In the discussion below, Pb is used as the heavy metal. Pb can simply be replaced by the heavy metal of interest.
Initial sample preparation
- Samples should be in plastic 13-15 ml test tubes (we have had very good service with Falcon brand polypropylene disposable centrifuge tubes with blue screw caps, and Fisherbrand test tubes with blue press caps). Have four 50 ml Falcon brand for two blanks and two standards.
- Add 0.5 ml of high-purity concentrated HNO3 to each of the unknown test tubes (2.5 ml to the blank and standard tubes). Also add 0.1 ml (0.5 ml to blanks and standards) of high-purity 30% H2O2*. Make sure that all insects are immersed in the solution.
- Leave overnight at room temperature. The hollow chitin exosleletons largely remain, though they become somewhat bleached and the acid turns yellow. Longer digestion times and other, more aggressive extraction techniques do not yield significantly more Pb in solution.
Concentrated Pb standard
To a 100 ml volumetric flask add 2.8 ml of high-purity concentrated HNO3 and 1 ml of 1000 ppm Pb stock standard. Dilute to volume and pour into a clean storage bottle.
| Concentrated standard | |
| Pb | 10 ppm |
| HNO3 | 2% |
| Total volume | 100 ml |
Diluting solution
Fill a 1000 ml plastic storage bottle with DI water and add 7 ml of concentrated, high-purity HNO3, 0.01 ml of 1000 ppm Bi stock solution. Bi is the internal standard. You can use a volumetric flask if you wish, but it adds more work and increases the likelihood of contamination. It doesn't matter so much what the diluting solution is, just that it is the same for all samples, blanks, and standards.
| Diluting solution | |
| Bi | 10 ppb |
| HNO3 | 0.5% |
| Total volume | 1000 ml |
Final dilutions for unknowns, blanks, standards
| Sample type | Add diluting solution, ml | Add concentrated standard solution, ml | Final volume | HNO3 | Pb | Bi |
| Unknowns (13 ml test tubes) | 10 | - | 10.5 ml | 3.8% | ? | 9.5 ppb |
| Blank (50 ml test tube) | 50 | - | 52.5 ml | 3.8% | ~0 | 9.5 ppb |
| Standard (50 ml test tubes) | 50 | 0.2 | 52.7 ml | 3.8% | 38.1 ppb* | 9.5 ppb |
| *Effective concentration taking into account that adding the 0.5 ml of concentrated standard dilutes the internal standard, and this dilution will be corrected automatically by the internal standard correction. | ||||||
Beware! Leftover insect parts can clog the sample introduction system, though we have found that serious clogs are rare (once in >3000 samples). To avoid insect parts, make sure the autosampler probe enters the solution close to the middle of the meniscus. Adjust the probe so the tip stays a few cm above the tube bottoms. After the analysis is done, inspect and replace parts of the sample introduction system where insect parts accumulate, especially where vinyl tubing meets glass or Teflon tubing.
* The H2O2 seems not to be strictly necessary for putting Pb into solution, but it has several advantages. it speeds digestion, reduces the foam-producing characteristics of the final solution, and produces exoskeleton fragments that are more likely to sink by the time analysis starts. Sinking fragments makes clogging of the sample introduction system less likely. DO NOT try to be clever and pre-mix HNO3 and H2O2 to reduce pipetting steps. The H2O2 quickly starts to decompose into O2, making pipetting an inaccurate mess. More slowly, the mixture evolves NO2 as well. Keep these samples in a hood.
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Schenectady, NY 12308 U.S.A. |