In the Laboratory
Determination of Pb in Biological Samples by Graphite Furnace Atomic Absorption Spectrophotometry
W
An Exercise in Common Interferences and Fundamental Practices in Trace Element Determination Thomas M. Spudich and Jennifer K. Herrmann School of Science, Penn State Erie, The Behrend College, Erie, PA 16563 Ronald Fietkau Department of Chemistry and Physics, Georgia College and State University, Milledgeville, GA 31061 Grant A. Edwards and David L. McCurdy* Division of Science, Truman State University, Kirksville, MO, 63501; *
[email protected] Trace- and ultra-trace-level elemental determinations (ppb concentrations) are common in environmental and industrial applications (1). Since graphite furnace atomic absorption spectrophotometry (GFAAS) is commonly used for these measurements, it is important for an undergraduate chemistry major to understand the general principles of the technique and have experience with the instrumentation. In addition, an experiment involving GFAAS allows the exploration of additional concepts that affect the measurement’s accuracy. Several experiments involving GFAAS have appeared in this Journal (2–5). Williamson emphasizes the selection of appropriate GFAAS furnace temperatures for the standard additions determination of copper in an instructor-prepared sample (2). Two experiments by Quigley and Vernon use GFAAS to determine elements in seawater or synthetic seawater. One experiment emphasizes analyte preconcentration prior to the standard additions determination of metals in seawater (3). The second uses matrix modification to reduce the NaCl-derived spectral scattering interferences in the GFAAS determination of Mn2+ in seawater (4). Mabury et al. recently published an experiment that used both GFAAS and ICP–AES to determine metals in environmental sediments, incorporating microwave extraction, the use of spiked samples and blanks for method validation (Quality Assurance兾Quality Control), and evaluation of the linear working range of both instruments as an emphasis (5). Though these experiments demonstrate the fundamentals of GFAAS, matrix modification, and calibration by standard additions, they do not illustrate the importance of D2 arc background correction for nonatomic spectral interferences, nor do they investigate the impact of the sample matrix on the formation of free, gaseous atoms within the furnace. Most of these experiments also provide only limited opportunities for students to learn important collateral concepts tied to the preparation of solid biological samples for ppb-level elemental determinations. This experiment uses GFAAS for the determination of trace-level Pb in samples of bovine liver or muscle and is intended for use in an instrumental analysis class or an integrated laboratory experience. The major focus is to demonstrate the impact of physical and spectral interferences 262
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in a trace-level elemental determination and illustrate the most common approaches to reduce errors in accuracy that interferences cause in the determination. If desired, the experiment can also easily introduce (i) preparation of solid samples for elemental analysis, (ii) common approaches for trace-level cleaning, (iii) the importance of the limit of detection (LOD), (iv) the proper use of reagent blanks, and (v) aspects of the validation of a chemical analysis (e.g., quality assurance). Experimental Procedure
Sample Preparation In performing sample decomposition, the student gains a basic understanding of the significant time required for sample preparation, approaches for trace-level cleaning, and an introduction to considerations to assess analyte losses–contamination in the decomposition steps. Moreover, issues that tie the instrument LOD to the method LOD can be emphasized (6). Each concept is important in successful real-world determinations. The sample preparation requires about four hours to complete. If time is limited, the instructor can prepare samples in advance or the decomposition can be easily done in a microwave sample preparation system (7). National Institute of Standards and Technology (NIST) certified bovine muscle (SRM 8414) or liver (SRM 1577 or 1577a) whose Pb content ranges between 300 and 500 ng Pb per g sample are used. NIST-certified materials allow a direct comparison of experimental results to a certified Pb content. They also heighten student awareness about how standard reference materials (SRMs) may be used to help validate chemical analyses. Sample decomposition is performed using trace-metal grade HNO3 (∼70% w兾w) and H2O2 (30% w兾w). The procedure is detailed in the Supplemental Materials.W All glassware and equipment are cleaned via standard approaches used in trace-level elemental determinations (8–10). Reagent blanks are included to assess contaminants added by the reagents and glassware. After decomposition and transfer to clean plastic bottles, the original solid sample is diluted about 1兾40, making the final Pb sample concentration between 3 and 10 ng Pb per g solution. If the GFAAS determinations
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In the Laboratory
List 1. GFAAS Instrument Conditions for Experimental Measurements Wavelength
283.3 nm
Bandpass
0.5 nm
Hollow cathode lamp current Signal measurement mode
Temperature/ °C
Dry
2 30 ng/g 20 µL
Sample injection volume Linear calibration range
Program
Time/ s
10 mA Peak area
Replicate measurements per sample Pb bulk standard concentration
Table 1. GFAAS Furnace Temperature Program for Experimental Measurements
Ash
6–900 pg Pb
D2 arc background correction
Off
Multiple sample injections (an inject, dry, inject sequence prior initiation of temperature program)
2
Atomize
Clean
Elapsed Total Time to End of Program Step /s
Ar Purge Flow Rate/ (L min᎑1)
85
5.0
5
3.0
95
40.0
45
3.0
120
10.0
55
3.0
400
5.0
60
3.0
400
1.0
61
3.0
400
2.0
62
0.0
2100
1.0
63
0.0
2100
2.0
65
0.0
2100
2.0
67
3.0
2500
2.0
69
3.0
cannot be performed immediately, the decomposed samples can be frozen to stabilize them. Frozen samples stored for as long as a week show no measurable changes in experimental results.
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A
Absorbance
0.3
0.2
0.1
0.0
-0.1 63.0
63.5
64.0
64.5
64.0
64.5
Time / s B 0.3
Absorbance
Calibration and Detection Limit Initial Pb determinations are made using aqueous standard calibration with no D2 arc background correction. The intent is to clearly demonstrate the impact of spectral interferences in GFAAS. A Varian SpectrAA-200 with GTA-100 graphite furnace and autosampler (Varian Instruments, Inc. Palo Alto, CA) is used. The furnace parameters and measurement conditions for the Pb determination are shown in List 1 and Table 1. Students prepare a single aqueous calibration standard of 30– 50 ng Pb兾g solution by serial dilution from a 1000 µg兾g stock standard Pb solution. Variable Pb concentrations for calibration are created using dilution of the single stock standard and an appropriately programmed autosampler. All sample and standard measurements are made using no D2 arc background correction. Students then measure the prepared bovine samples, reagent blanks, and at least six repeated measurements of distilled, deionized water. Once this has been completed, they determine the Pb content in their bovine liver or muscle and compare it to the NIST-certified value. They also evaluate the instrument LOD that, by definition, is the concentration of Pb that gives a signal three times the blank noise (6). Doing so allows the students to further compare GFAAS with other methods used for elemental determination. The GFAAS absorbance versus time trace for a bovine muscle sample compared to that of an aqueous standard of similar concentration are shown in Figure 1. The sample signal is typically in excess of six times that of an aqueous standard of similar concentration. Reagent blank results indicate that Pb contamination is not the cause of this high recovery provided the sample preparation is done correctly. These data, in addition to consultation with their instructor prior to pursuit of the next section of the lab, lead the students to con-
0.2
0.1
0.0
-0.1 63.0
63.5
Time / s Figure 1. Pb atomic absorbance versus time traces for (A) a Pb stock standard solution and (B) a decomposed sample of bovine muscle (NIST SRM 8414). Both samples are approximately 4 ng Pb per g solution in concentration.
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In the Laboratory Table 2. Typical Quantitative Results in Determinations of Pb in NIST Certified Bovine Muscle or Liver Samples Exp Pb level (µg/g) ± 90%a
NIST-Certified Pb level Recoveryd (%) (µg/g) ± 2 σb
Approach
Samplec
No correction
Bovine Muscle
2.8 ± 0.5
0.38 ± 0.24
640
D2 arc background correction
Bovine Muscle
0.07 ± 0.04
0.38 ± 0.24
18
Standard addition method
Bovine Muscle (at Truman State)
0.35 ± 0.07
0.38 ± 0.24
92
Bovine Muscle (at Penn State Erie)
0.37 ± 0.09
0.38 ± 0.24
97
Bovine Liver
0.35 ± 0.09
0.35 ± 0.08
100
a
Confidence limit.
b
2σ indicates twice the standard deviation of the NIST-derived data.
c
Bovine muscle samples is NIST SRM 8414 and the bovine liver sample is NIST SRM 1577.
d
Percent recovery is calculated as (experimental Pb)/(NIST-certified Pb) x 100.
sider potential interferences in GFAAS. In particular, students are prompted, during the discussion break, to think about the impact of spectral interference from nonatomic absorbance and the most common approach used to correct these elevated recoveries. Pb recoveries commonly observed for bovine muscle are detailed in Table 2 and compared to the NIST-certified values.
Influence of Background Correction D2 arc background correction is one of the most common approaches used to minimize the magnitude of molecular spectral interferences in GFAAS (11). This section allows students to investigate the influence of background correction on reducing the high recoveries obtained using aqueous standard calibration with no background correction.
Absorbance
0.3
nonatomic absorption (D2 background)
0.2
0.1
0.0
63.0
Pb atomic absorption 63.5
64.0
64.5
Time / s Figure 2. Absorbance versus time traces for the nonatomic absorption (measured using the D2 arc background correction lamp) and Pb atomic absorption (the total cathode absorption minus D2 background absorption) for bovine muscle.
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The experimental conditions of this section are identical to that in List 1, except that the D2 arc background correction lamp is activated and used on all measurements. Students again measure their samples and reagent blanks using conventional aqueous standard calibration and calculate the results upon completion of the measurements. The absorbance trace for the D2 arc background correction lamp showing absorbance of the spectral interference overlaid with that of the corrected Pb absorbance signal for a sample of bovine muscle is shown in Figure 2. Given the large difference between the signals, it is evident that nonatomic spectral interferences are a very significant portion of the overall hollow cathode absorbance and are responsible for the high Pb levels observed using no background correction. However, calculated Pb levels using background corrected measurements are typically less than 20% of the NIST-certified values, as shown in Table 2. Low Pb levels in GFAAS often indicate the presence of matrix components that can influence the formation of free atoms in a real sample. These results direct the perceptive student, in concert with a brief discussion with the instructor, to question other interferences that may be probable in GFAAS, particularly those related to the differences in sample matrix compared to that of the simple aqueous standards.
Physical–Chemical Interferences and Standard Additions A number of approaches are used to minimize the effects of matrix interferences, including matrix modification, extraction, and the use of standard additions (11). This section of the experiment is designed to illustrate the use of the method of standard additions to compensate for the matrix interferences inherent in real samples. The method of standard additions is useful when the analyte signal changes linearly with respect to concentration, and background correction is employed (12). Experimental conditions for standard additions are identical to those in List 1, with exception for the actuation of the D2 arc background corrector lamp. The analysis includes
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In the Laboratory
measurements of bovine muscle or liver samples as well as reagent blanks, using a calibration curve derived by using fixed quantities of sample into which varying quantities of a known concentration of Pb solution were added. Table 2 demonstrates the success of the standard additions calibration combined with background correction. The Pb recoveries fall within the NIST-certified range of values for each sample. Hazards Caution should be exercised when using concentrated nitric acid and 30% hydrogen peroxide, particularly when used as hot oxidants in the wet ashing of the biological samples. Solutions containing Pb should also be properly disposed after use. Summary This laboratory is successful in demonstrating several important fundamentals of GFAAS. It shows students the importance of understanding the sample content for decomposition during a trace-level analysis. It also demonstrates instrumental nuances, the inherent difficulties in furnace measurements owing to the sample matrix, and methods utilized to improve the measurement accuracy in real-world analyses. Finally, it is an excellent review of quantitative analysis skills such as dilutions or back calculations of analytical data. The procedure can take 3–12 laboratory hours to complete depending upon the approach taken by the instructor and the desired emphasis.
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Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Keller, R.; Mermet, J–M.; Otto, M.; Widner, H. M. Analytical Chemistry; Wiley-VCH: New York, 1998. 2. Williamson, M. A. J. Chem. Educ. 1989, 66, 261–263. 3. Quigley, M. N.; Vernon, F. J. J. Chem. Educ. 1996, 73, 671– 675. 4. Quigley, M. N.; Vernon, F. J. J. Chem. Educ. 1996, 73, 980– 981. 5. Mabury, S. A.; Mathers, D.; Ellis, D. A.; Lee, P.; Marsella, A. M.; Douglas, M. J. Chem. Educ. 2000, 77, 1611–1612. 6. Ingle, J. D., Jr.; Crouch, S. R. Spectrochemical Analysis; Prentice-Hall: Englewood Cliffs, NJ, 1988; pp 172–175. 7. Microwave Digestion Applications Manual; CEM Corporation: Matthews, NC, 1994; App. Note: BI-5. 8. Moody, J. R.; Lindstrom, R. M. Anal. Chem. 1977, 49, 2264– 2267. 9. Nalgene Nunc International. Trace Level Cleaning. http:// nalgenelab.nalgenunc.com/techdata/care/trace.asp (accessed Nov 2003). 10. Methods for Chemical Analysis of Water and Wastes; EPA/600/ 4-79-020; Environmental Monitoring an Support Laboratory: Cincinnati, OH, 1983. 11. Robinson, J. W. Atomic Spectroscopy; Marcel Dekker: New York, 1990; pp 106–108. 12. Varma, A. Handbook of Furnace Atomic Absorption Spectroscopy; CRC Press: Boca Raton, FL, 1990; p 25.
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