R. Steven Pappas

Part 1 of this tutorial addressed how environmental contamination may be minimized by using proper personal analytical practices, sample preparation environmental suggestions, avoidance of glass and low purity quartz contact with samples and standards during sample collection and preparation, and appropriate choices of high purity solvents during sample collection and preparation. Part 2 continues the discussion in terms of minimizing sample contamination issues by using high purity polymer materials for sample collection and preparation, as well as discuss the differences between instrument and method limits of detection (LODs). Accepted and appropriate procedures for calculating method LODs are related to variability in instrument response and unavoidable environmental contamination during even the cleanest sample preparation. Adherence to such practices will help analysts avoid false positives and report results with a high level of confidence.

Investigation of materials for trapping aerosols with minimum metal contamination compared to glass and low purity quartz fiber filters led to investigation of membrane filters manufactured from polymers. Unfortunately, polymer membrane filters (47 mm diameter or 60 mm diameter, 1.0 μm stated pore size) either could not meet ISO standard 20768 requirements for puff profile nor pressure drop across the filters, or they initially met the puff profile specifications, but clogged too quickly to collect adequate aerosol for analysis due to the excessive air flow restriction (1). Acid hardened ashless filter papers (chosen for lower metals contamination) were inadequate because aerosol breakthrough occurred even when three filters were placed together. Also, after a few standard 3 sec 55 mL puffs, the filters became wet and limited proper air flow, introducing deviations from requisite puff profile specifications (1). Although fluoropolymers are of much higher purity than glass, similar problems arose during method development. Thus, we developed a simple aerosol condensation trapping method using simply sufficient lengths of 3.97 mm i.d. high metals purity fluorinated ethylene propylene (FEP) tubing (Figure 1) (2,3). Tubing with smaller i.d. had a greater surface area to length ratio for more efficient condensation, but did not pass puff profile specifications due to resistance to air flow.

FIGURE 1: Acid cleaned fluorinated ethylene propylene (FEP) tubing (3.97 mm i.d., 5.18 meter length for ENDS with hydrophilic aerosols or 9.14 meter length for hydrophobic aerosols) met ISO Standard 20768 specifications for puff profile and requirements for negligible metal contamination of aerosol samples while providing quantitative metal oxide particle recovery. This image is CDC-sourced.

Impingers compatible with smoking machines are available from most tobacco product instrument manufacturers. Most of the impingers are made of glass with 25 to 50 mL volume, and used for measuring organic analytes. The simple fact that they are predominantly glass indicates the “experienced inorganic analyst” that these traps are not suitable for analyses of metals in smoke particulate nor aerosols from electronic nicotine delivery systems (ENDS).

Also, the trap volumes are not suitable for trapping metals in smoke particulate nor in ENDS aerosols because impingers should not be filled more than half full of liquid. A fraction of the liquid is lost, as it is bubbled out of the exit tube at standard puff air flow rates if impingers are more than half filled. Even a full 50 mL volume of dilute acid is insufficient to quantitatively trap neither cigarette smoke using commonly accepted smoke regimens nor ENDS aerosols using the ISO Standard 27068 regimen nor more aggressive smoking or aerosol generation regimens that may be representative of human puffing behaviors (1,4,5). Further, aqueous solvents are not appropriate solvents for smoke particulate that contains tar. Much of the tar is insoluble in dilute aqueous acid, and sticks inside the connection tubing and sides of impinger vessels rather than dissolving in the aqueous impinger liquid. Because tar is a heterogeneous mixture including very hydrophobic substances, such as polycyclic aromatic hydrocarbons (PAHs), as well as metals, neither hydrophilic nor hydrophobic solvents are ideal for collecting total particulate matter (TPM). We have used high purity quartz distilled 

-dimethylformamide (DMF) for a few applications because it is a highly polar solvent that dissolves metal ions as well as very hydrophobic substances in a single phase (6). The analyst must then either digest the solvent with dissolved or well-suspended particulate matter or dilute it into aqueous solutions for inductively coupled plasma–mass spectrometry (ICP-MS). Thus, neither tobacco industry labs nor public health labs commonly use impingers for collection of smoke particulate analyses of metals.

Some perfluoroalkoxy alkanes (PFA) impingers (60 mL) are commercially available (Savillex, Figure 2) and are suitable for trapping ENDS aerosols. The high purity PFA can be soaked and rinsed with ultrahigh purity dilute acids before use. One 60 mL impinger containing 30 mL dilute acid, however, is still insufficient for collection of aerosols using 55 mL puff volumes with a 3 sec rectangular puff profile (1). The air flow during this puff is 18.33 mL/sec, slightly greater than the average air flow rate using 35 mL puff volume and a 2 sec puff with a bell-shaped puff profile, as specified for ISO standard 3308, but less than the average air flow rate during a WHO Method 1 55 mL puff in 2 sec (4,5). We have used dimethyl sulfoxide for collection of mainstream cigarette smoke TPM using the ISO Standard 3308 and WHO Method 1 regimens with PFA impingers to provide the U.S. Food and Drug Administration Toxicology Lab dissolved TPM samples for cell culture studies. Three 60 mL impingers connected in tandem containing 30 mL liquid in each impinger were required to quantitatively collect smoke particulate matter from cigarettes using either smoking regimen. It is apparent that the 18.33 mL/sec air flow rate specified in ISO Standard 20768 would require approximately the same volume of liquid in three 60 mL impingers to quantitatively trap aerosol for analysis of metals as was required to trap smoke aerosol using the slightly lower ISO Standard 3308 average air flow rate and the higher WHO Method 1 air flow rate. For these reasons, it is apparent that 25 mL or 50 mL glass impingers were not manufactured with collection of metals in smoke or aerosol samples for analysis of trace metals in mind.

FIGURE 2: Half-filled perfluoroalkoxy alkane (PFA) 60 mL impingers connected in tandem with PFA tubing are acceptable materials for trapping aerosol for analysis of trace metals. One end is connected to the aerosol machine syringe pump connection, the other end to the ENDS device. After combining the contents of the three impingers, 10 mL of the acid used to collect aerosol may be used to rinse the final drops of liquid from the impingers and dilute to 100 mL in a dilute acid cleaned polymethylpentene (PMP), PFA, or polypropylene volumetric flask. This image is CDC-sourced.

may be defined as the persistence of a given elemental signal after the analysis of a standard or sample and a reasonable introduction system rinse time. Metals such as mercury are well known for memory effects, resulting in long introduction system rinse out times with nitric acid alone, since nitric acid is not a strong enough oxidizing acid to maintain mercury in oxidized form. The solution to this rinse out problem is the addition of an acid (such as hydrochloric acid), or a chelating agent that has a soft base anion ligand (such as sulfur) to the diluent and rinse solutions as previously discussed (7).

Modified polytetrafluoroethylene (TFM) vessel polymers are more rigid polymers than the PFA used to produce earlier generation microwave vessels. TFM vessels may be heated to higher temperatures without deformation, enabling more complete oxidation of organic substances. The TFM molecular polymer chains, like all molecules, have greater rotational motion at higher temperatures. It is possible that the more rapid motion of the PFA and TFM polymer chains at high temperatures permit metals to be captured in the surface polymer matrix. Therefore, PFA and even TFM vessels may superficially absorb metals whether the metals are known to exhibit memory effects when heated during a microwave digestion and may contaminate a subsequent digestion. The use of a hot nitric acid vapor cleaning system between analytical runs helps the problem, but does not eliminate it. Even after using two cycles with a hot acid vapor cleaning system to clean PFA vessels, we have observed trace metals carry over into digestion of a subsequent sample. This first became evident during digestion of urine samples for determination of uranium isotope ratios at low concentrations (8). It was noted that when urine samples containing spikes of reference materials or with known isotope ratios were digested in PFA vessels that previously contained a sample with a different isotope ratio, the resulting digested sample would have an isotope ratio intermediate between the previous sample and the subsequent sample, thus requiring blank digestions between samples to remove the metals absorbed by PFA vessels. We have subsequently observed similar effects with multiple metals from tobacco and other plant material digests when using higher temperatures in TFM microwave digestion vessels than were formerly used with PFA vessels. If a tobacco sample has a high concentration of any given metal, and a sample with a low concentration of the same metal follows in the same vessel, the resulting digestion yields a result that is slightly higher than expected. If the order is reversed, a tobacco sample that has a high concentration of any given metal following a sample that had a low concentration of the same metal in the same vessel will exhibit a less apparent effect. This demonstrates that even more rigid TFM vessels require blank digestions between samples that have significantly different metal concentrations before reutilization to avoid contamination from previous samples.

Memory effects are more troublesome if the metal is easily reduced in the presence of acid. For example, nitric acid catalyzes reduction of mercury as organic substances are oxidized. The reduced volatile elemental mercury is not quantitatively recovered. Some of the mercury is lost as vapor when the vessel is opened, and some is absorbed into the vessel walls, accumulating and causing increasing contamination of samples subsequently digested in the same vessel even after hot acid vapor cleaning. The addition of 1 mL hydrochloric acid to 9–10 mL nitric acid during microwave digestion will help remedy this problem, based on the same Pearson principle used to correct introduction system memory effects (4). Addition of sufficient soft base anion ligand such as chloride from hydrochloric acid chelates the Hg

. This complex ion is soluble in aqueous solvent. Since an equilibrium is formed between Hg

 complex, the equilibrium is shifted away from the Hg

 equilibrium (according to Le Chatelier’s principle), stabilizing mercury in soluble oxidized anionic complex form that does not have a memory effect, and minimizing contamination of subsequent samples in the same vessel.

The same considerations apply if a heating block is used. Figure 3 shows a heating block with a Teflon-coated surface for avoiding contamination and protecting the metal surface beneath. Note polypropylene tubes come from two sources, and PFA tubes come from another source. All need to be precleaned by heating the tubes with dilute nitric and hydrochloric acid, since heating causes the release of metals that were trapped in the polymer matrix. Note the discoloration of caps on the tubes on the left, demonstrating an attack of concentrated acids even on polymers.

FIGURE 3: Polypropylene (left and center) and PFA (right) tubes in a Teflon (PTFE) coated hot block digestion system. This image is CDC-sourced.

Instrument manufacturer instrument detection limits (IDLs) are useful only to compare signal to background ratio with other instruments. They are typically calculated as three times the standard deviation of a single analysis of a dilute ultrahigh purity acid blank that is run on a well-optimized instrument that has never been contaminated with samples with more complex matrices. Some analysts have published data with what appears to be a list of instrument manufacturer IDLs in supplementary files rather than true method detection limits (LOD), or with no description of determination of an LOD (9). An IDL is not relevant for reporting analytical data obtained using a validated method. Method LODs take into account instrument response determined under identical analytical method conditions developed for the analytes and matrices used. A real sample matrix is likely more complex than diluted ultrahigh purity nitric acid, and is measured with instrumentation that is no longer not pristine. Method LODs take into account trace metal contamination from microwave digestion vessels (if used), devices and vessels used for sample dilution, imperfectly prepared calibration standards and internal standards, minor pipetting inaccuracies, differences in instrument response from day to day, and all other aspects of sample preparation and instrument variability and error over a multiday period. The ideal method LOD would be useful to statistically determine the uncertainty of analytical method results at low concentration, and establish a concentration below which it is recognized that analytical results are insufficiently certain to report. True analytical results can then be distinguished from false positives resulting from trace metal contaminants of samples from previous vessel and instrument utilization, sample preparation and analysis, analytical interferences, and day-to-day differences in instrument response as optimization parameters change. It is apparent that using good analytical practices described in Part 1, as well as maintaining vessels used for sample collection and preparation as free of metal contamination as possible, will result in lower method LODs.

Although polypropylene bottles and 15 or 50 mL “centrifuge tubes” sold as sample containers generally have very low extractable metal concentrations, it is a good analytical practice to verify this. We tested a new batch of polypropylene tubes from a source for which we had formerly observed very low extractable trace metals, and observed significantly higher extractable aluminum in the new batch. We inquired about this, and learned that the vendor had changed the manufacturer to save on costs. Since aluminum was one of our hydrophobic aerosol method analytes, we had to change the source of sample tubes and check tubes from different vendors to determine the best source for the method. This illustrates that it is a good practice to screen a few containers every time a new batch of sample containers is received to avoid false positives from samples, even when using relatively high metal purity polypropylene tubes.

To minimize contamination of samples from containers as described above, it is a good practice to fill the tubes with 1% v/v ultrahigh purity nitric acid + 1% v/v ultrahigh purity hydrochloric acid, cap them, invert them a few times, and let them sit overnight before using them. This procedure cleans out most sample vessel impurities, unless they will be heated. Since the method LOD should incorporate variability in trace element background from the rinsed containers that we use, any trace metals that may remain have already been taken into consideration. Although acid rinsed sample containers result in contamination well below our method LODs, the following will illustrate how a well-validated method LOD prevents reporting false positives due to contamination. Polypropylene tubes (15 mL) obtained from three sources were prepared in triplicate with acid rinse solution, capped, inverted three times, and soaked overnight. Rather than discarding the overnight rinses, the rinses were analyzed according to our ENDS aerosol method (3).

It is evident from the results in Table I that nearly all of the polypropylene tube rinses were below the method LODs even before the tube prerinse. These tube sources had very low extractable elemental contamination. Two of the three nickel results in the tubes from our second source were below the method LOD. The nickel concentration obtained from only one tube was shown in the table to demonstrate that contamination from one tube in a batch of otherwise clean tubes may cause a false positive result. Since metals from only 3 of 100 tubes from each manufacturer were checked, it is possible that other tubes in the batch may also have low levels of contamination. It is also possible that tubes from the other sources had contaminated tubes that were not randomly selected. This confirms that prerinsing sample containers as a precaution is a good inorganic analytical practice to prevent contamination and false positive results.

There are several widely accepted ways to calculate method LODs. The simplest way is to include a sample in each analytical run for validation that has the same matrix as samples that will be analyzed with the method, and run it through the same sample preparation procedures as the samples. An appropriate sample should have very low analyte concentrations though remain in the same matrix as samples, in order to represent the higher relative variability that is analytically observed for low concentrations than high concentrations in the sample matrix. The same sample matrix is prepared independently a minimum of 14 times for 14 analytical runs per formed on 14 or more days. The standard deviation of the results obtained over a minimum of 14 days is multiplied by 3 to obtain approximately 99% certainty that the resulting concentration is above a level of response that would cause result uncertainty (9). This method may be taken a step further by analyzing 10 or more replicates of the same or similar sample with low analyte concentrations in the same run. The within run and between run standard deviations are then combined ( [

 ), since long term variability and uncertainty is usually greater than short term standard deviations (9,10).

The most well-known method for calculation of a method LOD is the Taylor method (11). This method requires multiple analytical runs as described in the simpler methods above. A minimum of seven or more samples are spiked with known analyte concentrations, analyzed according to the method procedure on separate days, and the standard deviations of the analytical results are plotted on the ordinate (

axis) versus concentrations on the abscissa (

 axis). The regression line through the plotted data is extrapolated to the ordinate at “0” analyte (abscissa) concentration. The apparent standard deviation at “0” analyte concentration is multiplied by 3 to determine the method LOD. This technique is a very good approximation, although it was likely intended for analyses in which there is negligible analyte concentration in the matrix, whereas samples are unlikely to have “0” metal concentration. When used for calculation of a method LOD with external standards, it is possible that the standards will be in a cleaner matrix than the samples, so a quality control sample (QCS) or certified reference material (CRM) that has characterized analyte concentrations and the same or very similar matrix should be included to add matrix effect to determination of the method LOD (12).

An additional level of care requires adding a statistical adjustment to account for potential overlap between false positives from samples with false negatives. This method requires determination of the regression using the Taylor method and inclusion of a sample blank along with the spiked samples, but statistically adjusting the results using the regression slope and ordinate intercept (12,13). Procedural blank and standard deviation are determined with analyses from multiple analytical runs, 

is the slope of the regression determined with the Taylor method, and 

is the intercept obtained using the Taylor method, displayed in equation 1 (11–13).

By properly calculating method LODs with an appropriate method, the analyst considers not only day-to-day variability of instrument response, calibration, and sample preparation, but trace contamination in digestion vessels (if used) and sample containers and other ancillary materials that contact samples as well. In this way, the analyst avoids reporting false positive and false negative results. The analyst determines “…the smallest measure, 

, that can be detected with reasonable certainty for a given analytical procedure” (9). In other words, “A common misconception is that the LOD is the smallest concentration that can be measured [with an instrument]. Instead, it is the concentration at which we can decide whether an element is present or not…” (9). A method LOD does not tell us the lowest possible ion concentration that produces ion counts in a secondary electron multiplier detector; rather, it reveals the concentration at which we can distinguish an elemental concentration in a sample from artifacts of variability from instrumentation response, calibration, and sample preparation procedures.

Trace metal contamination is unavoidable during preparation of samples for inorganic analyses. In order to minimize sample contamination when using these techniques, the analyst must take care to learn appropriate sample preparation practices and wisely choose ancillary vessels made from appropriate materials that contribute as little trace metal contamination as possible.

Method LODs should be calculated to take into account not only day-to-day instrument response variability, but variability of trace contamination acquired during independent sample preparation procedures as well. This will aid in avoiding reporting false positive and false negative results.

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

(1) International Standard Organization. Vapour Products — Routine Analytical Vaping Machine — Definitions and Standard Conditions. ISO Standard 20768:2018 Geneva, Switzerland. 

(2) Gray, N.; Halstead, M.; Valentin-Blasini, L.; Watson, C., Pappas, R.S. Toxic Metals in Liquid and Aerosol from Pod-Type Electronic Cigarettes. 

(3) Halstead, M.; Gray, N.; Gonzalez-Jimenez, N.; Fresquez, M.; Valentin-Blasini, L..; Watson, C.; Pappas, R.S. Analysis of Toxic Metals in Electronic Cigarette Aerosols Using a Novel Trap Design. 

(4) International Standard Organization. Routine Analytical Cigarette-Smoking Machine — Definitions and Standard Conditions. ISO Standard 3308:2012 Geneva, Switzerland, 

https://www.en-standard.eu/iso-3308-routine-analytical-cigarette-smoking-machine-definitions-and-standard-conditions/#:~:text=ISO%203308%20ISO%203308%3A2012%3A%20defines%20smoking%20parameters%20and,analytical%20smoking%20machine%20complying%20with%20the%20standard%20conditions

(5) World Health Organization. WHO TobLabNet Official Method SOP 01. Standard Operating Procedure for Intense Smoking of Cigarettes. World Health Organization 2012 Brussels, Belgium. 

https://www.who.int/publications/i/item/standard-operating-procedure-for-intense-smoking-of-cigarettes

(6) Fresquez, M.R.; Gonzalez-Jimenez, N.; Gray, N.; Valentin-Blasini, L.; Watson, C.H., Pappas, R.S. Electrothermal Vaporization-QQQ-ICP-MS for Determination of Chromium in Mainstream Cigarette Smoke Particulate. 

(7) Pappas, R.S. Sample Preparation Problem Solving for Inductively Coupled Plasma-Mass Spectrometry with Liquid Introduction Systems I. Solubility, Chelation, and Memory Effects. 

(8) Pappas, R.S.; Ting, B.G.; Paschal, D.C. A Practical Approach to Determination of Low Concentration Uranium Isotope Ratios in Small Volumes of Urine. 

(9) Thomsen, V.; Schatzlein, D.; Mercuro, D. Limits of Detection in Spectroscopy. 

(10) Pappas, R.S.; Ting, B.G.; Jarrett, J.M.; Paschal, D.C.; Caudill, S.P.; Miller, D.T. Determination of Uranium-235, Uranium-238, and Thorium-232 in Urine by Magnetic Sector Inductively Coupled Plasma-Mass Spectrometry. 

Quality Assurance of Chemical Measurements

(12) Gonzalez-Jimenez, N.; Gray, N.; Pappas, R.S,; Halstead, M.; Lewis, E.; Valentin-Blasini, L.; Watson, C.; Blount, B. Analysis of Toxic Metals in Aerosols from Devices Associated with Electronic Cigarette, or Vaping, Product Use Associated Lung Injury. 

(13) National Committee for Clinical Laboratory Standards (NCCLS). Protocols for Determination of Limits of Detection and Limits of Quantitation. National Committee for Clinical Laboratory Standards 2004 Wayne, PA, 24, 9–35.

 is a research chemist and inorganic analytical Team Lead. 

 is an inorganic analytical chemist. Both authors work in the Tobacco and Volatiles Branch of the Division of Laboratory Sciences at the National Center for Environmental Health, U.S. Centers for Disease Control and Prevention, in Atlanta, Georgia. Direct correspondence to: 

, the editor of the “Atomic Perspectives” column, is the principal of Scientific Solutions, a consulting company that serves the educational and writing needs of the trace element analysis user community. Rob has worked in the field of atomic spectroscopy and mass spectrometry for more than 45 years, including 24 years for a manufacturer of atomic spectroscopic instrumentation. He has authored more than 100 scientific publications, including a 15-part tutorial series, “A Beginner’s Guide to ICP-MS.” In addition, he has authored five textbooks on the fundamentals and applications of ICP-MS. His most recent book, a paperback edition of 

Measuring Heavy Metal Contaminants in Cannabis and Hemp, 

was published in December 2021. He is currently working on the 4th edition of his Practical Guide to ICP-MS: A Tutorial for Beginners textbook, which should be published in July 2023. Rob has an advanced degree in analytical chemistry from the University of Wales, UK, and is a Fellow of the Royal Society of Chemistry (FRSC) and a Chartered Chemist (CChem). Direct correspondence to 

Articles

Minimizing sample preparation issues requires users to consider method LODs as well as high purity polymer materials for sample collection and preparation. Here, we examine proper personal analytical practices one can take to avoid environmental contamination.

Contamination Control During Sample Preparation for Trace Element Analysis of Electronic Cigarette Aerosol with Inductively Coupled Plasma-Mass Spectrometry, Part 2

In this two-part article, the authors take a look at the major factors affecting background contamination in inductively coupled plasma-mass spectrometry (ICP-MS), particularly when measuring ultratrace elements in specialty applications such as aerosol matrices from electronic cigarettes and electronic nicotine delivery systems (ENDS). In order to obtain meaningful data, the analyst must understand and address the magnitude and variability of environmental contamination by ubiquitous trace metals during the sample preparation procedure. The impact of these contaminants on the procedural blank and the associated impact on the accuracy and precision of sample results can unfortunately lead to false positive results.

Liquid introduction with flow injection is the most widely used sample introduction method for inductively coupled plasma-mass spectrometry (ICP-MS). Nevertheless, background contamination issues are problematic for determinating trace or ultratrace analyte concentrations that are commonly investigated. This is particularly important in specialty applications such as aerosol matrices from electronic cigarettes (also known as vapes, electronic nicotine delivery systems [ENDS]). To obtain meaningful data, the analyst must understand and address the magnitude and variability of environmental contamination by ubiquitous trace metals during sample preparation for trace analyses. The impact of these contaminants on the procedural blank and the associated impact on the accuracy and precision of sample results can lead to false positive results. Part 1 of this tutorial addresses address how environmental contamination may be minimized by using good personal analytical practices, avoidance of glass and low purity quartz during sample collection and preparation, and choices of high purity solvents during sample collection and preparation.

Part 2 will discuss the use of high purity polymer materials during sample collection and preparation, elemental carryover during sample preparation, and how the inorganic analyst can avoid approaches and procedures that may cause unforeseen problems.

Preparation for Analysis of Organic Substances Versus Inorganic Substances

Sample preparation and analysis of samples for many organic substances may be based on the assumption that the organic analytes are not environmentally ubiquitous. For example, if an analyst is required to analyze nicotine in tobacco, tobacco smoke, liquids, or aerosols from ENDS, the analyst may reasonably assume that extractable nicotine concentrations on glass surfaces of clean beakers, flasks, impingers, filter materials, pipet tips, glass or polymer sample vials, and other ancillary materials are negligible. In such cases, any analytical signal detected in procedural blanks is likely due to poorly cleaned glassware, the presence of other compounds with the same nominal mass, or from instrumental electronic noise. This assumption, however, is not valid for sample preparation for analysis of trace metals in tobacco, tobacco smoke, liquids, or aerosols from ENDS with inductively coupled plasma-optical emission spectrometry (ICP-OES) or ICP-MS. Metals are ubiquitous in many, if not most, commonly used laboratory set-up and supplies, including (but not limited to) glassware, hood surfaces, dust particles, filter paper, tubing, and pipette tips. Different ancillary materials and a different skillset are required for sample preparation for analysis of trace metals in any matrix by ICP-OES or ICP-MS.

Factors That Interfere with Accuracy in Analyses for Inorganic Substances

The relative ease or difficulty of a given analysis depends on several factors, including concentration of the analyte relative to the sensitivity of the analytical instrumentation, the chemistry of the analyte, the matrix, isotopic interferences from other elements that have the same mass as the analyte (1), polyatomic ionic interferences formed in the plasma during the analysis that have the same mass as the analyte (2), and the potential for environmental contamination by extractable metals that are present to various degrees on all surfaces and most materials.

The following suggestions can be an effective means to reduce the background contamination issues, make a difference in levels of contamination associated with environmentally ubiquitous metals, and yield lower procedural blanks and method limits of detection (LOD), as well as false positive results:

Use powder-free gloves, nitrile if possible. Powder particles can contaminate samples.

Do not pick up sample tubes with a fingertip inside the tube opening.

Avoid contact of fingers or gloves with surfaces near the opening or inside the cap of any sample tube or solvent container.

Use pipets with polypropylene or fluoropolymer pipet tips, never glass pipets.

Use pipets that do not have external stainless steel tip ejectors (Figure 1; all figure images are CDC sourced). It is easy to touch a drop of liquid with the tip ejector and contaminate a sample with iron, chromium, nickel, and other metals. If pipets have external steel tip ejectors, it is advisable to remove the tip ejectors and remove the tips manually after use in sample preparation.

Never turn a pipet sideways while liquid is in the tip. Acid will corrode the pipet piston and degrade it, resulting in contaminated samples.

If a pipet tip touches skin or a laboratory surface, assume that it is contaminated. Learning not to lean back against a countertop in a laboratory also prevents contact and contamination of tips that may have been left protruding over the side of the countertop by a fellow laboratory worker who did not want the tip to touch the countertop surface.

Do not purchase acid in glass containers. Ultrahigh purity acids for analysis of metals are double distilled from fluoropolymer or high purity quartz stills and sold in perfluoroalkoxy (PFA) or fluorinated ethylene propylene (FEP) fluoropolymer bottles (Figure 2). Less expensive intermediate purity acids are available in polyethylene or polypropylene containers. Several purity grades can be obtained in polyethylene or polypropylene containers that are still higher purity than acids from glass bottles. The acids may be further purified by distillation in PFA or high purity quartz stills sold by some vendors. Depending on acid consumption in a laboratory, these stills can pay for themselves with savings on ultrahigh purity acids in approximately a year.

If using bottle top acid dispensers, avoid glass entirely. Use empty PFA, FEP, or polypropylene containers that have been well rinsed with dilute ultrahigh purity acid. Use dispensers with fluoropolymer only exposed to the path of liquid flow (Figure 2). A dispenser that has glass parts or that has a platinum coated valve ball is unsuitable for dispensing acids for trace metals analysis. For example, one of our team members obtained a dispenser that had an “inert” platinum coated valve ball in an otherwise all-fluoropolymer liquid path. This dispenser ruined several of our analyses before we realized that multiple metal impurities were leaching into acid from the platinum coated valve ball. The first few liquid pumps were low in metals concentrations, followed by metals concentrations that reached a peak as the acid that was in contact with the platinum reached the dispenser outlet, then declined again as subsequent pumps rinsed the metals out.

If the laboratory has high airborne particulate, use a plastic autosampler cover. This is a good idea even if the air particulate is not very high. Covers may be purchased with or without high efficiency particulate air (HEPA) filtered air units that blow filtered air over open sample containers.

Unless it is the only way to avoid inhalation of acid vapors, use a hood for diluting or transferring concentrated acids, but do not leave samples in a hood unless it has little or no corrosion and laminar flow with HEPA filtered air. If laboratory air has high particulate levels, a standard hood protects the analyst, but pulls contaminated laboratory air over open sample or solvent containers faster than air outside the hood. Polypropylene hoods with HEPA or ultra-low particulate air (ULPA) filtered air are available (Figure 2), but they require sufficient dedicated air flow. If the analyst works with hydrofluoric acid, a hood absolutely must be used for safety.

FIGURE 1: Pipet that is manufactured with plastic, not steel. Pipet tip ejector and glass volumetric flasks are used for mercury standards only. The use of glass containers for metals other than mercury is a poor analytical practice.

FIGURE 2: PFA ultra-high purity acid bottles that have been repurposed as high purity acid dispenser bottles. The bottletop dispensers also have 100% PFA liquid path. The vessels are inside a polypropylene laminar flow hood to prevent sample contamination during preparation.

Choice of Materials Used for Sample Collection, Preparation, and Analysis 

An analyst must consider and choose appropriate types of ancillary materials that will be used for transfer, containment, and digestion for dissolution of metals and elimination of organic substances before developing an analytical method for analysis of trace metals. As previously discussed, glass containers and transfer devices are appropriate choices for preparation of samples for the analysis of organic substances. Even though some glass microparticles and trace inorganic substances from glass may be extracted into the standard or sample solution, they are often eliminated during further organic sample preparation steps, or not detected during common chromatographic or mass spectrometric analyses. However, the use of glassware is a very poor analytical practice for analysis of trace metals with very few exceptions.

Mercury is a rare exception to the rule of avoiding glass for analysis of trace metals. Mercury compounds may adhere to glass silicate surfaces on the basis of polar attractions, but mercury is a Pearson soft acid cation metal (3,4) and is not strongly coordinated to surface oxygen atoms of glass silicate containers in the presence of a polar or acidic solvent. Mercury halides are readily extracted from some aqueous solvents into polar organic solvents in glass vessels (5). Because mercury is not strongly coordinated to silicates, it is usually found at very low concentrations in glass. Thus, glass tends to have very low mercury concentrations. Therefore, mercury standards may be prepared in glass volumetric flasks for analysis with a direct mercury analyzer (6,7). Low ng/L concentration mercury standards prepared in 0.2% v/v ultrapure nitric acid and 0.01% w/v high metals purity L-cysteine were stable when kept refrigerated in glass flasks for more than a year (Figure 1). Refrigeration was used between analyses to slow cysteine thiol oxidation beyond the disulfide form of sulfur that can coordinate soft acid cation metals like mercury to prolong the stability of mercury in oxidized form in solution.

Mercury is an exception to the common good analytical practice of avoiding sample and solvent exposure to glass when it is the lone analyte with a direct mercury analyzer. If mercury is not the lone analyte, the general good inorganic analytical practice of avoiding glass always applies, because other metal contaminants are likely to be extracted from glass by acidic solvents. Avoiding glass during trace metals analyses has been recognized as a good inorganic analytical practice for decades. We have found it necessary to briefly review the chemistry of glass related to metals analysis since the development of ENDS devices, and availability of grant funding to study their emissions has attracted many scientists with limited experience in the analysis of metals. In some states, regulatory requirements for aerosol de- vices include the analysis of toxic metals in liquids and aerosols of some devices. This has increased the demand for inorganic analytical chemists in commercial compliance laboratories as well, resulting in a shortage of competent inorganic analytical chemists. Often this demand is filled with analysts who may have experience performing analyses of organic substances but may benefit from a review of good inorganic analytical practices that differ from good organic analytical practices.

Avoidance of Glass for Inorganic Sample Collection, Preparation, and Analysis

Murphy (8) discussed the need to avoid glass during sample preparation for accuracy in trace element analyses in 1976, before greater sensitivity made possible by instrumentation developments since that time made lower contaminant concentrations even more apparent. In 1984, Knapp stated, “Glass is usually not suitable...” for trace element determinations (9). In 1996, Dulski discussed the avoidance of glass during analysis of metals (10). The inorganic analytical chemist needs to be aware of why these highly experienced chemists discussed the need to avoid glass during sample preparation and inorganic analysis.

The first reason is the mobile nature of ions in glass. Glass is manufactured from substances with low elemental purity, so glass is not a pure silicate substance. Thin glass membrane electrode glass is known to contain other impurities but is manufactured with high concentrations of some ions, such as sodium from soda ash. Whether the analyte intended to be measured is hydronium ion with a pH meter electrode or a specific metal with a selective ion electrode, the thin glass membrane analytical design is based on different potentials for two or more different hydronium or metal ions to migrate to and from the glass membrane silicate matrix (11). There are numerous selective glass electrode designs based on different glass membrane potential combinations for different metal ions, but the application of the pH meter demonstrates the migration of acid hydronium ions into the glass membrane in exchange for metal ions from glass silicate matrix. This is one reason that acidic samples are contaminated by metal impurities from glass.

If pure water were a sufficiently strong solvent for all metal ions, inorganic analytical chemistry would be much easier. However, most metals other than alkali metals require acids to prevent hydrolysis of the metal ions to the respective oxides or hydroxides, nearly all of which are insoluble in water at neutral pH (4). To illustrate problems with the use of acids in glass flasks as impingers for collection of aerosols for metals, four borosilicate glass laboratory flasks were rigorously precleaned overnight with ultrapure acid, and shaken with 10% v/v ultrapure concentrated nitric acid and 1% v/v hydrochloric acid that was discarded before five hour leaching periods in the same acid mix, corresponding to a procedure for trapping metals for aerosol analysis over a period of 5 d. Although five analyses in 5 d is insufficient for establishing a legitimate method limit of detection, the day-to-day variability and ranges of concentrations observed from leaching chromium, nickel, copper, zinc, tin, and lead were higher than sample results reported in the literature, with the exception of copper at elevated concentrations in some aerosols from a few devices (12). It is apparent that the use of glass vessels for sample collection for smoke or aerosol analysis will result in samples contaminated by metals leached from glass by acid, and produce unreliable results.

Before more advanced sample preparation devices became available, automated pipettors with glass barrels were used for dilution of samples such as urine into acids containing internal standard metal ions for analysis. The sample and internal standard solution uptake and delivery tubing were made from ultrahigh metals purity fluoropolymers such as PFA. However, the syringe barrels were made of glass and later from low purity quartz. When an analytical chemist began the workday, the syringe barrels were set to maximum volume and 10 to 20 syringe barrel volumes of dilute ultrapure acid solution were passed through the barrels to rinse out the metal ion contaminants that had accumulated from the glass barrels. When the analyst monitored the selected ions, they decreased with each barrel rinse. The acid-extracted metals were significant relative to metal concentrations in urine samples until the syringe barrels were well rinsed with acid, thus necessitating the daily preanalytical acid rinses of the glass barrels. Although the metal concentrations decreased by about half over a period of six months to a year, they never completely diminished. To make matters worse, the kinetics of ion exchange from the glass silicate matrix to the acid were fast. The analyst had to have all samples ready for dilution and continuously dilute one sample after another without stopping. If the sample preparation was stopped for even 5 min, the analyst would see an increase in metal concentrations that came from the glass barrels and contaminated the samples. Even after a 5 min delay, the analyst had to again rinse the barrel contents with the acid solution before resuming sample preparation. One manufacturer was aware of this problem and manufactured some quartz barrels for trial purposes. Unfortunately, the manufacturer used less expensive low purity quartz to manufacture the barrels rather than high purity fused silica quartz. The low purity quartz barrels had lower concentrations of metals leaching from the quartz silica matrix than from glass barrels over time, but the metals concentrations were still significant relative to metals concentrations in urine samples. Over a six-month period, the metals leached from low purity quartz barrels also diminished to concentrations lower than metals concentrations from glass barrels after six months, but the metals concentrations were still sufficiently high that the barrels still had to be daily rinsed out with acid prior to use and after any sample preparation delay. After years of intentional leaching from glass syringe barrels, leaching of metals by acids was still observed. This illustrates that glass cannot be “cleaned” sufficiently for use in trace metals analysis. The avoidance of contact of acidic solutions with glass is not limited to inorganic acids but applies to organic acids as well (13).

Glass fiber filters are commonly used for trapping mainstream smoke particulate for analyses of organic substances. These filters have high acid leachable metals concentrations and are inappropriate for aerosol sample collection from ENDS devices and from mainstream smoke for metals analyses. Low purity quartz filters (14) also released unacceptably high metal concentrations unless subjected to extensive soaking and elimination of fine particles with multiple hot acid rinses. Even after extensive soaking in dilute ultrahigh purity acids, only cadmium, lead, and thallium could be observed in mainstream smoke at concentrations sufficiently above the acid extractable metals backgrounds that remained in the “precleaned” quartz filters (14). Many metal ion concentrations are lower in ENDS aerosols than in mainstream smoke since no combustion occurs; and because ENDS devices are not constructed from some toxic metals and metalloids that are commonly present in tobacco, such as cobalt, arsenic, cadmium, and mercury. The metals concentrations obtained from using dilute acid to extract metals from ENDS aerosols trapped with low purity quartz fiber filters were too high and too variable from sheet to sheet of quartz filter material for use as ENDS aerosol traps (15). Metals that are used in manufacture of ENDS devices are often below sensitive analytical method limits of detection in liquids and aerosols from newly manufactured devices (16), but the metals become more elevated with age and use of the devices due to corrosion (17). Since most mainstream smoke metals concentrations and ENDS aerosol metals concentrations after device corrosion remain at trace or ultratrace levels, glass or low purity quartz fiber filters contribute significantly to aerosol background levels because of metals leached by acid from the glass or low purity quartz filters. Thus, such filter materials are not suitable for use as mainstream smoke and ENDS aerosol sample collection or preparation materials. Extraction of metals from glass is not limited to acidic solutions. A group affili- ated with Schott AG recently published a white paper on delamination of pharma- ceutical grade glass vials in basic solution (18). They discussed their work to produce glass vials that are more resistant to delamination, as well as earlier work on delamination of glasses of various compositions in alkaline solution. Delamination of glass is not unexpected, since silicates are more soluble in alkaline solution, permitting small flakes of glass (along with metals in the glass silicate matrix) to become liberated from glass surfaces as the silicate is dissolved. Significant solubilization of molded glass component surface elements was also determined when vials were stored containing neutral aqueous 15% w/v KCl solutions or other alkali metal salts (19). For the inorganic analytical chemist, this data on glass and metal contaminants in acidic, basic, and neutral high ionic strength solutions confirm that complete avoidance of the use of glass flasks, impingers, watch glasses, sample tubes, vials, glass fiber filters, and other ancillary glass materials is good inorganic analytical practice for avoiding sample contamination from metals in glass.

In Part 1 of this article, we have discussed good sample handling practices that will help minimize sample contamination during preparation for inorganic analyses. We reviewed the background and gave specific examples of sample contamination caused using glass and low purity quartz during sample collection and preparation.

In our next column, we will discuss the use of ancillary polymer and fluoropolymer materials that may be obtained with very low acid-leachable metals and that may be further cleaned for use in inorganic sample collection and preparation.

(1) Neubauer, K. Reducing the Effects of Interferences in Quadrupole ICP-MS. 

(2) May, T. W.; Wiedmeyer, R. H. A Table of Polyatomic Interferences in ICP-MS. 

; University Science Books, 2000, 191–228.

(4) Pappas, R. S. Sample Preparation Problem Solving for Inductively Coupled Plasma-Mass Spectrometry with Liquid Introduction Systems I. Solubility, Chelation, and Memory Effects. 

(5) Tandon, S. N.; Gupta, C. B. Liquid-Liquid Extraction Behaviour of Mercury (II) as Chloride. 

(6) Fresquez, M. R.; Gonzalez-Jimenez, N.; Gray, N.; Watson, C. W.; Pappas, R. S. High Throughput Determination of Mercury in Tobacco and Mainstream Smoke from Little Cigars. 

(7) Fresquez, M. R.; Pappas, R. S.; Watson, C. H. Establishment of Toxic Metal Reference Range in Tobacco from U.S. Cigarettes. 

Accuracy in Trace Analysis: Sampling, Sample Handling, Analysis. Volume I, Publication 422; National Bureau of Standards

(9) Knapp, G. Decomposition Methods in Elemental Trace Analysis. 

A Manual for the Chemical Analysis of Metals

. Analytical Sciences Library, Electrochemical Analytical Chemical Methods, 2023, 7.1. 

https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Supplemental_Modules_(Analytical_Chemistry)/Analytical_Sciences_Digital_Library/In_Class_Activities/Electrochemical_Methods_of_Analysis/02_Text/7%3A_Electrochemical_Analytical_Methods/7.1%3A_Ion-Selective_Electrodes#:~:text=The%20glass%20used%20in%20a%20pH%20electrode%20contains,distance%20into%20the%20glass%20and%20displace%20 sodium%20ions

(12) Ahmed, A, A.; Youssof, I. M. Attack on Soda-Lime-Silica Glass Bottles by Acetic, Citric, and Oxalic Acids. 

Measurement of Elemental Constituents of Cannabis Vaping Liquids and Aerosols with ICP-MS

Measuring Heavy Metal Contaminants in Cannabis and Hemp

(14) Pappas, R. S.; Polzin, G. M.; Zhang, L.; Watson, C. H.; Paschal, D. C.; Ashley, D. L. Cadmium, Lead, and Thallium in Mainstream Tobacco Smoke Particulate. 

(15) Lisko, J. G.; Hearn, B.; Pappas, R. S.; Stanfill, S.; Tyx, R.; Keong, L., et al. “Factors to Consider when Analyzing Electronic Cigarettes: Challenges and Solutions.” Society for Research on Nicotine and Tobacco Annual Meeting 2018, POS4–13. 

https://cdn.ymaws.com/www.srnt.org/resource/resmgr/conferences/2018_annual_meeting/65388_srnt_2018_abstract_fin.pdf

(16) Gray, N.; Halstead, M.; Valentin-Blasini, L.; Watson, C.; Pappas, R. S. Toxic Metals in Liquid and Aerosol from Pod-Type Electronic Cigarettes. 

 (4), 337–347. DOI: 10.1093/jat/bkaa185

(17) Halstead, M.; Gray, N.; Gonzalez-Jimenez, N.; Fresquez, M.; Valentin-Blasini, L.; Watson, C.; Pappas, R. S. Analysis of Toxic Metals in Electronic Cigarette Aerosols Using a Novel Trap Design. 

(18) Ennis, R. D.; Pritchard, R.; Nakamura, C.; Coulon, M.; Yang, T.; Visor, G. C.; Lee, W. A. Glass Vials for Small Volume Parenterals: Influence of Drug and Manufacturing Processes on Glass Delamination. 

(19) Haines, D.; Gober-Mangan, E.; Klause, M.; Rothhaar, U. Comparative Predictive Glass Delamination Study. 

 is a research chemist and inorganic analytical Team Lead in the Tobacco and Volatiles Branch of the Division of Laboratory Sciences at the National Center for Environmental Health, U.S. Centers for Disease Control and Prevention, in Atlanta, Georgia. Direct correspondence to: 

 is an inorganic analytical chemist who works for the Tobacco and Volatiles Branch of the Division of Laboratory Sciences at the National Center for Environmental Health, U.S. Centers for Disease Control and Prevention, in Atlanta, Georgia.

, the editor of the “Atomic Perspectives” column, is the principal of Scientific Solutions, a consulting company that serves the educational and writing needs of the trace element analysis user community. Rob has worked in the field of atomic spectroscopy and mass spectrometry for more than 45 years, including 24 years for a manufacturer of atomic spectroscopic instrumentation. He has authored more than 100 scientific publications, including a 15-part tutorial series, “A Beginners Guide to ICP-MS.” In addition, he has authored five textbooks on the fundamentals and applications of ICP-MS. His most recent book, a paperback edition of 

Measuring Heavy Metal Contaminants in Cannabis and Hemp,

 was published in December, 2021. He is currently working on the 4th edition of his 

Practical Guide to ICP-MS: A Tutorial for Beginners

 textbook, which should be published in July, 2023. Rob has an advanced degree in analytical chemistry from the University of Wales, UK, and is a Fellow of the Royal Society of Chemistry (FRSC) and a Chartered Chemist (CChem). Direct correspondence to 

Good sample handling practices that minimize sample contamination during preparation of inorganic analyses are discussed, using glass and low purity quartz as examples.

Contamination Control During Sample Preparation for Trace Element Analysis of Electronic Cigarette Aerosol with Inductively Coupled Plasma-Mass Spectrometry, Part 1

In inductively coupled plasma–mass spectrometry (ICP-MS), problems persist in the determination of analytes that are commonly investigated as well as in specialty applications for those seldom considered by most analysts. Understanding the chemistry that is common to different groups of analytes permits the development of successful approaches to rinse-out and elimination of memory effects. It also equips analysts for development of successful elemental analytical approaches with a broad spectrum of matrices and other analytical challenges, whether the sample is solid or liquid.  

Liquid introduction, in general, and flow injection, specifically, are the most widely used sample introduction methods for inductively coupled plasma–mass spectrometry (ICP-MS). Nevertheless, problems persist in the determination of analytes that are commonly investigated, as well as in specialty applications for those seldom considered by most analysts. Understanding the chemistry that is common to different groups of analytes allows the development of successful approaches to rinse-out and elimination of memory effects. This understanding also equips analysts for developing successful elemental analytical approaches in the face of a broad spectrum of matrices and other analytical challenges, whether the sample is solid or liquid.  

The majority of ICP-MS applications for elemental analysis utilize liquid sample introduction whether or not the original sample was a liquid. The relative ease or difficulty of a given analysis depends on several factors, including the matrix and the chemistry of the analyte.  

Probably the simplest matrix for elemental analytical purposes is water (that is, fresh water with low dissolved solids). Nevertheless, there are analysis problems with some analytes even in this matrix. General solubility rules state that alkali metal and ammonium ions are soluble in the presence of most anions. However, even in the presence of low concentrations of halide and polyatomic anions, many other metals hydrolyze and form poorly soluble hydroxides or oxides in water. When there is an absence of acid (or base in some cases), one may get an incomplete picture of the metal profile in a water sample because the poorly soluble hydroxide or oxide accumulates on walls of tubing, spray chambers, and nebulizers. An illustration of how such an incomplete profile may occur is presented in Figure 1. A total of 10 sequential water samples from an ultrapure water system were analyzed for 

 U intensity in counts per second (cps) in low resolution with a magnetic sector ICP-MS using a perfluoroalkoxy (PFA) 100 µL/min nebulizer and PFA double-pass spray chamber. If an analyst were to use uranium calibration standards diluted in ultrapure nitric acid before analyzing these samples, the uranium content would appear undetectable. A NIST 1643e water standard reference material (SRM) would give the appearance of validating the method accuracy. However, the NIST 1643 water SRM matrix contains 5% HNO

 to stabilize the characterized analytes in the water solution. 

Figure 1: Illustration of the accumulation of poorly water-soluble metal oxides on sample introduction system surfaces from 10 ultrapure water samples versus continuous rinse-out when the water is acidified. The decline in 238U counts per second from sample 11 to 16 occurs as nitric acid dissolves and mobilizes the accumulated U from the surfaces.			

Samples 11–20 in Figure 1 are acidified with 1% ultrapure nitric acid. It is obvious that the 

 U counts are elevated approximately 20-fold in all samples after the initiation of sample acidification. However, the approximate 100-fold increase in sample 11 demonstrates that 

 U from unacidified ultrapure water alone was accumulating in the introduction system. From that point on, the U continues to be mobilized and rinsed from the introduction system until it reaches a constant level. Thus, without proper sample preparation, sample carryover from accumulation could result in a false negative determination.   

Figure 1 illustrates that the solvent and rinse solution for a given method must account for the analyte's aqueous chemistry. Nitric acid (1–5%) is commonly used for metal dissolution and stabilization for ICP-MS analysis. Nitric acid is a strong acid, and general solubility rules suggest that nitrates are soluble. The hydronium counter-anion from nitric acid is nitrate, thus it would superficially seem to be the universal solvent for metals. It is an appropriate choice for many inorganic analytes. Dilute nitric acid also is commonly used in diluents for urine analysis for this reason. 

There are metals, however, that are well known for 

 when 1–5% nitric acid is used as a rinse solution. Memory effects are defined as the persistence of a given elemental signal after the analysis of a sample and a reasonable rinse time. A simple explanation for the causes of many memory effects can be made using the Pearson hard–soft acid–base (HSAB) theory as a model for coordination of a given metal (1). Though this dated model does not explain every case, it serves a useful purpose in general for approaching stabilization and rinse-out from an introduction system.  

In the Pearson model, thorium is a very hard acid cation metal. Hard acid cation metals are preferentially coordinated by hard base anion ligands. If solid thorium oxide is placed in dilute nitric acid, it will slowly dissolve. The affinity of thorium for the oxygen ligands in neutral thorium oxide is strong. Because of this affinity, dissolution and stabilization of thorium as a hydrated cation in dilute nitric acid is not completely successful. Even thorium halides tend to precipitate as neutral hydrate halides over time. However, the addition of a dilute fluoride ion to a solution (when possible) may improve the rinse-out characteristics of thorium. Fluoride is a harder base anion ligand than oxygen and a better nucleophile than water oxygen atoms or other halides. For these reasons, fluoride can effectively compete with oxygen for coordination to thorium. However, even ThF

 is poorly soluble. When a sufficient concentration of fluoride is present for formation of anionic complexes such as [ThF

, then the charged complex is quickly rinsed through the system without a memory effect. A rinse solution of 5% (v/v) nitric acid with 5% (v/v) hydrofluoric acid has been shown to eliminate thorium memory effects in urine at trace concentrations (2,3). Care must be taken because of the fluoride ion's neurotoxicity and the facile absorption via inhalation or dermal exposure. Safety concerns must be addressed when using sources of fluoride such as hydrofluoric acid or ammonium fluoride, and proper personal protection and appropriate fume hood ventilation must be used. In addition, the use of hydrofluoric acid necessitates an inert introduction system that includes a fluoropolymer nebulizer and spray chamber and a sapphire or alumina injector. 

Mercury is an example of an element that exhibits memory effects for multiple reasons. According to Pearson's HSAB theory, mercury is a soft acid cation metal. Soft acid cation metals are more readily coordinated by soft base anion ligands, such as sulfur and halides other than fluoride. Soft acid cations also generally have higher electronegativities and more easily deformable orbitals, and they more readily form bonds with greater covalent character than hard acid cation metals. Therefore, mercury and similar metal ions form strong metal-sulfide and halide bonds. Environmental mercury commonly occurs in organomercury forms such as dimethylmercury and methylmercury halide. Even mercury(II) chloride has covalent characteristics in metal–chloride bonds. Because mercury(II) chloride is also linear and nonpolar, it is readily soluble in organic solvents such as ethyl acetate, pentyl acetate, and diethyl ether (4). This covalent, and sometimes nonpolar, character suggests an explanation for one cause of mercury's memory effect: adhesion to nonpolar surfaces such as polymeric tubing. Another cause of mercury's memory effect is that it is easily reduced. Nitric acid does not coordinate well with mercury and catalyzes its reduction in the presence of a reducing agent. Elemental mercury is neither lipophilic nor hydrophilic (5,6). Presumably, it migrates into crevices to escape either organic or aqueous solvents. Chelation of mercury ions with soft base anion ligands is a solution for avoiding such memory effects. Bromide and iodide ions strongly coordinate with mercury, but they form insoluble mercury halides. Chloride is more soluble and therefore is a better choice, and it aids in long term stabilization against reduction; but, as stated above, mercury(II) chloride is readily adsorbed on hydrophobic surfaces. Only when sufficient hydrochloric acid or another source of chloride is present at concentrations approaching 1 M or greater will mercury(II) be found predominantly in the [HgCl

  water-soluble anionic form. Such an excessive concentration of chloride ions would form Cl

  species in the plasma and contribute to analyte signal suppression as a result of space-charge effects. In addition, such high concentrations of chloride may cause problems with rinse-out or stability of other elements such as thallium. Thus, though chloride from 1% HCl is sufficient to maintain long-term mercury stability in solutions, chelation with chloride may not be the most practical way to eliminate mercury's memory effect from a liquid introduction system.  

One approach to eliminating the mercury memory effect is illustrated in a practical application with analysis of mercury in blood in alkaline solution with a chelating agent. The discussion of this application also addresses the analysis of metals in a more complex matrix than water or even urine.  

Blood is a highly proteinaceous matrix containing hydrophobic lipids in cell membranes and in lipid transport proteins. Proteins generally do not tolerate acidic matrices well, specifically blood proteins. Acid causes precipitation and clumping of many blood proteins. Even slightly acidic solutions can have detrimental effects on blood consistency because the isoelectric point (pI) of many proteins, the pH at which the net protein charge is neutral, falls between pH 5 and 6. In this pH range, many proteins are as vulnerable to precipitation as in denaturing acidic solutions. In addition, most of the metals in blood are found in the cells or chelated by cell membrane functional groups. Therefore if the cells settle, or are not homogeneously dispersed, different analytical results could be obtained from the same blood sample. There are successful methods for elemental analysis of blood in acidic diluent, but additional precautions usually need to be made to ensure that nebulizers are not blocked and protein precipitate "strings" do not accumulate on or in the injectors.   

A rugged method for elemental analysis of blood will take into account these aspects of the blood matrix, as well as the metals being analyzed. Freezing the sample until the day of analysis is beneficial because the freeze–thaw cycle ruptures many cell membranes. This allows for equilibration of cytosolic metals with the extracellular matrix, thus decreasing possible differences in sampling precision caused by clumps of cells. Before sampling blood, it should be thoroughly vortexed to disperse the cells. Detergents such as Triton X-100 (Dow Chemical Co.) are often used in blood metals analyses to solubilize and disperse lipid membranes in blood samples (7–9). Detergents also aid in membrane protein solubilization and dispersion. Although many proteins respond poorly to both concentrated base and acidic diluents, dilute base is generally better tolerated than dilute acid. For this reason, tetramethylammonium hydroxide has been used as an effective diluent for blood, usually in conjunction with Triton X-100 detergent (7–9). Unfortunately, many metal ions are not soluble in base. Therefore, a chelating agent is necessary when using a dilute basic diluent to minimize surface losses in the introduction system. For mercury and other soft acid cations, a chelating agent with sulfur ligands such as pyrrolidinecarbodithioic acid ammonium salt (APDC) provides excellent rinse-out while maintaining mercury(II) in the oxidized state (9). The use of a water-soluble compound with a sulfur ligand, thiourea, is shown in Figure 2 as an example of using a soft base anion chelator to rinse-out several micrograms per liter of mercury(II) spiked into blood from a PerkinElmer AS93 autosampler. It is compared to the rinse-out obtained with another commonly used combination (AuCl

 + EDTA). By eliminating the need for a high soft acid cation gold concentration (added in excess to compete with mercury for the reduction or chelation with ligands that form poorly soluble compounds), signal suppression because of space-charge effect in the ICP-MS is decreased, while accomplishing the same rinse characteristics as with the use of excess gold. 

Figure 2: Hg washout: 0.01% thiourea, a soluble sulfur ligand (red), worked as effectively as 100 ppb gold(III) chloride + 0.01% EDTA (blue). The peristaltic pump was equipped with 0.45-Âµm i.d. PVC pump tubing. The liquid flow rate was 600 ÂµL/min.			

Fluoride is not the best chelating ligand for all hard acid cations, and sulfur is not the only chelating ligand that can be used with soft acid cations. However, to offer a rough approximation, one can say that hard acid cations are chelated better with nitrogen, oxygen, or fluorine ligands, and soft acid cations are better chelated with sulfur or chlorine ligands. Figure 3 shows the hard and soft cation or anion characteristic of the most common oxidation states of many elements for analytical purposes (adapted from Wulfsberg [1]). 

Figure 3: Cation acid and base anion characteristics for making appropriate choices of acid or chelating ligand in solutions and diluents. The charges shown in some cases are not a true representation of the form that would be found in solution. They represent only a common oxidation state.			

Not all matrices are liquids. Laser ablation has come a long way as a quantitative technique for many solid samples in the last 10 years, many elemental applications are still better performed by somehow liquefying the sample. Depending on the sample, this may involve alkaline decomposition, acid digestion, or borate fusion.  

Alkaline decomposition is sometimes used to liquefy biological tissues. Carbohydrate, protein, or other biopolymers may be decomposed under alkaline conditions. This is not an oxidative digestion. Carbon is not eliminated as CO

, but solid biological samples may often be dissolved with an alkaline solution such as tetramethylammonium hydroxide as biopolymers are broken down by the base (10). 

Alkaline decomposition rapidly decomposes even solid biological samples such as skin, nails, and hair (10). Unfortunately, it also degrades many organic chelating agents such as EDTA, DTPA, and APDC (11). Thus, when alkaline decomposition is used, analysts should be aware that organic chelating will need to be replaced after the decomposition or should be added after the dissolution of the matrix. It has been reported that free gadolinium (a hard acid cation) in tris hydrochloride buffer alone was lost during liquid chromatography–ICP-MS (LC–ICP-MS) analyses by accumulation in the size-exclusion column unless EDTA or DTPA (chelating agents with hard base anion oxygen ligands) was present. When hair was enzymatically dissolved near neutral pH, the chelating agents for gadolinium were not destroyed as they were during alkaline decomposition of hair (12). 

Microwave digestion is a common technique for bringing organic or inorganic solids into solution for analysis by ICP-MS. There are numerous variations on approaches to the acid mixtures used to accomplish oxidative digestions using a microwave system. There are too many to cite in a brief review, so only one good flexible model will be cited here. Environmental Protection Agency (EPA) Method 3052 is a very effective and flexible method for digestion of a wide variety of matrices. Typically, 100–250 mg of sample is digested at 180 °C for 9–20 min after a 5–10 min ramp. For simple matrices, the digestion is accomplished only with 9 mL of nitric acid. However, the option to add limited amounts of hydrogen peroxide, hydrochloric acid, and hydrofluoric acid depending on the matrix and the analyte requirements make it one of the most flexible methods available. One application, the digestion of smokeless tobacco samples, was accomplished using 9 mL of nitric acid, 0.5 mL of hydrogen peroxide, 0.5 mL of hydrochloric acid, and 0.5 mL of hydrofluoric acid (13). The additions were all within the limits of the method specifications. The principal acid, nitric acid, is an oxidizing acid for the destruction of organic matter, and one in which many metals will be very soluble as discussed earlier. However, it was noted that not all tobacco digestions were complete. Therefore, a small amount of hydrogen peroxide was added to support the complete oxidation of organic matter to CO

. Though colorless solutions indicated complete digestions, a white precipitate was noted; early work showed that lead and arsenic recoveries were low. The addition of 0.5 mL of hydrochloric acid produced a chloride ion to stabilize these analytes. The white precipitate was presumed to be insoluble silicates, because plants accumulate silicates for structural and other physiological purposes. The addition of 0.5 mL of hydrofluoric acid (see precautions described earlier) was sufficient to ensure that silicates were also dissolved in subsequent digestions. However, the addition of 0.75 mL or 1 mL of hydrofluoric acid caused another precipitation. Although calcium and magnesium are readily chelated by fluoride, their fluorides are not as soluble as those of some metals. Apparently, 0.5 mL of hydrofluoric acid was sufficient to dissolve the silicates, but an excess caused additional problems. This illustrates the importance of the optimum quantity of chelating ligand for effecting solubility, as described earlier for thorium. In the former case, the addition of too little HF would not sufficiently aid in the introduction system rinse-out, whereas in the latter case, too much caused precipitation of calcium and magnesium. These amounts had to be empirically determined. 

Before considering more challenging analyses and matrices, a fundamental understanding of the nucleophilicity of potential chelating ligands is often informative. In Table I, the chelation of several cations with EDTA is considered. As stated before, the charges on the cations are intended to represent the oxidation state and not the form in which they will be found in solution. Because the magnesium ion has a relatively low charge and large ionic radius relative to the other ions, its attraction to hydration-sphere water oxygen atoms is comparatively weaker than the attractions to oxygen in the other cases. Even though neither the carboxyl oxygens nor the tertiary amino groups of EDTA are strong nucleophiles, EDTA is readily able to compete with hydration-sphere water molecules for chelation of magnesium. Because of its relatively greater charge and smaller ionic radius, chromium(III) has a much greater attraction to oxygen atoms from water. The displacement of these ligands by EDTA or even by other water molecules has a half life of hours unless heated to increase the rate of coordination by EDTA. Titanium(IV) and silicon, however, are poorly chelated by EDTA. The binding of oxygen atoms by these oxides is very strong.  

Table I: Bond strength between metal and oxygen ligand from water or oxide affects relative facility of chelation by EDTA. Harder base or smaller, more nucleophilic ligands more readily compete with water oxygen or oxide ligands for chelation.			

Titanium(IV) oxide and silicon in silicates can be viewed together as examples of extremely hard acid cations — high oxidation states and very small ionic radii. Together, these characteristics make them challenging targets for dissolution whether they are considered analytes or as difficult matrix components. Because of their high oxidation states and effective nuclear charges, they have a very strong attraction to hard base anion ligands. In the compounds mentioned, the ligands are the oxygens in their three-dimensional structural forms. Water oxygens are not sufficiently nucleophilic to successfully attack the respective electrophiles and break the metal–oxygen bonds. Fluoride, usually from hydrofluoric acid (see precautions described earlier) is the most commonly used nucleophile for attacking the titanium(IV) or silicon ion because it is more strongly chelated as a hard base anion ligand for each cation in a water matrix than the water oxygens. Water would only be hydrolyzed by these hard acid cations anyway. An example of the use of hydrofluoric acid to dissolve silicates was described above in the analysis of tobacco. 

Hydroxide, the conjugate base of water, is also much more strongly nucleophilic than water. Basic conditions have been used to dissolve silicates because hydroxide may successfully attack the silicon-oxygen bonds. Thus, the analyst who cannot use hydrofluoric acid for instrumental or safety reasons has an alternative. However, the analyst must consider the solubility of many cations in base, and the fact that organic chelating agents are labile to concentrated hydroxide. 

An example of an application in which silica and other oxides were analyzed in a titanium(IV) oxide excipient matrix used an approach to aluminum oxide and silica dissolution with basic borate fusion (14). This approach includes heating with potassium hydroxide and borate from boric acid. As discussed, hydroxide is a strong nucleophile, but heating weakens the metal–oxygen bonds and increases the nucleophilic reaction rate. Even though the fusion breaks down the titanium, aluminum, and silicon oxide lattices, the addition of water permits some reformation of insoluble oxides after hydrolysis. Borate, however, is able to disperse between the various oxides at the elevated temperatures and act as an intervening ligand to prevent reformation of a tight lattice, rendering the fused metals more soluble. Many borates also are very soluble. For the analysis of other metals from the matrix, one would still need to dilute and add an appropriate chelating agent. However, this example illustrates the use of appropriate chemistry to effect a solution to a matrix problem. 

The most commonly used techniques for sample analysis with ICP-MS continue to be liquid introduction and flow injection. To utilize these standard techniques, analysts must render the matrix soluble and the analytes mobile in solution whether the sample was originally a solid or a liquid. 

To successfully develop and perform analyses of a broad spectrum of metal analytes in solution from a variety of matrices, a general understanding of the chemical principles involved for dissolution, stability, and rinse-out for matrix components and analytes is helpful. However, it is difficult to know the chemistry of every element and every oxidation state in the periodic table. Thus, adaptation of a general model to understand approaches to applications involving the dissolution, stabilization, and rinse-out of matrix components and analytes will aid in devising approaches for method development. The Pearson hard–soft acid–base principle was used as a general model to approach such problems in this review. 

In general, this approach relies on the use of soluble hard base anion ligands for coordination of hard acid cation metals, and soft base anion ligands for coordination of soft acid cation metals. 

Dissolution of matrix components may be accomplished utilizing techniques such as alkaline decomposition, microwave acid digestion, and borate fusions. 

Once analytes are dissolved and appropriately stabilized in solution, the most challenging part of method development for liquid introduction systems is accomplished.  

This tutorial was adapted from the first half of a course presented at the 7th International Conference on Sector Field Inductively Coupled Plasma Mass Spectrometry in 2008 and the 2012 Winter Conference on Plasma Spectrochemistry on sample preparation for liquid introduction systems. 

The findings and conclusions in this report are those of the author and do not necessarily represent the views of the Centers for Disease Control and Prevention. 

 is a research chemist and tobacco inorganics team lead in the volatile organics and tobacco branch, in the Division of Laboratory Sciences, at the National Center for Environmental Health, U.S. Centers for Disease Control and Prevention, in Atlanta, Georgia. Please direct  correspondence to: 

 (University Science Books, Sausalito, California, 2000), pp. 191–228. 

This tutorial on the chemical principles involved in dissolution, stability, and matrix components will help you successfully analyze a broad spectrum of metal analytes with ICP-MS using liquid introduction or flow injection.

Sample Preparation Problem Solving for Inductively Coupled Plasma-Mass Spectrometry with Liquid Introduction Systems: Solubility, Chelation, and Memory Effects