Second column confirmation

Test methods that analyze individual compounds (e.g., benzene-toluene-ethylbenzene-xylene mixtures and PAHs) are generally applied to detect the presence of an additive or to provide concentration data needed to estimate environmental and health risks that are associated with individual compounds. Common constituent measurement techniques include gas chromatography with second-column confirmation, gas chromatography with multiple selective detectors, and gas chromatography with mass spectrometry detection (GC/MS) (EPA 8240). 

Recognizing the fact that shifts in retention time of individual compounds may cause false negative results, laboratories use retention time windows for target analyte identification. Retention time windows are experimentally determined retention time ranges for each target analyte. To minimize the risk of false positive results, EPA methods require that chromatography analysis be performed on two columns with dissimilar polarities. This technique, called second column confirmation, is described in Chapter 4.4.3. It reduces the risk of false positive results, but does not eliminate them completely. 

Qualitative compound confirmation may be performed either with a second column or with a second detector. Second column confirmation technique consists of analyzing the sample on two columns with dissimilar polarities. Each column is calibrated with the same standards, and the same calibration acceptance criteria are applied. For the presence of a compound to be confirmed, its retention time values obtained from each column must fall into respective retention time windows. If a peak falls within the retention time window on one column, but not on the second column, the compound is not considered confirmed and should not be reported. 

Second column confirmation is an imperfect technique, prone to false positive detection, particularly if non-selective or low selectivity detectors are used. Even the most selective detectors may not be fully capable of correctly identifying target analytes in complex environmental matrices as illustrated in Example 4.12. 

Second column confirmation must be used in pesticide, PCBs, and chlorinated herbicide analyses by EPA Methods 8081, 8082, and 8151, respectively. In these methods, two columns with dissimilar polarities and two ECDs provide compound identification and quantitation. This technique produces a lower rate of false positive results, but does not eliminate them completely. This is particularly true for low concentrations of pesticides and herbicides, where non-target compounds, such as constituents of the sample matrix or laboratory artifacts listed in Table 4.3, produce chromatographic peaks on both columns. These interference peaks cannot be distinguished from the target analytes based on retention time only and cause false positive results. 

The complexities of the second detector and second column confirmation are illustrated in Example 4.13. 

Example 4.13 Second detector/second column confirmation... 

On the other hand, PCBs, which are also the mixtures of individual chemical constituents (congeners) and which also have characteristic and recognizable chromatographic patterns, require second column confirmation. This is because they are quantified using individual peaks, the presence of which must be confirmed on the second column. 

Was second column confirmation, if required by the method, performed for individual compound analysis with GC methods ... 

The chemist reviews results of each analysis and determines whether data qualification is needed. Typical deficiencies that turn definitive data points into estimated ones include insufficient surrogate standard recoveries the absence of second column confirmation and the quantitation performed outside the calibration curve. The chemist may even reject the data based on low surrogate standard recoveries. Example 5.6 shows how surrogate standard recoveries may affect the validity of analytical results. 

Initial and continuing calibration verifications and acceptance limits Raw data for each sample (including reanalysis and second column confirmation), blanks, LCS/LCSD, MS/MSD, and calibration standards Sample preparation bench sheets Gel permeation chromatography clean-up logs / (Summary only) ... 

Analysis on a single GC column (as opposed to situations requiring a second column confirmation) is acceptable if the required separation of all of the... 

The 2,3,7,8-TCDD toxicity equivalence of a sample is used in Sections 16 and 17 of this procedure to determine when second column confirmation or reextractions and reanalyses may be required. 

In the author s experience, such confirmation is not appropriate when the calibration range is greater than one order of magniffide or calibration points are not chosen carefully. The reason is that lower concentration levels of a calibration graph influence the correlation coefficient to a much smaller extent than higher concentrations. The hypothetical example of calibration results presented in Table 3 demonstrates this very simply. If the amount injected is correlated with the observed peak area in the second column in Table 3, the calibration graph in Figure 2 is obtained. 

The identification of the peak must be finally confirmed on a second GC column. This may be done either after performing steps 1 and 2 or by injecting the extract straight onto the second column (confirmatory GC column) without going through steps 1 and 2. 

Individual compound identification in all GC methods with the exception of GC/MS relies on the compound retention time and the response from a selective or non-selective detector. There is always a degree of uncertainty in a compound s identity and quantity, particularly when non-selective detectors are used or when the sample matrix contains interfering chemicals. To reduce this uncertainty, confirmation with a second column or a second detector is necessary. Analyses conducted with universal detectors (mass spectrometer or diode array) do not require confirmation, as they provide highly reliable compound identification. 

Second detector confirmation is another compound confirmation technique. Two detectors with selectivity to different functional groups are connected in series to one column or in parallel to two columns. For example, two detectors, a UV/VIS detector and a fluorometer, connected in series to the HPLC column are used in the EPA Method 8310 for analysis of PAH compounds. If the second detector is connected to a second column of a dissimilar polarity, then the confirmation becomes even more reliable. An example of such a configuration is organophosphorus pesticides analysis the samples may be initially analyzed with an NPD, and then confirmed on a different column with an ECD or a FPD. 

Confirmation with a second column of a dissimilar polarity and a second, selective detector is necessary for the correct identification and quantitation of BTEX and oxygenated additives. And yet, even when a dissimilar column and a second PID are used in confirmation analysis, false positive detection of MTBE often takes place. 

Second column and second detector confirmation are not always necessary. If the source of contamination is known and there is certainty that the contaminants are, in fact, present at the site, confirmation of compound identification may not be necessary. For example, the confirmation of pesticides in the samples collected at a pesticide manufacturing facility is not needed as the source of contamination is known. Confirmation of pesticide concentrations in this case is still necessary. 

Petroleum fuel analysis does not require second column or second detector confirmation. Fuels are identified based on their fingerprints or characteristic patterns of multiple peaks similar to ones shown in Figure 2.5. Each peak represents an individual chemical constituent, and each fuel has a unique combination of these constituents forming a characteristic pattern or a fingerprint. The fingerprints obtained... 

Request that the laboratory confirm all chromatography results with a second column or a second detector, when required by the method. (Laboratories do not automatically do this.)... 

Positive identification should be confirmed by a second column analysis or by confirmatory ions on the MS. 

The search of the literature carried out for this survey, allows us to estimate that approximately 90% of all toxaphene separation were carried out on non-polar stationary phases similar to 95% methyl-/5% phenylpolysiloxane and commercially available under the trade names DB 5, CP-Sil 8, HP-5, Ultra 2, Rtx 8, SE 52, etc. Two reports in the literature suggest the use of non-polar only. Alder et al. mentioned the decomposition of B8-1413 (P-26) and B9-1025 (P-62) on the polar DX-4 phase [145]. Baycan-Keller and Oehme tested four different stationary phases (capillary length 12 to 30 m) and found a remarkable decomposition of labile toxaphene compounds on 90% dicyanopropyl-/10% phenylcyanopropyl-polysiloxane (Rtx-2330) [146]. Therefore, the exclusive use of non-polar stationary phases was recommended for the quantification of toxaphene as well as a second column for confirmation of the results. A very non-polar stationary phase (CP-Sil 2) was suggested for the congener-specific separation of toxaphene [147,148]. 

It was stated earlier that formation of a first H-bond in a dimer will polarize both molecules. One can hence expect a dipole moment in the dimer that is larger than the vector sum of the individual moments of the isolated monomers. This supposition is confirmed by the SCF moments of these oligomers which are reported in the second column of Table 5.2. The third column lists the cooperativily, measured in a manner analogous to that for the energies. This property is approximately 1 D and increases with n. The average dipole moment per molecule in the chain is exhibited in the last column of Table 5.2 and echoes the cooperative nature of the electronic rearrangements within the chain. Later experimental measurements by Ruoff et al. indicated that the dipole moment of the linear trimer exceeds the vector sum of the three monomers by 1.8 D, quite close to the estimation of 2.0 D in Table 5.2 (i.e., twice the value listed for n = 3 in column three). 

Results obtained by use of an amino acid analyzer. Some of the results of the second column have been confirmed by independant methods. 

Solid wastes (Aroclors or congeners) Soxhiet extraction sulfuric acid/potassium permanganate cleanup HRGD/ECD confirmation on second column 57-70 pg/kg (soil) 62-125 (multiple lab) EPA 1995c (Method 8082) ... 

The velocities of bubbles of diameters between 0.1 and 2 mm are of the same order as those obtained from Eq. (10.45). Measured velocities are also presented in the second column of Table 10.1, taken from Schulze (1992), from which the Reynolds numbers are calculated (third column). The substitution of Reynolds numbers into Eq. (10.45) yields theoretical velocity values (column 4). The comparison of values of the second and fourth columns confirms the usefulness of Eq. (10.45). 

The results of the GC method coupled with its specific detectors must be confirmed on a second column with a different polarity. A flow divider can be used to automatically direct equal portions of the injected volume to two columns of different polarity (Table 23.10). 

Many analyses of complex environmental mixtures specify splitting an injection to two different capillary GC columns operating in parallel to separate detectors. An analyte is considered to be measureable only if it elutes with the appropriate RRT on both columns and the measured quantities of it on each column differ by less than a specified amount. The latter condition ensures that there is no significant coelution with yet another compound on one of the columns. The second column in such a pair is called the confirmation column. 

The shift factors calculated from the WLF equation are shown in the right-hand column of the table. These are measured from the curve that would in theory be obtained at Tg and should differ by a constant amount from those found with 70 °C as the reference T. Subtracting the values in the second column from those in the first leads to the approximate value 7.6 in all cases, confirming the applicability of the WLF equation. 

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