficiencies of analyte and standard. For internal standardization, a compound exhibiting close similarity in terms of ionization efficiency and retention time is added to the sample at a known level of concentration, e.g., an isomer eluting closely to the analyte or a homologue may serve for that purpose. It is important to add the standard before any clean-up procedure in order not to alter the concentration of the analyte without affecting that of the standard. For reliable results, the relative concentration of analyte and standard should not differ by more than a factor of about ten. 

The first calibration injection is used to identify the number of peaks and their retention times, this will vary throughout the experiments of a ruggedness study as the method conditions are altered. This information can then be used to identify the sample peaks. It is usual to start the ruggedness test by running the experiment with all factors set to their method values. This is because the analyst is familiar with these conditions from the rest of the validation study and thus can evaluate the suitability of... 

The effect of altering the acetonitrile concentration was observed as a reduction in retention times as the solvent strength increased, peak height increased correspondingly. The resolution between the peaks was slightly reduced with increased solvent strength but this was insufficient to cause peak overlap and hence deterioration of quantitative results. 

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