Quantitation peak area

As noted in the preceding table, elution of PAHs is detected by UV absorbance at two different wavelengths 280 nm and 365 nm. Fluorescence detectors are also applicable to the HPLC analysis of PAHs (9, 19). The UV detector monitors the sample simultaneously at two wavelengths, aiding in compound identification. For a specific compound, the ratio of absorbances at two different wavelengths is an intrinsic physical characteristic. Therefore, it is possible, in principle, to identify a sample analyte by this characteristic ratio. The chromatographic retention time of each of the specific peaks observed in the sample eluate is compared with those of known standard compounds for tentative analyte identification. For quantitation, peak areas of each standard, at each of six... 

Figure 9.50 Typical lysine 2,3-aminomutase run. (A) Initial incubation mixture (t = 0), (f ) Analysis after incubation for 135 minutes. (For quantitation, peak areas are determined the /B peak area is multiplied by 1.85, and total, (1.85 X 0) + a), normalized to 100%. (From Aberhart, 1988.)...

Processing of Data. The chromatogram obtained contains qualitative Ur and k ) and quantitative (peak area or height) information. The quantitative information can be evaluated manually or automatically (by an integrator or computer system). 

Similarly, known mixtures of the component halides, e.g. butyltin tribromide, dibutyltin dibromide and tributyltin monobromide, were made up, and plots were constructed of peak heights and areas against weight concentrations in the mixture. These calibration curves were sufficient to enable each component of the mixture to be determined quantitatively. Peak areas were measured as the products of peak height and width at half peak height (Scott and Grant ). 

For reasonable quantitative accuracy, peak maxima must be at least 4cr apart. If so, then Rs = 1.0, which corresponds approximately to a 3% overlap of peak areas. A value of Rs = 1.5 (for 6cr) represents essentially complete resolution with only 0.2% overlap of peak areas. These criteria pertain to roughly equal solute concentrations. 

A quantitative analysis for vitamin Bi was carried out using this procedure. When a solution of 100.0 ppm Bi and 100.0 ppm o-ethoxybenzamide was analyzed, the peak area for vitamin Bi was 71 % of that for the internal standard. The analysis of a 0.125-g vitamin B complex tablet gave a peak area for vitamin Bi that was 1.82 times as great as that for the internal standard. How many milligrams of vitamin Bi are in the tablet ... 

To produce a quantitative result, chromatographic peak areas of identified target compounds are compared with peak areas of the internal standards, which are of known concentration. 

Liquid chromatography was performed on symmetry 5 p.m (100 X 4.6 mm i.d) column at 40°C. The mobile phase consisted of acetronitrile 0.043 M H PO (36 63, v/v) adjusted to pH 6.7 with 5 M NaOH and pumped at a flow rate of 1.2 ml/min. Detection of clarithromycin and azithromycin as an internal standard (I.S) was monitored on an electrochemical detector operated at a potential of 0.85 Volt. Each analysis required no longer than 14 min. Quantitation over the range of 0.05 - 5.0 p.g/ml was made by correlating peak area ratio of the dmg to that of the I.S versus concentration. A linear relationship was verified as indicated by a correlation coefficient, r, better than 0.999. 

Analytical information taken from a chromatogram has almost exclusively involved either retention data (retention times, capacity factors, etc.) for peak identification or peak heights and peak areas for quantitative assessment. The width of the peak has been rarely used for analytical purposes, except occasionally to obtain approximate values for peak areas. Nevertheless, as seen from the Rate Theory, the peak width is inversely proportional to the solute diffusivity which, in turn, is a function of the solute molecular weight. It follows that for high molecular weight materials, particularly those that cannot be volatalized in the ionization source of a mass spectrometer, peak width measurement offers an approximate source of molecular weight data for very intractable solutes. 

Quantitative analysis of the peroxy group of macroinitiators is performed by iodometry [38] and that of the azo group is done by ultraviolet (UV) spectrometry. Recently, type II MAI composed of PU was determined of its azo concentration by UV [20]. When the UV absorption spectral peak of the azo group overlaps other peaks, DSC is available by determining the azo group from the exothermal peak area [1IJ. 

The quantitative determination of a component in gas chromatography using differential-type detectors of the type previously described is based upon meas urement of the recorded peak area or peak height the latter is more suitable in the case of small peaks, or peaks with narrow band width. In order that these quantities may be related to the amount of solute in the sample two conditions must prevail ... 

Peak area is commonly used as a quantitative measure of a particular component in the sample and can be measured by one of the following techniques. 

Data evaluation. It is, of course, necessary to correlate peak area with the amount or concentration of a particular solute in the sample. Quantitation by... 

Apparatus. A gas chromatograph equipped with a flame-ionisation detector and data-handling system. The use of a digital integrator is particularly convenient for quantitative determinations, but other methods of measuring peak area may be used (Section 9.4). 

The peak height is taken as the distance between the extended base line beneath the peak and the peak maximum. The peak height, under certain conditions, will be proportional to the mass of solute present in the peak and can, thus, be used in quantitative analysis. However, the most common measurement employed in quantitative analysis is the peak area. 

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. 

The refractive index detector, in general, is a choice of last resort and is used for those applications where, for one reason or another, all other detectors are inappropriate or impractical. However, the detector has one particular area of application for which it is unique and that is in the separation and analysis of polymers. In general, for those polymers that contain more than six monomer units, the refractive index is directly proportional to the concentration of the polymer and is practically independent of the molecular weight. Thus, a quantitative analysis of a polymer mixture can be obtained by the simple normalization of the peak areas in the chromatogram, there being no need for the use of individual response factors. Some typical specifications for the refractive index detector are as follows ... 

Quantitative estimates of the mass of a particular solute present in a sample are obtained from either peak height or peak area measurements. The values obtained are then compared with the peak height or area of a reference solute present in the sample at a known concentration or mass. In this chapter quantitative analysis by LC will be discussed but the procedures described should not be considered as entirely appropriate for other types of chromatographic analysis. Those interested in general quantitative chromatographic analysis including GC and TLC are referred to the book by Katz (4). 

There are two basic methods used in quantitative analysis one uses a reference standard with which the peak areas (peak heights) of the other solutes in the sample are compared the other is a normalization procedure where the area (height) of any one peak is expressed as a percentage of the total area (heights) of all the peaks. There are certain circumstances where each method is advantageous, and providing they are used carefully and appropriately all give approximately the same accuracy and precision. 

It is seen that there is not a great difference between the use of peak heights or peak areas for quantitative analysis, except possibly for very early peaks, where the results seem to indicate that peak height measurements might be more precise. However, it again must be emphasized that the measurements made by Scott and Reese were overall precision measurements that will include all variations in the chromatographic system. The difference between the two methods of measurement may well be significant, but the absolute values for precision will not, by any means, be solely dependent on the method of peak measurement. 

The factors chosen for study were the concentration of the ion-pairing reagent, the solution pH ( quantitative factors) and the acid chosen for pH adjustment (formic, acetic, propionic and trifluoroacetic acids) ( quahtative factor). The effect of these factors was assessed by using responses that evaluated both the HPLC (the number of theoretical plates and the retention time) and MS performance (the total peak area and peak height) for each of the four analytes studied, i.e. 1-naphthyl phosphate (1), 1-naphthalenesulfonic acid (2), 2-naphthalenesulfonic acid (3) and (l-naphthoxy)acetic acid (4). 

Successful use of modern liquid chromatography in the clinical laboratory requires an appreciation of the method s analytical characteristics. The quantitative reproducibility with respect to peak height or peak area is quite good. With a sample loop injector relative standard deviations better than 1% are to be expected. The variability of syringe injection (3-4% relative standard deviation) requires the use of an internal standard to reach the 1% level (2,27). 

The accuracy and precision of carotenoid quantification by HPLC depend on the standard purity and measurement of the peak areas thus quantification of overlapping peaks can cause high variation of peak areas. In addition, preparation and dilution of standard and sample solutions are among the main causes of error in quantitative analysis. For example, the absorbance levels at of lutein in concentrations up to 10 mM have a linear relationship between concentration and absorbance in hexane and MeOH on the other hand, the absorbance of P-carotene in hexane increased linearly with increasing concentration, whereas in MeOH, its absorbance increased linearly up to 5 mM but non-linearly at increasingly higher concentrations. In other words, when a stock solution of carotenoids is prepared, care should be taken to ensure that the compounds are fuUy soluble at the desired concentrations in a particular solvent. 

Quantitation of anthocyanins has become simple and fast since many anthocy-anin standards became commercially available as external standards in the past decade. When the standards are not available, individual anthocyanins or total monomeric anthocyanins can be determined by the use of a generic external standard such as commercial cyanidin-3-glucoside or other compound structurally similar to the analytes of interest. Individual and total peak areas are measured at 520 nm or their and quantified using external standards by which values are typically slightly different from those via the pH differential method. ... 

The aim of all the foregoing methods of factor analysis is to decompose a data-set into physically meaningful factors, for instance pure spectra from a HPLC-DAD data-set. After those factors have been obtained, quantitation should be possible by calculating the contribution of each factor in the rows of the data matrix. By ITTFA (see Section 34.2.6) for example, one estimates the elution profiles of each individual compound. However, for quantitation the peak areas have to be correlated to the concentration by a calibration step. This is particularly important when using a diode array detector because the response factors (absorptivity) may considerably vary with the compound considered. Some methods of factor analysis require the presence of a pure variable for each factor. In that case quantitation becomes straightforward and does not need a multivariate approach because full selectivity is available. 

Quantitation is performed by the calibration technique. A new calibration curve with anilide standard solutions is constructed for each set of analyses. The peak area or peak height is plotted against the injected amount of anilide. The injection volume (2 pL) should be kept constant as the peak area or peak height varies with the injection volume. Before each set of measurements, the GC or HPLC system should be calibrated by injection of standard solutions containing about 0.05-2 ng of anilide. Recommendation after constructing the calibration curve in advance, standard solutions and sample solutions are injected alternately for measurement of actual samples. 

Quantitative analysis is performed by the calibration technique. A new calibration curve with a standard solution of each diphenyl ether herbicides is constructed, plotting the peak area against the amount of standard solution injected. Each diphenyl ether herbicide in the sample is measured by using the peak area for each standard. Before each set of measurements, the GC and HPLC system is checked by injecting more than one standard solution containing ca 0.01-2 mg L of each compound. 

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