Peak-shape deteriorations

The low flow rate in the microbore column ensures sample volumes compatible with the secondary conventional column and permits the injection of a small volume onto the secondary column, making the transfer of incompatible solvents possible without peak shape deterioration or resolution losses [63], The possible disadvantage could be the lower sample capacity of microbore LC columns. However, in LCxLC, a sensitivity enhancement can be obtained if the formation of compressed solute bands at the head of the secondary column is achieved during the transfer from the first to the second dimension. Moreover, a larger volume can be injected into the first-dimension microcolumn, used as a highly efficient pre-separation step, and a limited decrease in efficiency due to a large injection volume can be tolerated. 

The general use of series (or parallel) coupled detectors has been to record different channels of independent information as a method of qualitative identification [391,392]. The use of detector ratios for compound identification has declined with the development of low cost mass spectrometers that allow the use of more reliable identification methods. There are also practical problems related to long term stability of response ratios and their system dependence. Although rarely addressed in the literature, the chromatograms recorded with series coupled detectors often show greater peak shape deterioration than predicted from consideration of the additional coupled detector volume. 

The LC peak shapes deteriorate for the latter peaks of interest... 

Some unexpected factors can affect the peak shape and migration in CE. For example, concentration of the analyte itself in the sample can have an effect on the peak shape, which can be remedied by dilution or by using a different buffer as illustrated in Fig. 1In this figure, hippuric acid at low concentrations shows a good peak shape in CE using borate or M-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer. However, when the concentration is increased tenfold, the peak shape deteriorates in the borate but not in the CHES buffer. 

When peak shapes deteriorate, examine yom inlet lines (change often). Redeactivate or replace if necessary at the same time, cut 0.5 m from the inlet end of the column prior to installation. 

Figures 19, 20, and 21 show the ethyl esters, n-butyl esters, and benzyl esters. The deterioration in peak shape with increased retention may be due to the decreased solubility of the esters in the mobile phase as well as to the band broadening that occurs with increased retention time.

Crop Extraction and Cleanup (OPA-MERC). GLYPH and AMPA were extracted and cleaned up prior to post-column fluorogenic labeling HPLC determination in exactly the same manner as for the FM0CC1 procedure with the exception that the concentrated highly acidic fractions from the 50W - X8 columns were adjusted with 10 M KOH to pH 3-8. This was necessary in order to prevent adverse shifting of the HPLC mobile phase pH and subsequent shifts in retention and deterioration of peak shape. 

The most common problem associated with analytical columns is column deterioration. Deterioration may appear as poor peak shapes, split peaks, shoulders, loss of resolution, decreased retention times, and high backpressure. These symptoms indicate contaminants that have accumulated on the frit or column inlet, or there are voids, channels, or a depression in the packing bed. Deterioration is more evident in higher efficiency columns. For example, a column with 3-pm packing is more susceptible to plugging than one with 5- or 10-pm packing. Proper column protection and sample preparation are essential to prolong a column s life and obtain its best performance. 

Aqueous samples are usually acidified prior to direct aqueous injection so that the organic acids are in their protonated form. This increases their volatihty, reduces adsorption effects and results in better peak shapes. However, some authors report that acidification leads to a deterioration of the films in capillary columns, whereas others did not observe any adverse effects.Some sample matrices require clean up and the resulting organic solvent extracts (diethylether or dichloromethane) are injected directly without further derivatization. 

One unfavourable characteristic of silica is its solubility at extreme pH values and at elevated temperatures, thus leading to reduced lifetime and deterioration of peak shape and resolution. With the advent of particles formed by the aggregation of silica sols, so-called sol-gel silicas, this property of silica has been significantly reduced. The recent development of hybrid silica particles also address this problem [59]. 

The problems described here are noticed more often when the sample contains so-called irregular components, discussed later. On the other hand, in isocratic systems a larger dead volume (= volume between the autosampler and the detector - without the column) leads only to broader peaks and thus to a deterioration of the resolution. Analog, with isocratic separations a longer column affects the retention time, peak shape, and resolution. Unlike gradient separations, the length of the column can change neither the selectivity nor the elution order ... 

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