Materials and Instrumentation
The ASV experiment was performed using a dedicated, commercially produced instrument - the Cogent/Modern Water PDV6000plus with VAS (voltammetric analysis system) software, but could be adapted for use by any lab in possession of a potentiostat and standard 3-electrode cell. The PDV6000plus is a portable device that can be used as a stand-alone instrument for field analysis or with the external VAS data analysis system for extended capabilities such as multiple metal analysis and more sophisticated analytical techniques. The software allows easy upload of new analytical methods into the PDV6000plus and provides comprehensive data display. By default, VAS automatically archives all acquired data and then calculates sample results based on a standard addition (or a simpler standard comparison) procedure, that can be exported into a spreadsheet. However, we require students to extract the raw current data (voltammetric peak height and peak area) and to perform their own calculations and regressions to extract the final results.
Several brands of cigarettes were purchased from local vendors for the student’s use. Students were allowed to bring their own samples if they preferred, adding a level of personal interest in the results. Concentrated nitric acid was obtained from our chemistry stockroom (we did not detect any lead or cadmium in our reagent grade HNO3, but ICP grade could be substituted if necessary). All the other chemicals used in the analysis, including 20 ppm standards for lead and cadmium, the mercury plating solution, the electrolyte (CLAC – a dilute acetic acid/acetate buffer with NaCl for ionic strength control) and the sample cups and electrode cleaning/polishing supplies were obtained from Modern Water/Cogent Inc. All of the reagents can alternatively be produced in-house by a typically-equipped chemistry stockroom (recipes available upon request).
Extraction of the samples was carried out in a nominally 1.5 M HNO3 solution, according to the following procedure: 1) two cigarettes were peeled and the tobacco was weighed; 2) tobacco leaves were placed into a clean 50-mL beaker with 20 mL of nitric acid (1:10 dilution of concentrated HNO3); 3) the beaker was placed in an ultrasonic bath (with no heating) for 15 minutes; 4) the sample was gravity filtered through a funnel with filter paper and transferred into a clean analysis cup; 5) a 1.00 mL aliquot of the sample extract from step 4 was combined with 20.00 mL of the CLAC electrolyte solution to produce the analytical sample, after obtaining a blank voltammogram as per the manufacturer's instructions (greater detail about our recommended analysis protocol is provided in the Appendix). Adding the sample aliquots to the electrolyte blank and spiking with the standards (Cd, Pb or a mixed standard) is facilitated by a ~0.5 cm hole in the side of the ASV cell in the Cogent cell design.
In principle, a sample blank should be prepared by following the extraction procedure above without adding tobacco and then transferring an aliquot to the CLAC electrolyte (as is done with the sample extracts). We determined that these were indistinguishable from the electrolyte blank described in the instrument operation procedure, so we opted for the use of the CLAC electrolyte as the blank. If it is suspected that there is metal contamination in any of the reagents or glassware, the former blank method could be used to test the hypothesis and eliminate the contamination or perform a background subtraction.
Sample Preparation (Active Learning Option)
Several more “open-ended” variants of this procedure are readily achieved by allowing students to select the system and design the actual sampling and work-up that is conducted. (The analytical procedure is more subject to problems when substantial modifications are tried and there is less parameter space to work in, so we do not recommend alteration of the ASV part of the experiment.) In addition to cigarettes, we have conducted experiments on chewing tobacco, eCigarette “juice”, shisha (for smoking in hookahs), and captured smoke from shisha and cigarettes. If a more student-directed experiment is desired, the students could be challenged to investigate the origin of the metals and to speculate on the likelihood that they would become biologically available as a result of the exposure route (chewing, vaping, smoking, etc.) They could then select a target for analysis and develop a sampling and extraction strategy. Simple extractions like the one used above are suitable for direct analysis of tobacco/shisha and for filter-collected smoke samples, but incorporation of hydrogen peroxide and/or microwave heating could improve the recovery of the metals. Replicate analysis can be used to indicate the variability of the sampling and extraction pre-treatment, while variation of the procedure (or spiking) can be used to investigate the percent recovery and its variability.
This change could give the students a more active role in the experiment and could extend it to two or more laboratory periods, if that is desirable. (We try to cover a wider array of methods and focus on writing in our one-quarter Instrumental Analysis laboratory.) In this case, a literature search for sampling and extraction methods may or may not be supplemented by provision of our procedure, which is included as an Appendix. The students might find this to be a useful starting point for their experimental design, in addition to published procedures from ASTM or NIOSH.
Nitric acid is a strong oxidizing acid that can cause severe burns. All of the sample handling during the extraction was performed in a fume hood and nitrile gloves were worn at all times. Cadmium is carcinogenic to humans. Lead is a poisonous metal that can target the nervous system. Waste was placed in an appropriate container for disposal, but typically falls below the ppb level that designates it as a hazardous aqueous solution. The 100 ppm mercury solution used for the plating of the working electrode is hazardous, but it can be reused several times according to the manufacturer’s recommendations. However, after the recommended number of usages (5 electrode platings) the Hg waste was placed in a separate heavy metal waste container. Cigarettes may become contaminated and should be used only for laboratory experiments and thus must be stored properly to prevent theft.
Instrumental Analysis Lab Course Context
Our laboratory sections are capped at 12 students that work in small groups (optimally 3 students per). Each group rotates their laboratory schedule over the term due to equipment limitations. The 3-student groups were able to finish the ASV experiment during the 5 hour lab period including: 1) validation experiments using three student-prepared challenge "unknowns" containing 20 ppb of the two metals alone and in a 1:1 mixture, all prepared from the 20 ppm standards, and 2) the one tobacco sample analysis; where each of the four samples was quantified by the method of Standard Additions, using 3 or 4 spikes per sample. A detailed procedure is provided in the Appendix. A journal-quality lab report describing the results of this experiment was prepared and submitted one week after the completion of the experiment.