Chromatographic baselines

FIGURE 4.18 Determination of data window. Final data window should span (include) data windows of new and old columns. It should also include a region on each end of the data window to establish a chromatographic baseline. The illustrations were created with Microsoft Excel . 

Electrolysis of mobile phase constituents will cause a continuous detector response (background current) resulting in a chromatographic baseline level that differs from the electrical detector zero-response level. The difference, baseline- offset, is an important analysis parameter, because baseline fluctuations (noise, drift) due to fluctuations in electrolysis conditions (potential, mobile phase flow rate, temperature) are proportional to baseline offset. See Figure 2-5 for an example of the influence of flow pulsation at different baseline offset... 

Application of Unical software also requires the selection of chromatographic baselines, thus selecting the specific data taken for further analysis. In the studies reported here, we choose to analyze only the polydisperse envelope from lignin elution, so that the distinct component which often elutes near V (the total column volume) was not included in the analysis. 

When we look at a separation to judge whether two peaks are separated, we look at the centers of the peaks, but more importantly, we look at the valley separating the peaks. An ideal separation is one in which all peak pairs are baseline separated the peak valleys all come down and touch the chromatographic baseline. If we were to draw a line connecting the two peak tops (Fig. 14.2, line 1-2), then drop a perpendicular line from the center of this connecting line to the baseline (A-B), the length of the resulting line would represent a standard of baseline separation for these two peaks. 

In the case of analytes with identical elemental formulas ( true isobars ) as positional or geometrical isomers (e.g. 11-hydroxycortisol and 21-hydroxycortisol or testosterone and epi-testosterone)—a discrimination of analyte and interfering compound is not possible with any mass analyzer—even if enabling highest mass resolution. Potentially, the disintegration pattern of isomers may be different, allowing analytical discrimination but in most cases chromatographic baseline separation of analyte and isomer prior to their MS/MS detection is required for unequivocal quantitative measurement. 

Is the method specific and stability indicating as shown by analysis of samples subjected to stressed stability studies (pH, light, heat, oxidation) Normally, specificity is determined through peak purity using ultraviolet (UV) diode array or liquid chromatography (LC)/mass spectrometry (MS) analysis. In methods for analysis of drug products, placebo formulations (and stressed placebos) must yield blank chromatographic baselines. The development... 

Fig. 7 Comparison of the rankmap results before ami after IVT treatment for a simulated two-component system, (a) Rankmap of the data, without adding any background, after IVT treatment, (b) Rankmap of the data without adding any background after IVT treatment. (c) Rankmap of the raw data with only chromatographic baseline drift added.

A highly conductive anion such as hydroxide (limiting equivalent conductance = 198 S cm eq ) would normally be used for detection of anions by indirect conductivity. This would establish a chromatographic baseline at a high conductance level. When a sample anion that has a much lower equivalent conductance passes through the detector, a peak of much lower conductance is observed. This occurs because the total ionic concentration (sample anion plus OH plus cations) remains constant but the zone containing the sample anion has a lower conductance that that of the background eluent. 

Figure 6.9 Chromatogram of DEHA in the olive oil extract. The portion of chromatogram presented corresponds to the region of elution of DEHA within the acetonitrile extract from the oil. The upper part represents the blank specimen and shows the chromatographic baseline at the retention time of the additive, whereas the bottom part shows the presence of the additive DEHA. Reproduced with permission from Simonceau and Hannaert, Food Additives and Contaminants, 1999,16, 5, 197 [27])...

Baseline Compensation Analysis—A baseline compensation analysis, or baseline blank, is performed exactly like an analysis except no injection is made. A blank analysis must be performed at least once per day. The blank analysis is necessary due to the usual occurrence of chromatographic baseline instability and is subtracted from sample analyses to remove any nonsample slice area fijom the chromatographic data. The blank analysis is typically performed prior to sample analyses, but may be useftil if determined between samples or at Ae end of a sample sequence to provide additional data regarding instrument operation or residual sample carry-over from previous sample analyses. Attention must be given to all factors that influence ba%line stability, such as column bleed, septum bleed, detector temperature control, constancy of carrier gas flow, leaks, instrument drift, etc. Periodic baseline blank analyses should be made, following the analysis sequence protocol, to give an indication of baseline stability. 

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