Sequential Determination of Cd, Cu, Pb,

Co and Ni in Marine Invertebrates by

Zeeman Graphite Furnace Atomic
Absorption Spectroscopy
Application Note

Atomic Absorption

Authors Introduction
Jens Kahle In marine biomonitoring, metal concentrations in different species of aquatic inverte-
brates are used to assess their role in the biogeochemical cycle within aquatic environ-
Birte Clason ments, to evaluate their suitability as monitors of the bio-available environmental supply
Gerd-Peter Zauke or to analyze the internal exposure to potentially toxic substances as a basis for an
Carl von Ossietzky Universität effect monitoring. Many environmental monitoring programmes record results of past
events without understanding the underlying physiological and ecological processes.
Oldenburg There is, however, an increasing demand for prospective approaches to detect potential
FB Biologie (ICBM) human impact on ecosystems, based on a sound understanding of these processes
Postfach 2503 [1,2,3]. Natural and anthropogenic metal inputs influence the bio-available metal supply,
which cannot be detected directly by routine analytical procedures for example, by mea-
D-26111 Oldenburg, Germany suring metal concentrations in the soluble phase. The bio-available fraction can only be
assessed by determining incorporated metal levels in organisms, which is the main goal
in biomonitoring [4,5]. This involves field investigations as well as bioaccumulation
experiments. In recent studies, these aspects have been evaluated in detail by investi-
gating various aquatic invertebrates and habitats such as marine zooplankton from the
Arctic [6,7,8,9] freshwater zooplankton and benthos from lakes [10], benthic inverte-
brates from German coastal waters and estuaries [11,12,13,14] and crustaceans from
the Antarctic marine environment [15]. Zooplankton organisms may contribute to the
transfer of metals to higher trophic levels and have been chosen, amongst others, as
recommended organisms in baseline studies for the marine environment for example,
within the scope of the Arctic Monitoring and Assessment Programme [16].

When determining zooplankton samples from the field or from bioaccumulation experi-
ments, there is normally only a limited amount of biomass available. This typically
requires the use of micro digestion procedures in combination with multi-element deter-
minations. In the past we employed such procedures, involving sequential multi-element
graphite furnace atomic absorption spectroscopy with deuterium background correction,
first using ammonium dihydrogen phosphate as matrix modifier for Cd and Pb [17] and
since some years a palladium nitrate – magnesium nitrate modifier [18,19].
This paper outlines the corresponding optimization of a dards (depending on linearity) prepared from a single bulk
graphite furnace (GFAAS) methodology for the Agilent standard using the auto-mix capabilities of the PSD
SpectrAA-880 Zeeman atomic absorption spectrometer, suit- (Programmable Sample Dispenser). Every ten samples a
able for the determination of the trace metals Cd, Co, Cu, Ni Reslope measurement was run automatically. The Reslope
and Pb in various aquatic invertebrates including zooplankton standard used was either the second or third standard from
and small zoobenthos. The certified reference materials the calibration. The elements were determined in the order of
Tort 2, Lobster hepatopancreas (National Research Council increasing ashing and atomization temperatures. After com-
Canada) and CRM No 278R, Mussel Tissue: Mytilus edulis pletion of one element, a tube clean was applied to ensure an
(Community Bureau of Reference) were used to optimize the appropriate Cal Zero value. Under normal conditions,
methods and assess the precision and validity of the meth- 300–600 firings could be completed before the tube had to be
ods. These certified reference materials were selected as changed. Nitrogen gas of grade 5.0 was used.
these samples are similar in matrix to the various aquatic
invertebrates being determined. The Rinse solution contained 0.002% Triton X -100 and
0.065% HNO3 (suprapure Merck). The Rinse solution was pre-
pared from 2–3 days old bidistilled water and the rinse vessel
Experimental was kept at least half full. The sample dispensing system was
bled before each automatic run to remove any gas bubbles in
Sample Preparation the syringe and capillary. The outside surfaces of the dispens-
The samples of marine organisms are normally freeze-dried ing capillary were frequently wiped with a soft tissue soaked
and then homogenized using a small boron carbide mortar with isopropyl alcohol to prevent adhesive effects due to the
and pestle or a ball mill made of agate. The certified reference matrix modifier used. Sample cups and the Reslope container
materials used in this study did not require this treatment were sealed with slitted lids to minimize evaporation and to
since they have already undergone these procedures. prevent the formation of drops on the capillary during the
determinations. With those lids on the sample cups, careful
Aliquots of about 10 mg dried material were digested for alignment of the dispensing capillary to the centre of the
3 hours at 80 °C with 100 µL HNO3 (65%, suprapure Merck) in sample cups was necessary.
tightly closed 2 mL “safe lock” Eppendorf reaction tubes. The
digests were allowed to cool down slowly and were subse- For the determinations of Cd, Pb, Ni and Cr, palladium and
quently made up to a volume of 2 mL using bidistilled water. magnesium nitrate modifiers were applied [22,23,24]. Both
After appropriate dilution, final sample and standard solutions modifiers must be kept in separate vessels on the PSD to
were adjusted to concentrations of 3.25% HNO3. prevent any chemical effects which may disturb the injec-
tion of samples in the graphite tube. Detailed information
Quality Assurance regarding the methods employed for the various elements
Quality assurance was performed in line with German GLP are listed in Appendices 1–5 as original printouts from the
regulations [20] using the following documented criteria: sta- automatic runs, including signal and calibration graphs.
bility of instrumental recalibration, precision of parallel injec- General instrument parameters are listed in Table 1. They
tions (normally showing a coefficient of variation of 1–5%) were optimized based on the recommended or default
and analytical blanks (also reflecting the digestion proce- instrument parameters provided in the software
dure). Furthermore, precision and validity was evaluated (Version 3.0) for the Agilent SpectrAA 880 Zeeman AAS.
using certified reference materials randomly allocated within
routine determinations. Limits of detection were calculated
as mean blank (eventually set to zero) plus 2.6 times the
standard deviation of a “low value” [21].

Instrument Parameters
Metal determinations were performed using an Agilent
SpectrAA-880 Zeeman atomic absorption spectrometer
equipped with the GTA-110 Zeeman graphite tube atomizer.
All elements were measured in the absorbance and concen-
tration calibration mode using wall atomization with Zeeman
background correction. Calibration graphs were obtained
using the New Rational calibration algorithm with 4–5 stan-

2
Table 1. General Instrument Parameters and Matrix Modifiers Used
Cd Cu Pb Co Ni Cr
PROMT PROMT PROMT PROMT PROMT PROMT
Measurement mode height area height area area height
Wavelength (nm) 228.8 327.4 283.3 242.5 232.0 357.9
Lamp current (mA) 4.0 4.0 10.0 7.0 4.0 7.0
Slit width (mm) 0.5 0.5 0.5 0.2 0.2 0.2
No. of calibration standards 5 5 5 5 4 4
Sample volume (µL) 5 10 20 40 20 10
Modifier 1* (µL) 5 – 5 – 8 5
Modifier 2** (µL) 5 – 5 – 4 5
Calibration range (µg/L) 1–5 5–25 20–150 3–12 2–10 6–25
* Modifier 1: 0.4 mg/mL Pd(NO3)2
** Modifier 2: 2 g/L MgNO3

Statistical Analysis
To assess the accuracy and the reproducibility of the methods,
quality control charts were produced, taking into account daily
measures of independent standard reference samples
(6–7 replicates per day). Furthermore, it was tested whether
the means between days differ significantly. First, the hypothe-
sis of normal distribution was tested using the Lilliefors test
provided in SYSTAT 8.0 for Windows [25]. Since this hypothesis
had not to be rejected in most cases (α = 0.01) further statisti-
cal evaluation was performed using BMDP Dynamic program
7d [26]. First, global null hypotheses (equality of means) were
tested either by classical ANOVA (assuming equality of vari-
ances) or by non-classical Welch Test (not assuming equality
of variances). The adequate procedure was selected after test-
ing equality of variances by Levene Test. Null hypotheses were
rejected at 95% significance level (P < 0.05). Second, hetero-
geneity was analysed in more detail using the
Student-Newman-Keuls Multiple Range Test (α = 0.05). The
robust NK procedure involves an adjusted significance level for
each group of ordered means.

Results
The measured results for the certified reference materials are
listed in Table 2. It is noted there is good agreement between
the measured and certified values. There were no certified
values listed for Co and Ni in the CRM No 278R, Mussel
Tissue: Mytilus edulis (Community Bureau of Reference). Also
included in the Table are the measured limits of detection [21].

The long term stability of the analytical methodology is

demonstrated in the quality control charts (Figures 1–2), which
display the daily measured results for both certified reference
materials. The statistical evaluation of the QA procedure is
summarized in Tables 3–5.

3
Table 2. Measured Results for the Certified Reference Materials
Measured
limits of Tort-2; CRM No 278R
detection lobster hepatopancreas Mussel tissue: Mytilus edulis;
Element (mg/kg) National Research Council Canada Community Bureau of Reference
(dry weight) Measured n Certified value % Recovery Measured n Certified value % Recovery
Cd 0.10 25.7 ± 0.92 45 26.7 ± 0.6 96 0.31 ± 0.01 54 0.348 ± 0.007 90
Cu 3.5 109 ± 4 50 106 ± 10 103 9.1 ± 0.4 53 9.45 ± 0.13 96
Pb 0.32 0.36 ± 0.04 47 0.35 ± 0.13 103 1.8 ± 0.1 51 2.00 ± 0.04 91
Co 0.13 0.55 ± 0.02 49 0.51 ± 0.09 107 0.34 ± 0.01 56 n/a n/a
Ni 0.39 2.30 ± 0.05 49 2.5 ± 0.19 92 0.94 ± 0.04 52 n/a n/a
Values listed are the mean value ± 95% confidence intervals (mg/kg d.w.) regarding the completed data set.
n: number of independent determinations.

Table 3. Statistical Analysis of Daily Measurements of Reference Materials

Tort-2 CRM No 278 R

Measurement Mean ± 95%CI N LIP Grp Measurement Mean ± 95%CI N LIP Grp
Cd 1 22.5 ± 4.1 6 0.182 | Cd 1 0.30 ± 0.03 7 0.249 |
Cd 2 27.2 ± 2.4 6 0.966 | Cd 2 0.30 ± 0.03 7 0.937 |
Cd 3 27.1 ± 2.1 6 1.000 | Cd 3 0.34 ± 0.05 7 0.816 |
Cd 4 25.5 ± 1.4 6 0.632 | Cd 4 0.32 ± 0.02 7 0.068 |
Cd 5 25.2 ± 1.1 6 0.420 | Cd 5 0.34 ± 0.04 7 0.018 |
Cd 6 25.4 ± 1.9 6 0.569 | Cd 6 0.32 ± 0.03 7 0.759 |
Cd 7 26.7 ± 4.3 7 0.801 | Cd 7 0.28 ± 0.04 8 0.378 |
Cu 1 98.0 ± 10.3 5 0.626 | Cu 1 8.7 ± 1.4 6 0.051 |
Cu 2 113 ± 12 5 0.948 | Cu 2 9.3 ± 0.3 6 1.000 |
Cu 3 112 ± 18 5 0.802 | Cu 3 9.1 ± 0.8 6 0.164 |
Cu 4 116 ± 8 6 1.000 | Cu 4 8.7 ± 0.9 6 0.553 |
Cu 5 109 ± 8 6 0.144 | Cu 5 8.9 ± 0.6 6 0.996 |
Cu 6 103 ± 14 6 1.000 | Cu 6 9.3 ± 2.3 6 0.002 |
Cu 7 111 ± 13 5 1.000 | Cu 7 9.6 ± 2.0 5 0.376 |
Pb 1 0.39 ± 0.21 6 0.088 | Pb 1 1.9 ± 0.4 7 0.009 |
Pb 2 0.27 ± 0.03 6 0.096 | Pb 2 2.0 ± 0.3 7 1.000 |
Pb 3 0.40 ± 0.13 6 0.659 | Pb 3 1.9 ± 0.1 7 0.915 |
Pb 4 0.37 ± 0.06 6 0.851 | Pb 4 1.8 ± 0.2 7 0.521 |
Pb 5 0.31 ± 0.11 6 0.282 | Pb 5 1.8 ± 0.1 7 0.494 |
Pb 6 0.33 ± 0.11 7 0.148 | Pb 6 1.5 ± 0.1 6 0.028 |
Pb 7 0.43 ± 0.10 7 0.426 | Pb 7 1.7 ± 0.2 6 1.000 |
Co 1 0.53 ± 0.08 6 1.000 | Co 1 0.36 ± 0.07 7 0.211 |
Co 2 0.56 ± 0.09 6 0.048 | Co 2 0.32 ± 0.04 7 0.827 |
Co 3 0.50 ± 0.02 6 0.801 | Co 3 0.34 ± 0.03 7 1.000 |
Co 4 0.54 ± 0.03 7 0.365 | Co 4 0.33 ± 0.02 7 1.000 |
Co 5 0.52 ± 0.06 7 0.135 | Co 5 0.32 ± 0.05 8 0.876 |
Co 6 0.57 ± 0.08 7 0.102 | Co 6 0.37 ± 0.03 8 1.000 |
Co 7 0.61 ± 0.08 7 0.690 | Co 7 0.35 ± 0.02 8 0.321 |
Ni 1 2.3 ± 0.3 6 0.948 | Ni 1 0.9 ± 0.1 7 0.720 |
Ni 2 2.3 ± 0.1 6 1.000 | Ni 2 1.0 ± 0.1 7 1.000 |
Ni 3 2.3 ± 0.1 6 1.000 | Ni 3 0.9 ± 0.1 7 0.515 |
Ni 4 2.3 ± 0.2 6 0.067 | Ni 4 0.9 ± 0.1 7 1.000 |
Ni 5 2.2 ± 0.1 6 1.000 | Ni 5 1.0 ± 0.2 6 0.021 |
Ni 6 2.4 ± 0.2 7 0.277 | Ni 6 1.0 ± 0.2 7 0.777 |
Ni 7 2.3 ± 0.2 7 0.637 | Ni 7 0.9 ± 0.1 6 0.065 |

Values listed are all shown as mg/kg dry weight.

Notes: 95% CI: 95% confidence intervals; N: number of independent samples; LIP: Lilliefors probabilities (2-tailed), test of normality (α= 0.01); Grp: homogeneous
groups according to the Student-Newman-Keuls multiple range test, means which do not differ significantly (α= 0.05) are marked by vertical bars.

4
Table 4. Test of Global Null Hypothesis for Daily Measurements of the Table 5. Test of Global Null Hypothesis for Daily Measurements of
Reference Material Tort–2 (Lobster Hepatopancreas) Reference Material CRM No 278 R (Mussel Tissue)

Element LS P Global null hypothesis P df Element LS P Global null hypothesis P df

Cd 3.83 0.005 WS 1.63 0.204 6; 16 Cd 1.08 0.388 F 2.11 0.072 6; 43
Cu 1.18 0.343 F 1.99 0.098 6; 31 Cu 2.32 0.055 F 0.40 0.873 6; 34
Pb 1.84 0.119 F 1.45 0.221 6; 37 Pb 1.89 0.107 F 2.24 0.059 6; 40
Co 1.33 0.269 F 1.70 0.147 6; 39 Co 4.51 0.001 WS 1.55 0.212 6; 20
Ni 1.62 0.170 F 0.89 0.511 6; 37 Ni 2.66 0.029 WS 2.25 0.088 6; 17
Notes: LS: Levene statistic; WS: Welch statistic; F: classical ANOVA; P: tail Notes: LS: Levene statistic; WS: Welch statistic; F: classical ANOVA; P: tail
probability (corresponding null hypotheses are rejected when P < 0.05); df: probability (corresponding null hypotheses are rejected when P < 0.05); df:
degrees of freedom (for LS = strata-1; total of determinations-strata). degrees of freedom (for LS = strata-1; total of determinations-strata).

Figure 1. Quality control charts for daily measurements of the reference material Tort-2 (Lobster hepatopancreas, LH; mg/kg dry weight).

5
Figure 2. Quality control charts for daily measurements of the reference material CRM No 278R (Mussel tissue; MT; mg/kg dry weight).

6
Discussion The utilization of slitted lids for the sample cups was also
important in this application, as this helped to reduce the
The graphite furnace methodology presented in this paper evaporation of the digest to a minimum.
yields accurate and long term stable results with a high preci-
sion. This is evident from the quality charts (Figures 1–2) and
the statistical analyses of daily measurements. The global null
Conclusion
hypotheses (equality of means) cannot be rejected in any The method presented in this paper is applicable to the analy-
case (Table 4-5) and consequently, all daily obtained means sis of various marine invertebrates (for example, zooplankton
are belonging to one group according to the or benthos) where often only small amounts of biomass are
Student-Newman-Keuls multiple range test (Table 3). available, especially when bioaccumulation experiments are
Furthermore, the Lilliefors probabilities (> 0.01) indicate that considered.
the hypothesis of normal distribution cannot be rejected for
each daily dataset. The overall recovery rate is high for all ele-
ments determined in both reference materials (Table 2) and References
the limits of detection are sufficiently low to make the
methodology suitable for the analysis of aquatic invertebrates. 1. P. S. Rainbow, 1993, “The significance of trace metal con-
This was likewise the case in previous studies employing centration in marine invertebrates”, in Ecotoxicology of
graphite furnace AAS with deuterium background correction metals in invertebrates. Edited by R. Dallinger, and P. S.
[8]. Somewhat surprising is that the current limits of detection Rainbow. Lewis Publishers, Boca Raton, USA, pp. 4-23.
are not lower than those previously reported despite the fact 2. P. S. Rainbow, 1995, Mar. Pollut. Bull., 31, 55-59.
the instrument used in this study (SpectrAA-880 Zeeman
3. G. P. Zauke, M. Krause, and A. Weber, 1996, Int. Revue
AAS) seems to be more sensitive than the instrument used
Ges. Hydrobiol., 81, 141-160.
before (Agilent AA-975 equipped with the GTA-95 graphite
tube atomizer). However since the method [21] relies on the 4. D. J. H. Phillips, and D. A. Segar, 1986, Mar. Pollut. Bull.,
variability of a “low value”, the micro-digestion procedure will 17, 10-17.
be the limiting factor due to unavoidable inhomogeneities in
5. P. S. Rainbow, and D. J. P. Phillips, 1993, Mar. Pollut.
the samples.
Bull., 26, 593-601.
It is noteworthy to mention that the peak appearance times 6. J. Ritterhoff, and G. P. Zauke, 1997, Polar Biol., 17, 242-250.
largely show good agreement between the calibration stan-
dards and both reference materials for all elements (see 7. J. Ritterhoff, and G. P. Zauke, 1997, Aquat. Toxicol., 40, 63-78.
Appendix 1–5) indicating that there were no severe matrix 8. J. Ritterhoff, and G. P. Zauke, 1997, Sci. Total Environ.,
effects. It was also noted that there was no significant drift 199, 255-270.
observed in the calibration or background signals, As a result,
determinations using the concentration calibration mode are 9. J. Ritterhoff, and G. P. Zauke, 1997, Mar. Pollut. Bull., 34,
preferred to the method of standard additions due to the simpli- 614-621.
fied analyses i.e. reduced analysis time and less material effort. 10. G. P. Zauke, J. Bohlke, R. Zytkowicz, P. Napiorkowski, and
This result is most likely due to the application of chemical A. Gizinski, 1998, Int. Rev. Hydrobiol., 83, 501-526.
modifiers and Zeeman background correction [23,27,28].
11. G. P. Zauke, R. von Lemm, H. G. Meurs, and W. Butte,
In order to obtain as most information as possible from small 1995, Environ. Pollut., 90, 209-219.
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sequences within one automatic run is necessary. This Toxicol., 34, 351-369.
reduces analytical effort and prevents digests from ageing.
However, the concentration ranges of calibration curves, 13. D. Bernds, D. Wübben, and G. P. Zauke, 1998,
sample volumes and volume reduction factors have to be Chemosphere, 37, 2573-2587.
selected carefully. Thus, drying and ashing stages must be 14. B. Clason, and G. P. Zauke, 2000, Can. J. Fish. Aquat. Sci.,
adjusted to the different sample volumes. To prevent any pos- 57, 1410-1422.
sible over range signals occurring, it is strongly recommended
that the concentration range of samples being determined
should be approximately known.

7
15. G. P. Zauke, and G. Petri, 1993, “Metal concentrations in For More Information
Antarctic Crustacea. The problem of background levels”
in Ecotoxicology of metals in invertebrates. Edited by R. For more information on our products and services, visit our
Dallinger, and P. S. Rainbow. Lewis Publishers, Boca Web site at www.agilent.com/chem
Raton, USA, pp. 73-101.
16. AMAP assessment report, 1998, “Arctic pollution
issues”, Arctic Monitoring and Assessment Programme
(AMAP), Oslo, Norway.
17. G. P. Zauke, H. Jacobi, U. Gieseke, G. Sängerlaub,
H. P. Bäumer, and W. Butte, 1986, “Sequentielle
Multielementbestimmung von Schwermetallen in
Brackwasserorganismen” in Fortschritte in der atom-
spektrometrischen Spurenanalytik, Edited by B. Welz.
VCH Verlagsgesellschaft, Weinheim, Germany, 543-560.
18. X. Yin, G. Schlemmer, and B. Welz, 1987, Anal. Chem., 59,
1462-1466.
19. G. P. Zauke, M. Krause, and A. Weber, 1996, Int. Revue
Ges. Hydrobiol., 81, 141-160.
20. “Chemikaliengesetz: Gesetz zum Schutz vor gefährlichen
Stoffen. Anhang 1 (zu § 19a Abs. 1) - Grundsätze der
guten Laborpraxis (GLP)” in Handbuch angewandte
Limnologie, Chapter IX-5, 7. Erg.Lfg. 4/99. Edited by
C. Steinberg, H. Bernhardt, W. Calmano, H. Klapper and
R.-D. Wilken, ecomed, Landsberg am Lech.
21. J. R. Büttner, R. Borth, H. J. Boutwell, P. M. G.
Broughton, and R. C. Bowyer, 1980, J. Clin. Chem. Clin.
Biochem., 18, 78-88.
22. T. M. Rettberg, and L. M. Beach, 1989, J. Anal. At.
Spectrom., 4, 427-431.
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Anal. At. Spectrom., 7, 1257-1271.
24. J. Sneddon, and K. S. Farah, 1994, Spectrosc. Lett., 27,
257-267.
25. L. Wilkinson, Ed., 1998, “SYSTAT 8.0 Statistics”, SPSS
Inc., Chicago, IL., USA, 711-713.
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manual (Version 7.0) Volume 1”, University Press of
California, Berkeley, CA., USA.
27. G. Dulude, 1992, Advances in Atomic Spectroscopy, 1,
125-160.
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8
Appendix. Instrumental Parameters, Calibration Graphs and Selected Signal Graphics
Appendix 1. Method: Ni (Zeeman)
Instrument mode Absorbance
Sampling mode Automix
Calibration mode Concentration
Measurement mode PROMT height
Replicates standard 3
Replicates sample 3
Total volume 37 µL
Sample volume 20 µL
Volume reduction factor 5
Bulk conc 20.00 µg/L
Modifier 1 mode Co inject
Modifier 1 volume 8 µL
Modifier 2 mode Co inject
Modifier 2 volume 4 µL
Furnace parameters
Step Temp (C) Time (s) Flow (L/min) Gas type Read Signal storage
1 100 5.0 3.0 Normal No No
2 115 40.0 3.0 Normal No No
3 130 10.0 3.0 Normal No No
4 800 5.0 3.0 Normal No No
5 800 1.0 3.0 Normal No No
6 800 1.3 0.0 Normal No Yes
7 2700 1.4 0.0 Normal Yes Yes
8 2700 1.2 0.0 Normal Yes Yes
9 2800 1.0 3.0 Normal No Yes
Calibration
Sample id Conc (µg/L) %Prec Mean abs
Cal zero 0.00 m 40.6 0.0017
Standard 1 6.00 m 5.1 0.0532
Standard 2 12.00 m 0.4 0.1048
Standard 3 18.00 m 0.7 0.1520
Standard 4 25.00 m 0.1 0.2094
Curve fit = New rational
r = 1.0000

Figure 4. Signal graphics for standard 2.

Figure 5. Certified reference material CRM 786R (MT).

Figure 3. Calibration graph.

Figure 6. Certified reference material Tort-2 (LH).

9
Appendix 2. Method: Pb (Zeeman)
Instrument mode Absorbance
Sampling mode Automix
Calibration mode Concentration
Measurement mode PROMT height
Replicates standard 3
Replicates sample 3
Total volume 30 µL
Sample volume 20 µL
Volume reduction factor 5
Bulk conc 25.00 µg/L
Modifier 1 mode Co inject
Modifier 1 volume 5 µL
Modifier 2 mode Co inject
Modifier 2 volume 5 µL
Furnace parameters
Step Temp (C) Time (s) Flow (L/min) Gas type Read Signal storage
1 90 10.0 3.0 Normal No No
2 110 45.0 3.0 Normal No No
3 150 5.0 3.0 Normal No No
4 1000 5.0 3.0 Normal No No
5 1000 5.0 3.0 Normal No No
6 1000 1.5 0.0 Normal No Yes
7 2200 0.6 0.0 Normal Yes Yes
8 2200 1.0 0.0 Normal Yes Yes
9 2650 0.3 3.0 Normal No Yes
10 2650 0.8 3.0 Normal No Yes
Calibration
Sample id Conc (µg/L) %Prec Mean abs
Cal zero 0.00 m 14.6 0.0029
Standard 1 5.00 m 2.5 0.0606
Standard 2 10.00 m 2.7 0.1305
Standard 3 15.00 m 0.1 0.1880
Standard 4 20.00 m 1.6 0.2448
Standard 5 25.00 m 1.5 0.3045
Curve fit = New rational
r = 1.0000

Figure 8. Signal graphics for standard 2.

Figure 9. Certified reference material CRM 786R (MT).

Figure 7. Calibration graph.

Figure 10. Certified reference material Tort-2 (LH).

10
Appendix 3. Method: Co (Zeeman)
Instrument mode Absorbance
Sampling mode Autonormal
Calibration mode Concentration
Measurement mode PROMT height
Replicates standard 3
Replicates sample 3
Total volume 40 µL
Sample volume 40 µL
Volume reduction factor 5
Bulk conc 10.00 µg/L
Furnace parameters
Step Temp (C) Time (s) Flow (L/min) Gas type Read Signal storage
1 100 5.0 3.0 Normal No No
2 110 20.0 3.0 Normal No No
3 130 8.0 3.0 Normal No No
4 800 5.0 3.0 Normal No No
5 800 1.5 3.0 Normal No No
6 800 1.2 0.0 Normal No Yes
7 2300 0.9 0.0 Normal Yes Yes
8 2300 1.4 0.0 Normal Yes Yes
9 2500 2.0 3.0 Normal No Yes
Calibration
Sample id Conc (µg/L) %Prec Mean abs
Cal zero 0.00 16.4 0.0066
Standard 1 2.00 1.7 0.0440
Standard 2 4.00 2.6 0.0990
Standard 3 6.00 0.4 0.1497
Standard 4 8.00 2.3 0.2028
Standard 5 10.00 1.0 0.2480
Curve fit = New rational
r = 1.0000

Figure 12. Signal graphics for standard 2.

Figure 13. Certified reference material CRM 786R (MT).

Figure 11. Calibration graph.

Figure 14. Certified reference material Tort-2 (LH).

11
Appendix 4. Method: Cu (Zeeman)
Instrument mode Absorbance
Sampling mode Autonormal
Calibration mode Concentration
Measurement mode PROMT area
Replicates standard 3
Replicates sample 3
Total volume 20 µL
Sample volume 10 µL
Volume reduction factor 5
Bulk conc 100.00 µg/L
Furnace parameters
Step Temp (C) Time (s) Flow (L/min) Gas type Read Signal storage
1 100 5.0 3.0 Normal No No
2 110 35.0 3.0 Normal No No
3 120 10.0 3.0 Normal No No
4 800 5.0 3.0 Normal No No
5 800 5.0 3.0 Normal No No
6 800 1.0 0.0 Normal No Yes
7 2300 1.1 0.0 Normal Yes Yes
8 2300 2.0 0.0 Normal Yes Yes
9 2650 2.5 3.0 Normal No Yes
Calibration
Sample id Conc (µg/L) %Prec Mean abs
Cal zero 0.00 20.5 0.0045
Standard 1 20.00 0.4 0.0655
Standard 2 50.00 0.5 0.1469
Standard 3 80.00 0.3 0.2254
Standard 4 110.00 0.5 0.3027
Standard 5 150.00 0.0 0.3940
Curve fit = New rational
r = 1.0000

Figure 16. Signal graphics for standard 2.

Figure 15. Calibration graph.

Figure 17. Certified reference material CRM 786R (MT).

Figure 18. Certified reference material Tort-2 (LH).

12
Appendix 5. Method: Cd (Zeeman)
Instrument mode Absorbance
Sampling mode Autonormal
Calibration mode Concentration
Measurement mode PROMT height
Replicates standard 3
Replicates sample 3
Total volume 15 µL
Sample volume 5 µL
Volume reduction factor 5
Bulk conc 5.000 µg/L
Modifier 1 mode Co inject
Modifier 1 volume 5 µL
Modifier 2 mode Co inject
Modifier 2 volume 5 µL
Furnace parameters
Step Temp (C) Time (s) Flow (L/min) Gas type Read Signal storage
1 80 5.0 3.0 Normal No No
2 110 25.0 3.0 Normal No No
3 600 5.0 3.0 Normal No No
4 600 7.0 3.0 Normal No No
5 600 1.0 3.0 Normal No Yes
6 1800 0.8 0.0 Normal Yes Yes
7 1800 1.0 0.0 Normal Yes Yes
8 2400 0.3 0.0 Normal No Yes
9 2400 0.8 3.0 Normal No Yes
Calibration
Sample id Conc (µg/L) %Prec Mean abs
Cal zero 0.000 36.6 0.0050
Standard 1 1.000 3.3 0.0609
Standard 2 2.000 1.5 0.1198
Standard 3 3.000 e 1.3 0.1617
Standard 4 4.000 1.3 0.2123
Standard 5 5.000 e 2.5 0.2625
Curve fit = New rational
r = 1.0000

Figure 20. Signal graphics for standard 2.

Figure 21. Certified reference material CRM 786R (MT).

Figure 19. Calibration graph.

Figure 22. Certified reference material Tort-2 (LH).

13
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Printed in the USA
November 1, 2010
AA129