How Does the IS Mode of Instrument Calibration Work and Why is It Increasingly Important to TEQA?

The IS mode is most useful when it has been determined that the injection volume cannot be reproduced with good precision. This mode is also preferred when the instrument response for a given analyte at the same concentration will vary over time. Both the analyte response and the IS analyte response will vary to the same extent over time; hence, the ratio of analyte response to IS response will remain constant. The use of an IS thus leads to good precision and accuracy in construction of the calibration curve. The calibration plot is usually established by plotting the increasing ratio of the analyte response to the fixed IS response versus the increasing concentration of analyte. In our THM example, 1,2-dibromopropane (1,2-DBP) is often used as a suitable IS. The molecular formula for 1,2-DBP is similar to each of the THMs, and this results in an instrument response factor that is near to that of the THMs. The concentrations of IS in all standards and samples must be identical so that the calibration curve can be correctly interpolated for the quantitative analysis of unknown samples. Refer to the THM example above and consider the concentrations cited above for the six-point working calibration standards. 1,2-DBP is added to each standard so as to be present at, for example, 200 ppb. This mode is defined as such since 1,2-DBP must be present in the sample or is considered internal to the sample. A single-point or multipoint calibration curve is usually established when using this mode.

The IS mode to instrument calibration has become increasingly important over the past decade as the mass spectrometer (MS) has replaced the element-selective detector as the principal detector coupled to gas chromatographs in the contemporary practice of TEQA. The mass spectrometer is somewhat unstable over time. The IS mode of GC-MS calibration quite adequately compensates for this instability.

Consider the determination of clofibric acid (CF) in wastewater. Clofibric acid or [2-(4- chlorophenoxy)-2-methyl-propanoic] acid is the bioactive metabolite of various lipid-regulating prodrugs. After chemically derivatizing CF to its methyl ester, a plot of the ratio of the CF methyl ester peak area to that of the internal standard 2, 2', 4, 6, 6'-pentachlorobiphenyl (22'466'PCBP) against the concentration of CF methyl ester in ppm is shown in Figure 2.4. An ordinary (or unweighted) least squares regression line was established and drawn as shown (we will take up


least squares regression shortly). The line shows a goodness of fit to the experimental data points. This plot demonstrates adequate linearity over the range of CF methyl ester concentrations shown. Any instability of the GC-MS instrument during the injection of these calibration standards is not reflected in the calibration. Therein lies the value and importance of the IS mode of instrument calibration.

For a single-point calibration approach, a relative response factor is used:

Quantitative analysis is then carried out by relating the ratio of analyte instrument response for an unknown sample to that of IS instrument response to the ratio of unknown analyte concentration to IS concentration according to

Equation (2.4) is then solved for the concentration of analyte / in the unknown sample, C'unknown. Refer to the quantification equation for IS in Table 2.2 and A'ls are allowed to vary with time. This is what one expects when using high-energy detectors such as mass spectrometers. The ratio AmfapHTT I A'is remains fixed over time. This fact establishes a constant RR'F and hence preserves the linearity of the internal standard mode of instrument calibration. Equation (2.4) suggests that if RR'f is constant, and if we keep the concentration of IS to be used with the /th analyte, C‘ls, constant, the ratio A'unknown / A'ls. varies linearly with the concentration of the /th analyte in the unknown. C[mknown.

The manner in which one uses internal standards in preparing calibration standards, ICVs, matrix spiked samples, and other QC samples will have a significant impact on the analytical result. Three strategies, shown in Figure 2.5, have emerged when considering the use of the IS mode of calibration." In the first strategy, internal standards are added to the final extract after sample prep steps are complete. The quantification equation for IS shown in Table 2.2 would yield an analytical result for C'unknown that is lower than the true concentration for the /th analyte in the original sample since percent recovery losses are not accounted for. This strategy is widely used in analytical method development. The second strategy first calibrates the instrument by adding standards and ISs to appropriate solvents, and then proceeds with the calibration. ISs are then added in known amounts to samples prior to extraction and cleanup. According to Budde:" The measured concentrations will be the true concentrations in the sample if the extraction efficiencies of the analytes and ISs are the same or very similar. This will be true even if the actual extraction efficiencies are low, for example, 50%.

The third strategy depicted in Figure 2.5 corrects for percent recovery losses. Again, according to Budde:"

The system is calibrated using analytes and ISs in a sample matrix or simulated sample matrix, for example, distilled water, and the calibration standards are processed through the entire analytical method ... [this strategy] is sometimes referred to as calibration with procedural standards.

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