5 The Liquid Skin Pen
5.5 An experiment to determine the most efficient solvent for metal extraction using
EDTA has extensive medical, industrial, agricultural and engineering applications and is a widely used chemical compound found in cosmetic preparations and cleaning, processed foods, skin care and personal care products. High purity nitric acid is commonly used for metal analysis in mass spectrometry whereas water is used as part of everyday life. Due to its function as a hexadentate ligand and chelating agent, EDTA has the ability to capture metal ions such as Fe3+ and Ca2+. After being bound to EDTA, metal ions remain in solution but exhibit reduced reactivity. At a very low pH (very acidic conditions), the fully protonated H2EDTA2+ form is prevalent whereas at very high pH (very basic conditions) the fully deprotonated form EDTA4- is predominant. EDTA4- usually binds to metal cations through its four carboxylates and two amines with the resulting coordination adopting an octahedral geometry. Metals are extensively enveloped by EDTA resulting in the suppression of their catalytic properties. Since complexes of EDTA4- are anionic they are likely to be highly soluble in water.
The aim of this experiment was to identify which of three readily available solvents were best suited as the LSP sampling solvent (SS) in order to recover a known concentration of Mn from a target surface. In addition, a further objective was to investigate the efficiency of the LSP. The three solvents investigated were 0.1M EDTA, 2% HNO3 and DI H2O. H0: there was no difference in the amount of Mn recovered from a nitrile glove, using the LSP, using the three sampling solvents. i.e. H0: SS (x) = SS (y) = Ss (z).
5.5.1 Experimental Outline
1 LSP was used to collect a total of 15 samples. This included 4 samples (1 sample from the spiked area of Mn and the 3 subsequent wash samples from the exact location) and 1 LSP pen blank for each of the 3 sampling solvents used. One drop of 100 µg.mL-1 Mn was initially weighed after which it was added onto the surface of a fresh (out of the box), unused nitrile glove using a 20 µL pipette. The drop was allowed to sit on the surface and dry for 30 minutes.
Figure 5.7. Experimental procedure for sample solution testing using the LSP for solvent comparison SAMPLE ANALYSIS
SAMPLE COLLECTION
Spot target area with 1 drop of SRM Sample target area with LSP (SS = EDTA) Dilute samples with 10ml 2% HNO3 ICP-MS
Analysis Data Output
Conc Recovered Re-sample target area (x3) Allow spot To dry (30 mins) PRE SAMPLING SRM 1000 → 100 → 10 µg.ml-1 0.1M EDTA (Di-Na), 2% HNO3 & DI H2O Collect LSP Pen Blank Connect vials to LSP Storage at -20° C Thaw all samples Report Clean LSP with 2% HNO3 Collect LSP Pen Blank
Spot target area with 1 drop of SRM Sample target area with LSP (SS = 2% HNO3) Re-sample target area (x3)
Spot target area with 1 drop of SRM Sample target area with LSP (SS = DI H2O) Re-sample target area (x3) Allow spot To dry (30 mins) Clean LSP with 2% HNO3 Collect LSP Pen Blank Allow spot To dry (30 mins)
A clean, fresh out of the box nitrile glove was used for each solvent sample test (3 nitrile gloves used in total). EDTA samples were collected first, followed by 2% HNO3 samples and finally DI H2O samples. Each sample was collected within 60 seconds once the face of the sampling base covered the target sample area. All samples were collected on the same day and stored at -20°C until the day analysis.
5.5.1.1 Sample Analysis
Samples were removed from storage after 2 days, allowed to thaw at room temperature for 30 minutes and then homogenised using a vortex mixer. For all 15 samples, 1 ml of collected sample was diluted with 4 ml 2% HNO3 (1:5 dilution) in 15 ml fresh, unused, polypropylene centrifuge tubes (Table 5.3).
All samples were analysed on the same day they were removed from storage. Elemental detection of Mn was performed using ICP-MS (Chapter 1 section 1.3.5). Each sample was analysed in triplicate and sample concentrations were semi quantified by taking the ratio of intensity obtained for the known Mn concentration of 100 µg.mL-1 against those obtained for samples. Comparing the concentrations of samples with the known concentration of the drop enabled the recovery to be calculated as well as the sampling efficiency of the LSP (sampling solvent transfer from the delivery to collection vial) (supplementary information Table SI.5.2).
5.5.2 Results and Discussion
A total of 15 samples obtained using the LSP, plus a sample containing 100 µg.mL-1 Mn which was analysed for semi quantitation purposes, were successfully analysed on the same day. Mn was successfully extracted from the target surface using all three delivery solvents of interest.
5.5.2.1 Recovery of Mn using the LSP
Theoretical 100% recoveries for each sample were calculated using the same procedure as that used in section 5.4.2.1. Once again, three phases were conducted. Step 1, where a known amount of a Mn sample was added to the target surface. Step 2, a known amount of sampling solvent was used to sample the surface using the LSP. Finally step 3, in order for the sample to be analysed using ICP-MS analysis, the collected sample was diluted with a suitable matrix.
Theoretical Concentration STEP 1
The average mass of one drop of Mn used was 0.010 g (σ = 0.0001, n = 5) with a calculated concentration of 90.7877 µg.mL-1. Therefore, an estimate of 0.010 g of Mn was added onto the target surface of an unused, fresh latex glove.
STEP 2
Approximately 1.5m l of sampling solvent was delivered and collected over the target area, where the drop of Mn had been placed, using the LSP. Using Equation 5.1, the concentration of this dilution was calculated for each sample (Table 5.4). C1 = 90.7877, V1 = 0.010, V2 = 1.5, and C2 = unknown. For most samples this concentration was approximately 60 ng.mL-1. STEP 3
Finally, 1 ml of sample collected was diluted with 4 ml 2% HNO3, in a 15 ml polypropylene tube, enabling ICP-MS introduction. In this step, C1 = 60 ng.mL-1, V1 = 1 ml, V2 = 4 ml and C2 was the unknown concentration for a 100% theoretical recovery. These calculated values can be viewed in the step 2 column of Table 5.4. For most samples, 100% recovery would yield a concentration of approximately 11 ng.mL-1 Mn.
The experimental concentrations for each of the samples are also shown in Table 5.4. For the first sample using the LSP for each sampling solvent, Mn concentrations of 6.5, 4.6 and 2.9 ng.mL-1 were observed for the 2% HNO3, EDTA and DI H2O, respectively. Subsequent samples using the LSP, for each sampling solvent, showed a decrease in concentration obtained showing that the first sample obtained extracts the largest amount of Mn. For the first sample, the highest concentration achieved was with the LSP using 2% HNO3 as the sampling solvent, followed by EDTA and finally DI H2O. This can be seen in Figure 5.8 along with the decreasing trend in concentration for subsequent samples using each of the three sampling solvents. Very little of the Mn sample was extracted from the target surface following the first two samples using the LSP.
5.5.2.2 Recovery
Using Equation 5.2 and both the theoretical and experiment concentrations calculated previously, the relative recovery for each sample was calculated (Table 5.4). For the primary sample, the highest recovery of Mn was observed when using the sampling solvent 2% HNO3
with a recovery of 55.7%. The next highest achieved was when using EDTA as the sampling solvent and then DI H2O with relative recoveries of 39.9% and 18.6%, respectively. For the first wash sample, the highest recovery was attained when using 2% HNO3 (32.6%) as the sampling solvent followed by EDTA (19.7%) and finally DI H2O (12.0%). The LSP pen blank recovery when using 2% HNO3 as the sampling solvent was 12.4%, for EDTA was 4.3% and DI H2O was 1.2%. The last two wash samples using the LSP were lower in recovery compared to each of the blanks for the relevant sampling solvents and this trend is shown in the insert in Figure 5.8.
Table 5.4. Calculated theoretical (CTHEORETICAL) and experimental concentrations
(CEXPERIMENTAL) (ng.mL
-1
Mn) and recovery (%) of Mn obtained from the three sample solutions collected using the LSP
Sample CTHEORETICAL CEXPERIMENTAL Recovery (%)
Step 1 Step 2 (final) µ (n=3) RSD (%)
EDTA Blank 57.36 11.86 0.514 4.75 4.3 Sample (B) 56.31 11.46 4.573 3.28 39.9 Sample wash 1 (B1) 58.48 11.81 2.328 4.41 19.7 Sample wash 2 (B2) 57.84 11.88 0.516 1.13 4.3 Sample wash 3 (B3) 55.27 11.26 0.482 3.25 4.3 Total (B to B3) 68.2 2% HNO3 Blank 60.18 12.02 1.486 4.45 12.4 Sample (B) 58.49 11.60 6.465 3.40 55.7 Sample wash 1 (B1) 60.69 12.43 4.057 3.00 32.6 Sample wash 2 (B2) 61.21 12.52 1.480 3.90 11.8 Sample wash 3 (B3) 58.95 12.18 1.474 4.02 12.1 Total (B to B3) 112.3 DI H2O Blank 57.29 11.55 0.144 3.33 1.2 Sample (B) 77.71 15.48 2.871 3.98 18.6 Sample wash 1 (B1) 60.63 12.25 1.473 5.72 12.0 Sample wash 2 (B2) 51.81 10.49 0.100 3.67 1.0 Sample wash 3 (B3) 59.44 11.97 0.088 3.10 0.7 Total (B to B3) 32.3
Intriguingly, the total Mn recovery from the samples and washes yielded 112.3% when using 2% HNO3 as the sampling solvent whereas total recovery when using the sampling solvents EDTA and DI H2O were calculated to be 68.2 and 32.3%, respectively. If it is considered that only a maximum of 100% should be theoretically recovered, there is an extra 12.3% recovery of Mn using 2% HNO3 as the sampling solvent. A number of factors should be considered to explain this extra recovery. The 2% HNO3 samples were the second batch of samples collected, using the same LSP, following sample collection using EDTA as the sampling solvent. It may have been possible that the LSP may not have been thoroughly cleaned and there were contaminants of the Mn from the previously collected batch resulting in memory
effects of Mn. The high blank sample recovery could point to this. Another factor could be extraction of traces of indigenous Mn from the nitrile glove itself.
5.5.2.3 Significance Testing
ANOVA was performed to investigate significant differences between the first samples obtained from the LSP of each of the three solvent samples used (Appendix Table 8.13). The calculated probability of F (346.02) was much larger than the critical value of F (5.14), suggesting evidence of significant differences between the first sample collected using each sampling solvent.
Table 5.5. Intensity (counts per second), mean (µ), standard deviation (σ) and relative standard deviation (%) of Mn signal from the three sample solutions collected using the LSP. Probability score for test of variance (F) and difference sample means of the previous sample collected (T) are also given where values P < 0.05 show evidence of statistically significant differences in the mean
Sample Intensity (cps) RSD (%) F (P) T (P)
1 2 3 µ σ (n = 3)
EDTA
Blank 51959 54551 57144 54551 2593 4.75 Sample (B) 500856 469013 484935 484935 15921 3.28 0.05 3.35E-04 Sample wash 1 (B1) 257746 246854 235963 246854 10891 4.41 0.64 7.24E-05 Sample wash 2 (B2) 54716 55332 54100 54716 616 1.13 0.01 6.88E-06 Sample wash 3 (B3) 52739 49421 51080 51080 1659 3.25 0.24 4.92E-02
2% HNO3
Blank 164553 150520 157537 157537 7017 4.45 Sample (B) 708881 685541 662201 685541 23340 3.40 0.17 2.49E-04 Sample wash 1 (B1) 443086 430187 417287 430187 12900 3.00 0.47 3.82E-04 Sample wash 2 (B2) 150832 163078 156955 156955 6123 3.90 0.37 8.75E-05 Sample wash 3 (B3) 150060 162615 156337 156337 6278 4.02 0.98 9.09E-01
DI H2O
Blank 14732 15748 15240 15240 508 3.33
Sample (B) 292296 316554 304425 304425 12129 3.98 0.00 2.06E-06 Sample wash 1 (B1) 147289 156225 165162 156225 8936 5.72 0.70 1.24E-04 Sample wash 2 (B2) 10567 10179 10955 10567 388 3.67 0.00 9.40E-06 Sample wash 3 (B3) 9322 9033 9611 9322 289 3.10 0.71 1.33E-02
Between sample 1 of delivery matrix
EDTA & 2% HNO3 0.64 2.51E-04
EDTA & DI H2O 0.73 9.81E-05
2% HNO3 DI H2O 0.43 1.50E-05
A t-test to compare the two experimental means was conducted to investigate whether these differences occur between the first samples obtained from each of the sampling solvent. Table 5.5 shows the probability of the t-test and implies there is evidence of significant differences between the first samples obtained using the LSP for all three sampling solvents. The probability obtained between the first sample when using the solvent samples EDTA and 2% HNO3 was 0.000251, much less than the 0.05 threshold, and thus there is evidence present of statistically significant differences. Evidence of statistically
significant differences (95%) were also present between sampling solvents EDTA and DI H2O (T = 0.0000981) and 2% HNO3 and DI H2O (T = 0.000015). As shown in Table 5.5, t-tests between results from consecutive samples showed statistically significant differences in all cases except between the final 2 washes of the samples using 2% HNO3.
5.5.2.4 Efficiency
Using the data collected for these samples, the relative efficiency of the LSP was determined by taking the amount of solvent sampled over the target surface (solvent collected) and subtracting from this the initial amount of solvent delivered (delivery solvent), from the delivery vial, over the target sample area. Multiplying this by 100 produces the relative efficiency (equation 6.3). The relative efficiency for all samples obtained can be found in supplementary Table SI.5.2.
( Equation 5.3
where E = efficiency
SCOL = solvent collected SDEL = solvent delivered
On average, the efficiency for samples that had been collected using EDTA was 99.9%, using 2% HNO3 was 100.3% and DI H2O achieved 95.9% efficiency. The 100.3% average using a delivery solvent of 2% HNO3 suggests that extra solvent has been picked up from previous samples. A closer look at the breakdown shows that the blank efficiency was 102.3%. This may be due to traces of the sampling solvent from previous testing, in possibly the tubes of the LSP, following cleaning of the device prior to collecting the blank sample. Also, the first sample (B) collected using the sampling solvent of DI H2O showed an efficiency of 78.2%, very low in comparison to all other recoveries. For this to occur, some of the solvent delivered would not have been transferred to the collection vial via the target sampling site. It may have been possible that some of the sampling solvent could have been left in the transfer tubes or if a seal had not been retained at the sampling base of the LSP, the sample could have leaked out around the target surface and lost. Previously, some solvent had been observed to go into the collection vial via one needle but would form a drop at the end of the needle, a drop big enough to then touch the needle that led to the peristaltic pump outlet. This would then suck up the drop over this needle and the collected sample would go through the peristaltic pump tubing to waste.
The observed differences in collection efficiency were not due to different uptake from the delivery vial.
1
2
1
Figure 5.8. Mn concentration (ng.mL-1) for the comparison of the three different sample solutions; 2% HNO3 (green); EDTA (pink); and DI H2O (blue). Insert: Recovery (%) of the three different sample solutions. B = reference sample, B1 = wash sample 1, B2 = wash sample 2 and B3 = wash sample 3
H0 was rejected, as a statistically significant difference was found in the recovery of a Mn sample from a nitrile glove using the LSP, between sampling solvents (H0: SS (x) ≠ SS (y)). A pre-selected sample was extracted using three sampling solvents with highest first recoveries observed for 2% HNO3 (55.7%) followed by EDTA (39.9%) and finally DI H2O (18.6% recovery). Although samples collected using the sampling solvent 2% HNO3 produced the best recoveries, this solvent is not suitable for use on human skin. However, using EDTA as the sampling solvent produced acceptable recoveries and is better suited to collecting samples from the surface of human skin. EDTA is a good chelating agent that can extract metals from the skin surface better than DI H2O and is less hazardous than 2% HNO3.