Absolute response factor

To calculate the response factor Kt of a compound t, it is essential, according to equation (4.7), to know the injected quantity. However, it is difficult to know precisely the injected volume, which depends on the injector or injection loop or the precision of the syringe. Moreover, the absolute response factor K, (not to be confused with the partition coefficient) depends on the tuning of the chromatograph. This factor is not an intrinsic property of the compound. This is why most chromatographic methods for quantitative analyses, whether they are pre-programmed into an integrating recorder or software, do not make use of the absolute response factor, Kj. 

There is no widely used detector that has predictable absolute response factors. This is why predicted rather than calibrated response factors are only used when calibration is not practical, such as for unknowns, or when a closely related compound has been calibrated, allowing extrapolation to the desired response factor. 

This technique, employing the absolute response factors, yields very reliable results with chromatographs equipped with an auto-sampler a combination of a carousel sample holder and an automatic injector. This permits numerous measurements to be made without interruption, to the condition that no change in the apparatus tuning is made between injections. 

The external standard method uses absolute response factors, the internal standard method is calibrated in terms of response ratios. In both calibration methods, each peak is calculated independently. In external standard methods, the amount of sample injected must be highly reproducible. The method is well suited for automatic mechanical methods of injection. Optimum system performance must be maintained by frequent checks and regular recalibration. The internal standard method is independent of sample size and compensates for any slight instrumental drift. When used properly, it is the most accurate... 

External Standardization Technique (EST). This method requires the preparation of calibration standards. The standard and the sample are run as separate injections at different times. The calibrating standard contains only the materials (components) to be analyzed. An accurately measured amount of this standard is injected. Calculation steps for standard (1) for each peak to be calculated, calculate the amount of component injected from the volume injected and the known composition of the standard then (2) divide the peak area by the corresponding component weight to obtain the absolute response factor (ARE) ... 

Calculation Step for Sample. For each peak, divide the measured area by the absolute response factor to obtain the absolute amount of that component injected ... 

ARABS ARF angle resolved Auger electron spectroscopy absolute response factor DA diode array... 

Accuracy (systematic error or bias) expresses the closeness of the measured value to the true or actual value. Accuracy is usually expressed as the percentage recovery of added analyte. Acceptable average analyte recovery for determinative procedures is 80-110% for a tolerance of > 100 p-g kg and 60-110% is acceptable for a tolerance of < 100 p-g kg Correction factors are not allowed. Methods utilizing internal standards may have lower analyte absolute recovery values. Internal standard suitability needs to be verified by showing that the extraction efficiencies and response factors of the internal standard are similar to those of the analyte over the entire concentration range. The analyst should be aware that in residue analysis the recovery of the fortified marker residue from the control matrix might not be similar to the recovery from an incurred marker residue. 

Establish control charts of instrumental performance. Day-to-day variations in pump flow rate, relative response factors, absolute response to a standard, column plate counts, and standard retention times or capacity factors are all useful monitors of the performance of a system. By requiring that operators maintain control charts, troubleshooting is made much easier. The maintenance of control charts should be limited to a few minutes per day. 

Sample preparation, injection, calibration, and data collection, must be automated for process analysis. Methods used for flow injection analysis (FLA) are also useful for reliable sampling for process LC systems.1 Dynamic dilution is a technique that is used extensively in FIA.13 In this technique, sample from a loop or slot of a valve is diluted as it is transferred to a HPLC injection valve for analysis. As the diluted sample plug passes through the HPLC valve it is switched and the sample is injected onto the HPLC column for separation. The sample transfer time typically is determined with a refractive index detector and valve switching, which can be controlled by an integrator or computer. The transfer time is very reproducible. Calibration is typically done by external standardization using normalization by response factor. Internal standardization has also been used. To detect upsets or for process optimization, absolute numbers are not always needed. An alternative to... 

Generally, different components possess different response factors, application of which not only compensates for different detector response for different components but also take into consideration the other factors inherent with the procedure. However, these factors may be calculated by preparing a synthetic mixture absolutely identical to what is expected in the sample, and subsequently carrying out the gas-chromatography of this mixture exactly under idential experimental parameters as described in the method of analysis. Thus, we have ... 

It is critical when performing quantitative GC/MS procedures that appropriate internal standards are employed to account for variations in extraction efficiency, derivatization, injection volume, and matrix effects. For isotope dilution (ID) GC/MS analyses, it is crucial to select an appropriate internal standard. Ideally, the internal standard should have the same physical and chemical properties as the analyte of interest, but will be separated by mass. The best internal standards are nonradioactive stable isotopic analogs of the compounds of interest, differing by at least 3, and preferably by 4 or 5, atomic mass units. The only property that distinguishes the analyte from the internal standard in ID is a very small difference in mass, which is readily discerned by the mass spectrometer. Isotopic dilution procedures are among the most accurate and precise quantitative methods available to analytical chemists. It cannot be emphasized too strongly that internal standards of the same basic structure compensate for matrix effects in MS. Therefore, in the ID method, there is an absolute reference (i.e., the response factors of the analyte and the internal standard are considered to be identical Pickup and McPherson, 1976). 

If a calibration curve is not made and a data system is used to make the calculations, a slightly different procedure is followed. A calibration mixture prepared from pure standards is made by weight and chromatographed. Absolute calibration factors, equal to the grams per area produced, are stored in the data system for each analyte. When the unknown mixture is run, these factors are multiplied times the respective areas of each analyte in the unknown resulting in a value for the mass of each analyte. This procedure is a one-point calibration, as compared to the multipoint curve described before, and is somewhat less precise. Note also that these calibration factors are not the same as the relative response factors used in the area normalization method. 

To quantitatively determine the absolute amounts of certain esters present in the oxidation products, the mass spectrometer responses to these compounds were compared with the response to the internal reference, octadecane. Using authentic samples of the esters of interest, relative mass spectrometric response factors were determined. From these response factors, listed in Table I, the amounts of esters present in the oxidative degradation products were calculated using an INCOS subroutine. 

Comparison of the HPLC Technique with Clay—Gel Chromatographic Separation. A number of vacuum gas oils were analyzed by preparing solutions in n-heptane at a concentration near 100 mg/mL. These samples were injected into the HPLC equipment and the concentration of saturates and aromatics calculated from the response factors shown in Table II. The absolute percentages of saturates and aromatics are shown in Table III along with the polar aromatics obtained by subtracting the sum of these from 100%. 

Thus, by substitution in this equation for the peak areas from the chromatogram, the relative response factors, derived from the calibration analysis, and the concentration of the internal standard added to the sample, the concentration of the components in the sample can be calculated. Since this method involves ratios of peak areas rather than absolute values, it should be noted that the precision of analysis is not dependent on the injection of an accurately known amount of sample. However, the accuracy does depend on the accurate measurement of peak area. Assay and quantitation by the internal standard is often the preferred method as it takes account of variable compound response and removes potential errors due to variation in sample injection. The 80 and 200 mg% (mg per 100 ml) standard solutions are used to confirm the linearity of response over this concentration range. 

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. 

For quantitative interpretations of the chromatogram the detected compounds must be identified, and response factors have to be apphed to transform peak areas into weights. Automatic identification of peaks may be accomplished by numbering the peaks, on the basis of absolute retention time bands or by retention times relative to an internal time standard. Since retention times relative to one standard are not constant enough for reliable identification especially in long chromatograms, multistandard... 

Proeedure. Inject a sample of reference standard gas of known composition at absolute pressure of 700 mm Hg. Repeat at least onee, and calculate response factors for each component in the reference standard gas. Error shall be less than 1% for each component response factor. In the same operating condition inject the sample gas at absolute pressure of 700 nun Hg, repeat at least once and obtain peak areas for each component. In order to obtain the unnormalized composition of the sample gas, multiply the peak areas of eaeh eomponent by the appropriate response factor derived from the reference standard gas by using the above equation. 

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