Chromatographic peak, processing

Manually processing each chromatographic peak is not only time and labor intensive but difficult to reproduce. To overcome these problems and to provide a consistent data format that was independent of retention time, a number of data-processing subroutines were automated to produce a single representative cellular protein spectrum. 

Computer software is used to improve spectral quality. The most widespread procedures deal with averaging and background subtraction. The averaging process is rather obvious. The intensities of ions peaks at each m/z, recorded along the analyte chromatographic peak profile, are summed in several spectra and divided by the number of spectra used. Averaging minimizes, for example, spectral skewing problems. 

Another factor that needs to be considered is the dilution of the chromatographic peak during the separation process. The extent of chromatographic dilution can be... 

Despite these advancements in chromatographic data processing, peak detection and integration algorithms were crude, the user interface was cumbersome, and there was very little flexibility in the types of reports that these systems could generate. 

The differential volatilization of neat kerosene components from a liquid phase, directly into the atmosphere during volatilization up to 50% (w/w), is presented in Fig. 8.8. Ten kerosene components were selected, and their composition was depicted as a function of gas chromatograph peak size (%), which is linearly related to their concentration. It may be seen that the lighter fractions evaporate at the beginning of the volatilization process. Increasing evaporation causes additional components to volatilize, which leads to a relative increase in the heavier fractions of kerosene in the remaining liquid. 

The reports from the integrator consist of retention time and sample amount for each integrated peak. These are transmitted to a small computer. The integrator has the capacity to process up to 250 peaks in a run. However, because of the limited memory space of the computer, we had to decrease the number of peaks processed. Chromatographic runs with more than 150 peaks were reduced to 150 peaks by elimination of those with the smallest area. The reduced reports were then stored on tape. 

A decongestant syrup was basified with ammonia and extracted into ethyl acetate, thus ensuring that the components extracted were in their free base forms rather than their salts, which is important for obtaining good chromatographic peak shape. Salts of bases will thermally dissociate in the GC injector port but this process can cause a loss of peak shape and decomposition. 

Because a chromatographic peak is assumed to result from the spreading of the sample zone with a Gaussian distribution of sample concentrations in the mobile and stationary phases, the calculation of the column efficiency is related to the associated variance, a2. The efficiency of the process is thus the ratio t2/a2 a2 is the variance in time units (in this case). When dr is measured on the recorder chart, the ratio d2la2 is considered, with a in distance units. 

Integration is a process of determining the area under a chromatographic peak for the quantitation of the analyte concentration. There are several scientifically valid techniques that may be used to integrate peaks. Modern chromatography computer software performs peak integration with a minimum of human intervention. Once an... 

Figure 1 is a comparison of the analysis of a complex mixture using 0.32 and 0.10 mm I.D. columns. In this example, the more efficient 0.10 mm column, produces almost equivalent resolution to the 0.32 mm column in approximately one third the analysis time. Unfortunately there are practical limitations to decreasing column diameter. With diameters equal to or less than 0.10 mm, sample introduction becomes difficult due to high inlet pressures. Sample capacity is limited and signal processing of the fast chromatographic peaks requires specialized A/D conversion. 

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