Peak counting chromatograms

Method a(counts) KxlO a (counts" ) Calibration Curve Slope at Peak of Chromatogram... 

A series of computer-simulated chromatograms has been generated to test the validity of a procedure derived from the statistical model for calculating the number of randomly distributed components when many of them are obscured by overlap. Plots of the logarithm of the peak count versus reciprocal peak capacity are used for this purposTI TRese plots are shown to provide reasonable estimates of the total number of components In the synthetic chromatograms. 

Two methods of peak counting were used In this study. Both methods were based on visual Inspection of the synthesized chromatograms. The criteria used to differentiate between peaks were baseline separation and resolution between maxima. The former was used to test directly the validity of the model Independently of any empirical adjustment. The simulated chromatograms were synthesized with a flat and clearly discernible baseline before the first and after the final peak (see Figure... 

A statistical analysis of the peak counts obtained from the simulated chromatograms was made as follows. We changed the random number sequence, by means of the seed change previously described, to generate random changes In component retention times and amplitudes while holding constant component number, zone width, and peak capacity. This procedure. In essence, mimicked the Injection of different samples with the same component number and zone width onto a column. A mean peak count and standard deviation at each of the different peak capacities were calculated. The means and standard deviations of the peak counts were fit by a least squares analysis to Equation 11 with a proper transformation of the standard deviations from an exponential to a linear function (5). From the value of the least squares slope and Intercept, an estimated component number was calculated. 

Tables I and II are compilations of the total number of components estimated by using Equation 11 for the five data sets described above. Input data were the peak counts from a series of simulated chromatograms at five different n values for the three different component numbers. The results of Table I were obtained by a counting of baseline resolved peaks. Because these counts are nearly independent of component amplitude, these data are a rather direct test of the validity of the model and procedure. The results of Table II are, by contrast, based upon a counting of peak maxima and upon a calculation of an empirical resolution R. Table III contains data for a comparison of baseline peal counts from the simulated chromatograms to those predicted by theory.

Band width, and plate number, 38 Baseline peak, computer-simulated chromatograms, I8,20f Baseline separation, peak-counting methodology, 17,18 Benzenethiol, RPLC, 99f Benzo(e)pyrene, excitation spectra, I9lf,l92f Bls(chloromethyl) ether, air monitor, 20>lf... 

Most GPC columns are provided with vendor estimates of the plate count of the column and a chromatogram of a series of test peaks. These plate count estimates are usually obtained using small molecule analytes that elute at the total permeation volume (Vp) of the column. The Gaussian peak shape model... 

When John Phillips, in 1991, presented the practical possibility of acquiring a real comprehensive two-dimensional gas chromatographic separation (33), the analytical chemists in the oil industry were quick to pounce upon this technique. Venkatramani and Phillips (34) subsequently indicated that GC X GC is a very powerful technique, which offers a very high peak capacity, and is therefore eminently suitable for analysing complex oil samples. These authors were able to count over 10 000 peaks in a GC X GC chromatogram of a kerosine. Blomberg, Beens and co-workers... 

Having chosen the test mixture and mobile diase composition, the chromatogram is run, usually at a fairly fast chart speed to reduce errors associated with the measurement of peak widths, etc.. Figure 4.10. The parameters calculated from the chromatogram are the retention volume and capacity factor of each component, the plate count for the unretained peak and at least one of the retained peaks, the peak asymmetry factor for each component, and the separation factor for at least one pair of solutes. The pressure drop for the column at the optimum test flow rate should also be noted. This data is then used to determine two types of performance criteria. These are kinetic parameters, which indicate how well the column is physically packed, and thermodynamic parameters, which indicate whether the column packing material meets the manufacturer s specifications. Examples of such thermodynamic parameters are whether the percentage oi bonded... 

FIGURE 5 A chromatogram showing a peak from a column with plate count of 2916. 

Figure 3 shows a gas chromatogram obtained from soil with plants of one barley cultivar, Etu. It is possible to count over 400 peaks, and the figure illustrates the very complex pattern of volatiles in the rhizosphere. If we compare chromatograms obtained from samples of Etu, Tellus and soil only, no specific difference can be seen. Most of the peaks seem to be present in all three chromatograms, although the intensities of the peaks vary. The necessity of computer help with the pattern recognition is obvious. 

First, the sample was examined by GPC, for which four columns of styragel of 106,10s, 104 and 103 A nominal pore size were used. The total number of theoretical plates as determined by acetone at a flow rate of 1 ml/min was ca. 26,000. The eluent was tetrahydrofuran. The chromatogram is shown in Figure 9, which indicates two peaks at ca. 21 and 24 counts. The former may be assigned to the tetra-chain, star-draped component, and the latter to the precursor. However, no complete separation of the two peaks was observed. For another comparison, velocity ultra-centrifugation was performed for the sample at 59,780 rpm using a 6-solvent for polystyrene, cyclohexane. The operation temperature was established at 35 °C, the 6-temperature, to minimize the concentration dependence of sedimentation velocity and other effects. A sedimentation pattern taken by UV-absorption is shown in Figure 10. It is seen that the separation of S-A sample into the two components was quite difficult even at a very low polymer concentration, 0.077 g/dl. 

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 marked improvement in results presently obtained lies in the elimination of sources of error now recognized as being present in the prior procedure. A major source of error was the extrapolation from the log-log plot of half-count vs. intrinsic viscosity for the four eluates which lay about the peak of the chromatogram. It is this extrapolation that supplied the necessary intrinsic viscosities of the remaining more... 

Figure 1.8 Variation of the theoretical peak capacity (rip of a chromatogram with the plate count (A/), for three ranges of capacity factors (a) 0.2k<10 and (c) 0.5

Davis and Giddings [105] have argued that the theoretical peak capacity is usually not even approached. Instead, they conclude that in order to provide a 90% probability for a compound of interest to appear as a pure peak in the chromatogram, the available peak capacity should exceed the theoretically required value (eqn.1.25) by a factor of 20. If we consider the same range of capacity factors this results in an excess plate count of about a factor of 400. 

We note that m is a statistical number related to basic constant A it may differ slightly from the true component number m. However, because of peak overlap, the true m cannot be obtained by counting the number of peaks appearing in the chromatogram. Thus m, if obtainable, becomes our best approximation for m. 

An example of the application of this procedure to a complex chromatogram is shown in Figure 6.13 [34]. The sample is a mixture of polynuclear aromatic hydrocarbons from river sediments fractionated by capillary gas chromatography in the laboratory of Dr. M. L. Lee [40]. A count of peak maxima yields p = 145. Component numbers estimated from the slope and intercept give m = 234 and 267, respectively, yielding an average estimate of m = 250 for the number of components, which exceeds the number of peaks... 

Once a separation is developed, several pieces of information about the sample can be ascertained from the chromatogram. First, by counting the peaks, one can estimate how many components are present in the mixture. Second, by the use of standards, both the identity and concentration of each compound present can be obtained. Lastly, if the mixture is totally unknown, the peaks can be collected and the identity confirmed by other instrumental methods of chemical analysis (e.g., infrared, nuclear magnetic resonance, or mass spectroscopy). 

Fig. 8.4 shows the chromatogram from the original l(X) mg injection of crude sample overlaid with the chromatogram from the injection of the purified 40 mg of sample. This clearly shows that not only was the purification successful, but both injections gave main peaks of the same area count for UV response. The collected fraction from this second injection (i.e. the injection of 40 mg of pure compound) yielded almost 40 mg of material upon recovery from the liquid fraction. The combination of these two results... 

---