Quantitation peak height

Quantitation Peak height Peak area Method used Calibration method Number of calibration standards Linear range of calibration. 

The slab is enlarged in an enlarger (9A), and with a light spot planimeter the curves are integrated for quantitation. Peak height is not as accurate because many peaks are un-symmetrical."... 

If the purity of the synthetic standards is questionable, the following is suggested use the (impure) ng/yl standards to obtain retention time and the detector linear range. Then, during the analysis, add the appropriate phenyltin chloride directly to a test tube and derivatize with lithium aluminium hydride, eliminating the usual extraction and concentration steps. Since the conversion occurs quantitatively, peak heights from these derivatization standards can be used to construct a standard curve. The procedure calls for... 

The external reflection of infrared radiation can be used to characterize the thickness and orientation of adsorbates on metal surfaces. Buontempo and Rice [153-155] have recently extended this technique to molecules at dielectric surfaces, including Langmuir monolayers at the air-water interface. Analysis of the dichroic ratio, the ratio of reflectivity parallel to the plane of incidence (p-polarization) to that perpendicular to it (.r-polarization) allows evaluation of the molecular orientation in terms of a tilt angle and rotation around the backbone [153]. An example of the p-polarized reflection spectrum for stearyl alcohol is shown in Fig. IV-13. Unfortunately, quantitative analysis of the experimental measurements of the antisymmetric CH2 stretch for heneicosanol [153,155] stearly alcohol [154] and tetracosanoic [156] monolayers is made difflcult by the scatter in the IR peak heights. 

Quantitative Calculations In a quantitative analysis, the height or area of an analyte s chromatographic peak is used to determine its concentration. Although peak height is easy to measure, its utility is limited by the inverse relationship between the height and width of a chromatographic peak. Unless chromatographic conditions are carefully controlled to maintain a constant column efficiency, variations in... 

Of the six parameters shown in Figure 13.18, the most important are peak height and return time. The peak height is related, directly or indirectly, to the analyte s concentration and is used for quantitative work. The sensitivity of the method, therefore, is also determined by the peak height. The return time determines the frequency with which samples maybe injected. Figure 13.19 shows that when a second sample is injected at a time T after injecting the first sample. 

Most flow injection analyses use peak height as the analytical signal. When there is insufficient time for reagents to merge with the sample, the result is a split-peak, or doublet, due to reaction at the sample s leading and trailing edges. This experiment describes how the difference between the peak times can be used for quantitative work. 

To unambiguously identify the presence of a peak and, in addition, be able to give some proximate estimation of its size for quantitative purposes, the peak height needs to be at least 5 times the noise level. The detector sensitivity, or the minimum detectable concentration, (Xd), is defined as that concentration of solute that will give a signal equivalent to twice the noise level and, consequently, the concentration of solute at the limiting (k ) value must be 2.5Xd. 

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. 

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 ... 

For quantitative measurements peak heights (expressed in mm) are usually measured of the long-wave peak satellite of either the second- or fourth-order derivative curves, or for the short-wave peak satellite of the same curves. This is illustrated in Fig. 17.16(a) for a second-order derivative DL is the long-wave peak height and Ds the short-wave peak height. Some workers11 have preferred to use the peak tangent baseline (DB) or the derivative peak zero (Dz) measurements [Fig. 17.16(h)]. 

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. 

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. 

Quantitation in high performance liquid chromatography, as with other analytical techniques, involves the comparison of the intensity of response from an analyte ( peak height or area) in the sample under investigation with the intensity of response from known amounts of the analyte in standards measured under identical experimental conditions. 

When making quantitative measurements, shouid peak height or area be used ... 

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). 

This technique detects substances qualitatively and quantitatively. The chromatogram retention time is compound-specific, and peak-height indicates the concentration of pollutant in the air. Detection systems include flame ionization, thermal conductivity and electron capture. Traditionally gas chromatography is a laboratory analysis but portable versions are now available for field work. Table 9.4 lists conditions for one such portable device. 

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 highest separation standards are set for preparative separations beeanse the resolution has to be at least 1.5 (6 o), ensuring that peaks are separated eompletely (Figure 5.1). It is based on the faet that the whole substance peak is intended to be recovered to the highest degree of purity. In contrast, for quantitative purposes a resolution of 1.0 (4 a) is sufficient, because then a peak is pure at its maximum and can be evaluated by peak height. 

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. 

Quantitation is performed by the calibration technique. Prepare a calibration curve by injecting pyrithiobac-methyl standard solutions, equivalent to 0.2,0.5,1.0,2.0,3.0 and 4.0 ng, into the gas chromatograph. Measure the heights of the peaks obtained. Plot the peak heights in millimeters against the injected amounts of pyrithiobac-methyl in nanograms. 

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