Area, peak measurement

Equation (5.13) can only be used in this way when the various factors are multiplied together. If the factors 

The process of combining uncertainties will have to be done in separate stages. In this example, the overall uncertainty of (C — B) must be calculated by using Equation (5.11). This nncertainty, expressed in relative terms, can then be combined in quadrature with the relative uncertainties of Y and E. Combination of uncertainties will be discnssed further in Section 5.8, where uncertainty bndgets are discnssed. 

If we take the first channel on each side of the peak beyond what we consider as being the peak region as representative of the background, then the gross (or integral) area of the peak is  

It is stiU possible to find incorrect expressions for the calculation of peak area uncertainty in the literature. The confusion arises because of a failure to appreciate that unlike single background counts where the variance of the count is numerically equal to the count itself, the variance of a peak background depends upon the number of background channels used. The offending expressions are variation of the form  

These expressions are certainly correct for a single count plus background count, for example, from a simple beta counter. They are not valid for peak area calculations where Equation (5.40) must be used, resulting in the correct expression  

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In addition, even if peak area measurements are used, a separation of 4a will usually... 

The fact that we have peaks within a 2D space implies that where no peak is found represents a true detector baseline or electronic noise level. In a conventional petroleum sample, a complex unresolved mixture response causes an apparent detector baseline rise and fall throughout the GC trace. It is probably a fact that in this case the true electronic baseline is never obtained. We have instead a chemical baseline comprising small response to many overlapping components. This immediately suggests that we should have more confidence in peak area measurements in the GC X GC experiment. 

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). 

The area of a peak is the integration of the peak height (concentration) with respect to time (volume flow of mobile phase) and thus is proportional to the total mass of solute eluted. Measurement of peak area accommodates peak asymmetry and even peak tailing without compromising the simple relationship between peak area and mass. Consequently, peak area measurements give more accurate results under conditions where the chromatography is not perfect and the peak profiles not truly Gaussian or Poisson. 

Unfortunately, neither the computer nor the potentiometric recorder measures the primary variable, volume of mobile phase, but does measure the secondary variable, time. This places stringent demands on the LC pump as the necessary accurate and proportional relationship between time and volume flow depends on a constant flow rate. Thus, peak area measurements should never be made unless a good quality pump is used to control the mobile phase flow rate. Furthermore, the pump must be a constant flow pump and not a constant pressure pump. 

The Relative Precision of Peak Height and Peak Area Measurements... 

CALIB.dat Calibration measurements at eight concentrations (double determinations) using a GC peak area measurements in [mV sec] vs. weight... 

Analytical Techniques. Sessile drop contact angles were measured with a NRL C.A. Goniometer (Rame -Hart, Inc.) using triply distilled water. The contact angles reported are averages of 2-8 identically treated samples with at least three measurements taken on each sample. ESCA spectra were obtained on a Kratos ES-300 X-ray Photoelectron Spectrometer under the control of a DS-300 Data System. Peak area measurements and band resolutions were performed with a DuPont 310 Curve Resolver. 

Table 4.12 Precision of methods of peak area measurement Method of measurement Relative precision, %...

Rarely will it be possible to draw conclusions directly from the raw data of analytical measurements and it is usual for some refinement of the data to be carried out. In its simplest form this could merely comprise background corrections, but it is often much more complex, requiring corrections for a number of factors as in mass spectrometry, X-ray fluorescence and electron probe microanalysis. More complex routines made available by computers include spectrum smoothing, stripping one component from a spectrum or making peak area measurements from chromatograms. 

Changing the attenuation is not required, and (Hi) Offers highest precision in peak-area measurement. 

The relationship between the concentration of the solute and the peak produced in the chromatogram is, strictly speaking, only valid for peak area measurements, but in most instances it is more convenient to measure peak height. Such peak height measurements should only be used when all the peaks are very narrow or have similar widths. The tedium and lack of precision associated with non-automated methods of peak area measurements may be overcome using electronic integrators, which are features of most modern instruments. 

FIGURE 7 Comparison of two peaks with signal-to-noise ratio (S/N) of 50 and 10. The random distribution of noise in the noisy peak controls the variation of the peak area measurement. 

A comprehensive review of electrothermal atomization devices has been published (94). The review includes a discussion of commonly encountered problems such as atom loss through non-pyrolytic graphite, non-isothermal conditions, differences in peak height and peak area measurement, etc. 

Have you had an opportunity to evaluate the results from peak heights versus peak area measurements ... 

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. 

Contrary to peak height measurement, there are a number of techniques used for peak area measurement. Some of these are manual techniques and others make use of instrumental accessories to provide an area measurement. The discussion that follows will consider all of these techniques from the manual through the instrumental, in that order. 

CALCULATION OF RESULTS. Peak area measurement was done by triangulation. The equation below would be used for the calculation of the amount of steroid in 20 cm3 urine ... 

There are a variety of other factors that influence the accuracy of quantitative analysis. Noise, in the form of baseline disturbances and baseline drift, affects area more than it does height, as it can cause area to be lost at the tailing edges of the peaks where they are widest. Peak asymmetry and detector saturation or nonlinearity, however, have a more detrimental effect on peak height. Figure 7.6 shows a calibration curve comparing peak height measurements with peak area measurements.13... 

Fig. 10.1 shows the chromatogram of a mixture of lOmg L 1 of chloride sulphate, nitrate, phosphate and carbonate obtained with the flow colorimetric detector by elution with 60% acetone water. As can be seen from this figure phosphate could be separated from the strong acid anions and carbonate. The RS value between the strong acid anions and phosphate was about 1.7. This RS value suffices for the quantitation of phosphate by the peak area measurement with a computing integrator. 

There are several conclusions that can be drawn from these results. First, the peak areas have a pooled RSD of about 5%, a value which is much better than that expected for densitometric scanning of electrophoresis gels. Second, note that the standard deviation of the peak area measurements was essentially independent of the volume injected. Thus, the relative standard deviation of the peak area dramatically decreases as the injection volume increases. An injection volume of at least 10 nL is required to obtain good precision (2-3%, excluding a single poor replicate... 

There are other major problems with peak assignment on the basis of the areas. These problems relate to the reproducibility of peak area measurements under widely varying conditions. Ideally, the area of a peak remains constant even if its capacity factor varies. However, varying the conditions may affect the peak areas. If the column temperature is changed in GC, then the flow rate may be affected. Peak areas will change (by a constant factor) if concentration-sensitive detectors such as the hot wire detector (H WD katharo-meter) are used, but not with mass flow sensitive detectors (such as the flame ionization detector, FID). 

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