Peak retention times, computing

Preparation of the reference mixture in the same matrix as the wastewater samples, namely the wastewater itself, was investigated for minimizing sample differences and their effect on peak retention-time behavior. The same series of 17 reference compounds was added to a sample of Oxy-6 gas condensate. A sufficient amount of each reference was added to swamp any nearby wastewater headspace peak. Triplicate analysis were run, and the computed retention indices for the reference compounds and the unknown wastewater headspace peaks were stastically compared. The results (Table 1, column V Fig. 2, peaks with asterisks) show a substantially improved correlation. Fourteen of the 17 reference compounds, including all the methyl- and ethyl-pyridine isomers, were correlated with wastewater peaks. Since 7 of the correlated peaks were among the 22 peaks proviously shown (see Section 32) to be common to the 8 wastewater samples (Table 1), these compounds may be presumed to be present in all the waters 2- and 3-methyl- 2-, 3-, and 4-ethyl- 2,4-dimethyl- and 2,6-dimethyl-pyridines. Furthermore, the 37 peaks cotmnon to Oxy-6 gas condensate and Oxy-7 and 8 gas condensate (see Section 32) included 11 of the... 

The GC results are computed automatically with the aid of a calculator. The natural rubber butadiene-styrene rubber butadiene rubber ratio in the sample material is printed out together with the initial data on pyrolysis product peak retention time, peak area, etc.). This system has been used successfully by Coulter and Thompson [69] for over 2 years in industrial analysis. As a result, the quality of the end product (tyres) was drastically improved. A similar device can be developed using a furnace-type pyrolyser. 

Another major use of computers for HPLC data should be varietal identification (see later). Qualitatively, cereal storage proteins vary little within genotypes but significantly among different varieties, so they provide characteristic fingerprints. Varietal identification can be automated by computer comparisons with stored standard data. Scanlon et al. [80] showed that normalization of peak retention times provided sufficient precision for cultivar identification. Resulting data based on peak heights and times could be used in an automated library search to identify wheat varieties [81]. 

Now, we are in a position to compute peak retention times in terms of their measured thermodynamic coefficients, the column dimensions, the carrier-gas type, the pressure drop, and the column temperature. We get the following relationship by combining Equations 4.10 and 4.11 ... 

A2.2.1.3 Record the partial pressure and operate the gas sampling valve to place Ae sample onto the column. Record the chromatogram, integrator/computer peak areas, and peak retention times. 

Fig. 2. Amino acid analysis by automated ion-exchange chromatography. Standard column, 4.6 mm ID x 60 mm Ninhydrin developer. Computer print out indicates retention time (RT), height and area of peaks, and the ratio of the height of an amino acid in the sample to the height of a standard amino acid.

A range of concentrations of the two substances were inserted in equation (6) and a curve constructed relating retention time of the composite peak (calculated by means of a computer) to mixture composition. The results are shown in Figure 7. 

Let the distance between the injection point and the peak maximum (the retention distance on the chromatogram) be (y) cm and the peak width at the points of inflexion be (x) cm. If a computer data acquisition and processing system is employed, then the equivalent retention times can be used. 

Using equation (10), the efficiency of any solute peak can be calculated for any column from measurements taken directly from the chromatogram (or, if a computer system is used, from the respective retention times stored on disk). The computer will need to have special software available to identify the peak width and calculate the column efficiency and this software will be in addition to that used for quantitative measurements. Most contemporary computer data acquisition and processing systems contain such software in addition to other chromatography programs. The measurement of column efficiency is a common method for monitoring the quality of the column during use. 

From a quantitative perspective, each peak is defined by two parameters, i.e. the position of its baseline and the retention time boundaries, with those derived by the computer system being shown in Figure 3.27. It is not the intention of this present author to discuss how these have been determined but simply to point out that their positions may have a significant effect on the accuracy and precision of any quantitative measurements, especially, as in Figure 3.27, when the baseline is not horizontal and the signals from each of the components are not fully resolved. It is usual for the software to allow the analyst to override the parameters chosen by the computer to provide what they consider to be more appropriate peak limits and/or baseline positions. 

Xianren, Q., Chong-yu, X., and Baeyens, W., Computer-assisted predictions of resolution, peak height and retention time for the separation of inorganic anions by ion chromatography, ]. Chromatogr., 640, 3, 1993. 

Experiment A is a non-adsorption experiment through the core, performed to measure the time for emergence of the peak. A 1.3% (concentration higher than the standard) acidified brine is loaded into the sample loop to be used as a sample medium. This particular experiment is carried out to measure the retention time by recording the time required before the peak is observed. The retention time can also be used to compute the exact porosity of the core, under the assumption of zero adsorption of salts from the brine. 

TIC) for each urine sample. For comparison of TICs, Agilent ChemStation software allows one easily to stack (overlay) several chromatograms in the same computer window (Fig. 2.2). This feature helps to identify compounds with common retention times among several samples, but does not contain any mass spectral data for verification of a common identity for these peaks and is most useful for comparison of only a few TICs or a few compounds of interest. In these cases, mass spectral comparisons can be made quickly by visual inspection of the relevant spectra. 

A data processor plots the chromatogram, automatically integrates the peak areas and prints retention times, percent areas, baseline drift and attenuation for each run. It also computes blank values, constant factors and relative average elemental contents. 

The modern analytical laboratory employing instrumental chromatography uses a computer data collection system and associated software to acquire the data and display the chromatogram on the monitor. Parameters important for qualitative and quantitative analysis, including retention times and peak areas, are also measured and displayed. The software can also analyze the data to determine resolution, capacity factor, theoretical plates, and selectivity. 

Pigments were separated on a normal (150 X 4 mm i.d., particle size 5 /tin) and on a microbore ODS column (150 X 2 mm i.d., particle size 4 jttm) using gradient elution. The steps of gradient elution are shown in Table 2.7. Carotenoids were detected at 440 nm. Columns were not thermostated and separations were performed at room temperature (20 2°C). The mean and the relative standard deviation of retention time and peak area were computed from three parallel measurements. The carotenoids capsanthin, zeaxanthin and j0-carotenein and the extracts were tentatively identified comparing their retention time with those of authentic standards. 

Fenvalerate Data. Calibration data for the GC measurement of Fenvalerate were furnished by D. Kurtz (17). Average responses for five replicates at each of five standard concentrations are given in Table III. It should be noted that the stated responses are not raw observations, but rather on-line computer generated peak area estimates (cm ). (Had we started with the raw data [chromatograms], the problem would actually have been two-dimensional, including as variables retention time and concentration.) The stated uncertainties in the peak areas are based on a linear fit (o a+bx) of the replication standard deviations to concentration and the "local slopes" [first differences] in the last column of Table III are presented... 

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. 

Retention time calibration. In spite of all effort to obtain reproducible retention time values these varied for the same component between different chromatographic runs, mainly because of different sample amounts. To solve this problem all retention time values were calibrated. Values for a limited number of peaks, that could easily be found in all the chromatograms in a set, were manually entered into the computer. For each of these reference peaks the mean value M j) over all the runs was calculated. New retention tim.es, Rtcal(i), for the peaks in the data set were then calculated by the straight-line expression ... 

When a particular component eluting at a certain retention volume is to be estimated, this approach can be outlined as follows. Since SEC is extremely reproducible, the peak shape, peak width and peak height are dependent on the amount of the species in the sample volume injected, sample volume and retention time. From these factors the SEC peaks can be simulated or elution pattern of any species within the separation range can be plotted as a function of mass vs. retention volume. The analysis data supplies the concentration of this particular species over two or more 0.5 ml intervals. A match-up computer program has to be developed so that it can pick up the peak shape and concentration based on 3 or 4 data points at known Intervals. 

---