Calibration normalizing peak areas

An important feature of modern high-performance liquid chromatography (HPLC) is its excellent quantitation capability. HPLC can be used to quantify the major components in a purified sample, the components of a reaction mixture, and trace impurities in a complex sample matrix. The quantitation is based on the detector response with respect to the concentration or mass of the analyte. In order to perform the quantitation, a standard is usually needed to calibrate the instrument. The calibration techniques include an external standard method, an internal standard method, and a standard addition method. For cases in which a standard is not available, a method using normalized peak area can be used to estimate the relative amounts of small impurities in a purified sample. 

Quantitative analysis usually requires the use of standards and/or certified reference materials (CRMs), the selection of an appropriate elemental optical emission line, and, in most cases, the selection of a normalization line, used as an IS. Calibration curves are then constructed using the normalized peak area versus concentration, as previously described for calibration using an IS. When there is a dominant matrix component for which the concentration will remain approximately constant across the calibration set, it is best to use an emission line from that matrix element for normalization. This approach helps minimize effects due to changes in plasma conditions caused by shot-to-shot fluctuations in laser intensity. Alternatively, chemometric correlation analysis of the entire observed spectrum with the concentration of the analyte can be used to construct calibration curves automatically. In general, RSDs of 5%-10% are readily achievable. To improve quantitation, sample preparation methods such as pressing pellets may improve results for soils and sediments and fusion with salts to convert the sample into a glass bead can eliminate matrix effects. Fusion was discussed in Chapter 1 and is used extensively in XRF analysis (Chapter 8). 

Again, calculate the peak area of the indium melt and include the onset temperature in the calculation (Fig. 4). Observe that the limits of the calculation are set on the flat portion of the baseline before and after the melting peak. Use the onset temperature as the melting point of indium and use the normalized peak area (AB) as the enthalpy value for indium. Indium is the purest material available for calibration of a DSC and should always be... 

GPC analyses were performed with a Waters Model 244 chromatograph using Microstyragel columns. Both differential refractive index and UV (254 nm) detectors were used. THF was the eluant with a flow rate of 2 ml min-1. A benzene internal standard was employed to correct for flow variations and for normalization of the integrated peak areas. The column set was calibrated using nearly monodispersed polystyrene standards and all molecular data are reported as polystyrene-equivalent molecular weights. 

Concentration in the sample (c). This is normally calculated using both peak areas and peak heights as it is a good idea to postpone the selection of a calibration technique until after the ruggedness study. Mean number of theoretical plates, N, there are several methods to calculate N. The following calculation is often employed due to its convenience as it uses values which are previously collected as part of the data handling. 

Thompson and Hatina (135) showed that the sensitivity of a fluorescence detector toward unesterified vitamin E compounds under normal-phase conditions was at least 10 times greater than that of a variable-wavelength absorbance detector. The relative fluorescence responses of the tocopherols at 290 nm (excitation) and 330 nm (emission), as measured by HPLC peak area, were a-T, 100 /3-T, 129 y-T, 110 and 5-T, 122. The fluorescence responses of the corresponding to-cotrienols were very similar to those of the tocopherols, and therefore tocotrienol standards were not needed for calibration purposes. The fluorescence detector also allows the simultaneous monitoring of ubiquinone derivatives for example ubiquinone-10 has been detected in tomato (136). 

Quantification can take place by the normalized percent method or by standard addition using the integrated peak areas of the fluorosilanes, taking account of the substance-specific correction factors. Calibration and checks of linearity were carried out using mixtures of siloxanes of known purity, e.g., methyltris(trimethylsiloxy)silane (M3T) in octamethylcyclotetrasiloxane (D4). 

The basic operation of the instrument (3) consists of the Ionization of the sample in the cell by a timed electron beam which Is followed, after a short Interval, by an RF pulse applied to the plates of the cell. This pulse coherently excites all ions In the cell into cyclotron motion. The motion continues after cessation of the pulse, and the resonance is detected by the plates of the cell, amplified and the data stored In the computer. The excltatlon/detection cycle is repeated numerous times and the collected data summed. The data Is then subjected to fourler transformation and the frequency spectrum resulting converted into a mass spectrum. The spectrum Is normalized to the major peak. For quantitative work, the calibration can be based either on peak height or peak area. Here, major considerations will Include the resolution chosen and the relative concentrations of the constituents under Investigation. 

The calculation of the relative characteristic peak areas on the chromatograms of the volatile pyrolysis products, using an external standard irrespective of the pyrolysis procedure, permits one to take into account the sensitivity of the detector, with easy computation of the ratio between the peak areas of the component of interest and the standard which, under normal conditions (sample size, carrier gas flow-rate, pyrolysis temperatures, etc.) are proportional to the absolute amounts of the pyrolysis products. This method of calculation is essentially a modification of the absolute calibration method in gas chromatography, which had never been used before in Py—GC.To facilitate comparison of the results obtained at different times or on different instruments, the results of individual measurements should preferably be presented in terms of specific yields (or relative characteristic peak areas), i.e., the yield of the volatile pyrolysis products must be calculated per 1 mg (or g or ng) of the pyrolysed sample with respect to 1 mg (or g or Mg) of the external standard. Such a calculation makes sense in the range of sample sizes which affect only insignificantly the specific yield of light pyrolysis products. 

Four techniques are commonly used to convert peak heights or areas into relative composition data for the sample. These are the normalization method, the external standard method, the internal standard method and the method of standard additions [264,284]. In the normalization method the area of all peaks in the chromatogram are summed and then each analyte is expressed as a percentage of the summed areas. All sample components must elute from the column and their responses must fall within the linear operating range of the detector. This method will always lead to totals representing 100%. If the detector response is not the same for all compounds then response factors are required to adjust the peak areas to a common scale. Response factors are usually determined as the slope of the calibration curve and converted to relative response factors since these tend to be more stable than absolute values. 

The photo peaks from a well behaved detector are gaussians. Thus the FWHM value determines the peak shape. The energy scale is calibrated against standard isotopes and is normally almost linear. In most applications only the area below the photo peak but above the underlying continuum is used as a measure of activity. The total efficiency (based on photo peak area) varies usually with energy in the way illustrated by Figure 8.20. 

A further complication is encountered where the analyte line may be partially overlapped by a second line with a peak at [ [Fig. 9.3(b)]. The best solution in this case is to estimate the contribution of i at p, either by calibration or by means of a blank, and to determine the intensity ratio of i at Op and By assuming that this ratio Ki is constant, the background at Op can be taken as Kjli. In the case of energy-dispersive spectrometers where peak areas are normally used, a simple mathematical solution based on area interference can be employed. However, in practice it is more common to employ a computer-applied least-squares fitting technique (see Chap. 6). 

Most integrators perform area percent, height percent, internal standard, external standard, and normalization calculations. For nonlinear detectors, multiple standards can be injected, covering the peak area of interest, and software can perform a multilevel calibration. The operator then chooses an integrator calibration routine suitable for that particular detector output. 

Generally, it is usually better to prepare a calibration curve by a plot of peak area (or height) against the concentrations of known standards. Such a plot will normally be a straight line, but a perfectly valid calibration plot may deviate from linearity, especially at lower concentrations. 

As is apparent from the table, it is not easy to select the most appropriate method for quantification. The plot of peak height (peak area) against concentration is linear only within a narrow range of concentrations and, therefore, the application of a 5-point calibration covering the total range of concentrations is highly recommended. Similarly, the usefulness of the internal standard method relates mainly to those analytical tasks requiring complex sample preparation procedures. Area normalization can not be used for the evaluation of the TLC results. 

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