13 PFAS Testing & Analysis The separation and detection were performed using an Avantor® ACE® Excel® 3
C18, 100 x 2.1 mm, with the mobile phase A: 5 mM ammonium acetate in H2O and B: methanol (MeOH).
The fl ow rate was set to 0.4 mL/min, and the column thermostat set at 40 °C. Table 2 details the gradient conditions employed. A PFAS delay column (Avantor® ACE® PFAS Delay Column, 50 x 2.1 mm) was employed, and installed prior to the injector loop. [19]
Table 2. Gradient conditions employed in the separation of 18 PFAS compounds
Time (mins) 0
0.1 8.5
10.5 10.6
% B 5
20 95 95 5
A SCIEX QTRAP® 6500+ system was used to detect the compounds of interest, running in negative ESI mode with a source temperature of 450°C and an IonsprayTM source voltage of -4500 V.
Figure 1 shows an example chromatogram obtained by injecting a 1 µL sample at a concentration of 1000 ng/L [19].
11,12,13
1.00e4 2.00e4 3.00e4 4.00e4 5.00e4 6.00e4 7.00e4 8.00e4 9.00e4 1.00e5
0 1.0 2.0 3.0 4.0 17,18
Figure 2 Extraction of trace residual PFAS present in a variety of common laboratory consuables
Figure 2. Extraction of trace residual PFAS present in a variety of common laboratory consuables 8 9,10
19,20,21 23
2,3 1 4,5 6 7
14 15,16
22 24 25
As well as looking for possible contamination within general laboratory and its consumables, PFAS can also be found in solvents and in tubing associated with the instrumentation [21]. PFAS originating from the mobile phases or components on the HPLC system prior to the autosampler will also result in chromatographic peaks, since it will build up at the front of the column during the low elutropic phase of the gradient elution, and when the gradient reaches a critical concentration, the PFAS components will start to elute, resulting in inaccurate concentration determination of the individual PFAS.
5.0 Time, min 6.0 7.0 8.0 9.0 10.0
Figure 1 Example chromatogram obtain with 18 PFAS compounds and selected stable isotope labelled internal standards
Figure 1. Example chromatogram obtain with 18 PFAS compounds and selected stable isotope labelled internal standards
When developing the method it was noted that there was a signifi cant amount of background contamination, which subsequently led to an intensive investigation to systematically determine the source and levels of contamination. A variety of possible sources were identifi ed. Initially, the sample vials and caps were the focus, with a simple solvent extraction being applied to determine if there were any extractable PFAS present. Each possible source and alternatives were extracted using 300 µL of methanol. The resulting solution was injected as a 10 µL aliquot. For the early eluting compounds this resulted in some poor peak shapes which is the combined effect of injection of a larger volume of strongly eluotropic diluent, weakly retained analytes being more susceptible to system dispersion, as well as being present at low concentration levels and close to the instrument’s limit of detection. Due to the use of the qualifying extracted ion chromatograms, this facilitated the identifi cation/qualifi cation of contaminants without having to introduce another possible contamination source e.g. from a pre-concentration step and the use of a blow down evaporator.
The data, given in Table 2, is not quantitative, but the peak areas do indicate that there are detectable levels of PFAS. In this case two septa, both based on a polyimide silicone material compared to the polypropylene material used in the other septa, were identifi ed as having ADONA (3H-perfl uoro-3-[(3-methoxy-propoxy)propanoic acid]) present at detectable levels.
Table 3. Extraction of trace residuals present in caps, septum or vials demonstrates that some vial caps do contain PFAS components
Vial Manufacturer Vial material
Cap Manufacturer Septum material ADONA
Other PFAS (18 cpds)
Blank run N/A N/A N/A N/A û û
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000
0 1 2
PP PP 1
2 3
PP 3
500 1000 1500 2000 2500 3000 3500 4000 4500
0 7.0
1000 2000 3000 4000 5000 6000 7000 8000
7.2 7.4 û 0 7.0 7.0 7.2 7.4 7.6 3 7.6 7.8 Time, min 8.0 8.2 8.4 1
PP PP 1
7.8 Time, min
PP PP PI/Si PI/Si PP û û ü14930 ü2341 û û û
7.2
Additional to the testing of the vials and caps, the analysis also looked at other possible sources of contamination as highlighted in Figure 2 [20]. The extraction process employed was also qualitative for this set of experiments and involved taking a small proportion of the sample and inserting into a test tube, adding 2 mL of methanol and shaking for a short period of time and then injecting 10 µL of the
1000 2000 3000 4000 5000 6000 7000 8000 9000
0 6.6 6.8 7.0 7.2 7.4 Time, min 7.6 7.8 8.0 7.4 7.6 7.8 û û 8.0 Time, min
1000 2000 3000 4000 5000 6000 7000 8000 9000
0 6.6 6.8 7.0 7.2 8.2 8.4 8.6 8.8
1000 2000 3000 4000 5000 6000 7000 8000
0 7.0 7.2 7.4 7.6 7.8 8.0 Time, min Background PFAS 8.0 8.2 8.4 3
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000
0
500 1000 1500 2000 2500 3000 3500 4000 4500
0 7.0 7.2 7.4 7.6 7.0 7.2 7.4 7.6 Time, min 7.8
The approach to help alleviate this is to use a delay column which is placed in line before the injector, thus any PFAS components that are in the system will build up on the delay column preferentially before eluting onto the main analytical system. Once the gradient composition reaches a critical amount the PFAS components that have been retained on the delay column will be eluted onto the analytical column. The delay column is chosen so that it will introduce a retention time difference between the system derived PFAS and sample derived PFAS components. Figure 3 shows an example chromatogram; in this scenario the system is contaminated with a range of PFAS. It can be seen in the chromatogram that there are two peaks present. The fi rst peak relates to the PFAS in the sample and the second broader peak relates to the PFAS in the system. The poor shape of the chromatography is caused by the PFAS reagent going through two columns and also the build-up effect that the PFAS has as more solvent is passed through the column. The separation of the system PFAS components and the sample derived components means that accurate quantifi cation of analytes within the sample becomes feasible. However, it should be stated that this does not preclude inaccurate quantifi cation of the PFAS levels due to other sources of contamination within the sample itself, and as has already been shown, PFAS is ubiquitous within a laboratory environment. If this approach was not employed, then the system PFAS would elute at the same time as the sample PFAS, due to the nature of the focussing effect when using gradient chromatography. This would obviously result in the inaccurate quantifi cation of the amount of the individual analytes in the sample. As mentioned previously, the ubiquitous nature of PFAS means that system contamination can come from a variety of sources from the solvents to the tubing, whereas sample contamination can come from a myriad of sources contained within workfl ow materials & consumables, sample prep workfl ows etc.
10000 15000 20000 25000 30000 35000
5000 0
resulting solution into the LC-MS using the method specifi ed previously. It can be seen that there are a range of PFAS compounds that have been identifi ed. The profi les are different for the different sample types. It is interesting to note that the two sources of paper tissue that were being analysed were the same brand but bought on different dates, suggesting that the manufacturing process may involve variable amounts of PFAS material that is added due to the recycling of the paper, or the possibility of the production line having contamination issues.
Nitrile Glove Sticky tape White tape Laboratory Tissue Laboratory Tissue 2
Background PFAS
0e0 1e4 2e4 3e4 4e4
8.0 8.2 8.4 Background PFAS
0e0 1e4 2e4 3e4 4e4
7.8 Time, min 8.0 8.2 8.4 Background PFAS 8.2 8.4 8.6 8.8
0e0 1e4 2e4 3e4 4e4
0e0 1e4 2e4 3e4 4e4
0 7.4 Time, min 7.6 7.8 8.0
Blank run No delay column
0.5 ng/mL PFAS sample No delay column
Blank run Delay column installed
0.5 ng/mL PFAS sample Delay column installed
2 4
PFOA system background
PFOA from system co-elutes with sample PFOA
tR of Sample PFOA
Using delay column, system PFOA is delayed
Sample PFOA
System PFOA
6 Time, min
Figure 3 Examples of how the PFAS delay column effectively separates system PFAS from sample PFAS
Figure 3. Examples of how the PFAS delay column effectively separates system PFAS from sample PFAS
8
System PFOA is separated from sample PFOA
10
Intensity, cps
PFOA
PFDA
PFOS
PFNA
Peak area Intensity, cps
NEtFOSAA PFBS PFDA
PFHpA PFHxA PFNA PFOA PFOS PFTA
PFTrDA PFUnA
HFPO-DA
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