Peak area data processing

Note S— The total area for each carbon number can be measured by either pre-programming the int rator to sum the area of the peaks within the appropriate retention time wiinlows or aiialyzing the peak area data after the peak int iation process is complete. 

Recent advances in sample preparation techniques now make analysis of off-flavor metabolites simple, fast, accurate, and sensitive. With the SPME-MS-MVA technique, a mass intensity list representing all the volatiles in the milk sample is the basis for shelf-life prediction—not GC peak area data. SPME-MS-MVA prediction of processed milk shelf life is based on measurement of volatiles and semivolatiles present in milk after a pre-incubation period. 

Ethanol concentration in the fermentation broth is determined by using gas chromatography (HP 5890 series II with HP Chemstation data processing software, Hewlett-Packard, Avondale, PA) with a Poropak Q Column, and a Hewlett-Packard model 3380A integrator. A flame ionisation detector (FID) is used to determine ethanol. The oven temperature is maintained at 180 °C, and the injector and detector temperature are maintained at 240 °C. The sample taken from the fermentation media has to be filtered and any internal standard must be added for analysis based on internal standard methods otherwise, the area under the peak must be compared with known standard samples for calculation based on external standard methods. 

The mixture is identical in each example. The peaks are shown separated by 2, 3, 4, 5 and 6 (a) and it is clear that a separation of 6a would appear to be ideal for accurate quantitative results. Such a resolution, however, will often require very high efficiencies which will be accompanied by very long analysis times. Furthermore, a separation of 6o is not necessary for accurate quantitative analysis. Even with manual measurements made directly on the chromatogram from a strip chart recorder, accurate quantitative results can be obtained with a separation of only 4a. That is to say that duplicate measurements of peak area or peak height should not differ by more than 2%. (A separation of 4a means that the distance between the maxima of the two peaks is equal to twice the peak widths). If the chromatographic data is acquired and processed by a computer, then with modem software, a separation of 4a is quite adequate. 

Modem data processing software includes routines that can be used to obtain accurate quantitative results from chromatograms where the components of the sample are incompletely resolved. The routines, in fact, deconvolute the individual peaks from the composite envelope and calculate the area of the individual de-convoluted peaks. Such algorithms can be used very effectively on peaks that are entrained in the tail of a major peak but are not so accurate for composite envelopes containing many unresolved peaks. 

GC-Computer System Nowadays, a large number of data-processing-computer-aided instruments for the automatic calculation of various peak parameters, for instance relative retention, composition, peak areas etc., can be conveniently coupled with GC-systems. A commercially available fairly sophisticated computer system of such type are available abundantly that may be capable of undertaking load upto 100 gas-chromatographs with ample data-storage facilities. In fact, the installation such as multi GC-systems in the routine analysis in oil-refineries and bulk pharmaceutical industries, and chemical based industries have tremendously cut-down their operating cost of analysis to a bare minimum. 

Some extensions are not essential for CC, but greatly improve its capabilities. Interfaces to a data storage device and to a hard copy unit are valuable. Some facility for data processing afterwards (baseline correction and peak area determination) is desirable. 

Gas chromatographic data was obtained on a Tracor Model 220 gas chromatograph equipped with a Varian Model 8000 autosampler. The analysis column was a 1.7 m "U column, 4 mm id, filled with 3% SP-2250 packing (Supelco, Inc., Bellefonte, PA) held at 200 C. The injection temperature was 250 and the nitrogen carrier gas flow rate was 60 mL/min. The detector temperatures were 350 for electron capture and 190 for flame photometric. Detector signals were processed by a Varian Vista 401 which gave retention times and peak areas. 

The OPA reagent for HPLC is prepared according to the method of Benson and Hare (21). The fluorescence reaction is performed in a 55 C water bath. OPA reacts with the guanidino group of TTX presumably to form a fluorescent product, l-alkylthio-2-alkylisoindole (22, 23). TTX is monitored at 453 nm with 332-nm excitation. Peak areas are calculated by a data processing system of the analyzer. 

It is seen that for base-line resolution the peak maxima must be six standard deviations (6a) apart. But for accurate quantitative analysis, employing peak heights measurements, a separation of (4o) is usually quite adequate. Even when peak area measurements are employed, a separation of (4o) will usually provide adequate accuracy, particularly if computer data acquisition and processing is employed with modern software. Therefore, throughout this book, whenever dealing with resolution, or column design, a resolution of (4o) will be assumed. 

GC-MS runs were stored as files by the data system on discs FORTRAN routines were written to compare selected parameters in file sets and to reduce the data to summary tables for hard copy output. These routines facilitated the determination of peak areas of components in extracted ion current profiles (EICP) for both total and selected ion chromatograms, calculated the removal of components of interest (e.g., those containing halogen isotopes) by treatment processes (GAC, CI2) or derivatization, summarized the occurrence of new components of interest in treatment or derivatization, and calculated the percent of the total ion current represented by a given component. The programs allowed operator discrimination between major and minor components in a file set by preselection of an ion current threshhold for data reduction. For data summarized herein, components were >4000 ion counts, which corresponds to a level >5 of the internal standard (decachlorobiphenyl) response. 

The data in the memory of the y-ray spectrometer was punched on 8-channel paper tape, converted to punched cards, and processed through a rather primitive computer program which provided both a count per channel output plus a not too reliable routine for peak finding and integrating net area. All results were hand calculated from net peak areas and... 

For each set of Chromarods (normally ten per rack), five are considered as one unit and are spotted with one sample or standard solution to provide five replicates. The average peak area derived from each unit is considered as one data point. One rack (two units) can be processed and analyzed at a time. 

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