J . Sci. Food Agric. 1986,37,366-372

Comparison of Techniques for Chromium Sesquioxide Analysis in Marker Studies

Julian Lee, Murray T. Fisher and Beatrice Mare

Applied Biochemistry Division, DSIR, Private Bag, Palmerston North, New Zealand

(Manuscript received 15 September 1985)

Titrimetric, atomic absorption and plasma emission spectrometry methods are compared in the estimation of chromium from an alkaline fusion solubilisation of chromium sesquioxide (Cr203) digestibility marker in rat faeces. Considerable differences in the between-batch precision for the methods were noted. The ferrous ammonium sulphate titration showed the least imprecision (0.67% RSD) compared with 2.1% RSD for the inductively coupled plasma emission spectrometry and 4.7% RSD and 5% RSD for the atomic absorption methods of the nitrous oxide-acetylene and air-acetylene flames respectively. The atomic absorption methods were found to be highly sensitive to interferents, chromium oxidation state and changes in flame chemistry with respect to operating conditions.

Keywords: Chromium sesquioxide, analysis, atomic absorption, titration, plasma emission spectrometry, faeces.

1. Introduction

Chromium sesquioxide (Cr203) is often used in nutrition studies as a continuous, non-absorbed, non-toxic, dietary marker. The measured recovery of chromium as Cr203, in faeces in particular, determined by atomic absorption methods has been at variance with determinations carried out by inductively coupled plasma emission spectrometry (i.c.p.e.s.).'

This study was undertaken to investigate possible sources of variation between four available methods, namely atomic absorption spectrometry (air-acetylene and nitrous oxide-acetylene flames), i.c.p.e.s. and a titrimetric method, for the determination of chromium from an alkaline fusion of Cr203.2 In particular the effects of flame stoichiometry, chromium oxidation state and interferences on the shape and response of calibration curves were considered for both air-acetylene and nitrous oxide-acetylene flames in the atomic absorption method. Other workers have shown the close dependence of air-acetylene flame chemistry on numerous interelement and oxidation state effectss7 and to a lesser extent similar effects for the nitrous oxide-acetylene flame also.839 However, the inductively coupled plasma is relatively free from chemical interferences, offering an independent assessment of the flame effects in atomic absorption methods.

2. Methods and material

Four methods of measuring chromium content were tested on alkaline fusions of pure Cr203 and rat faeces containing cr203 at approximately 10 mg g-'.

2.1. Akaline fusion Chromium sesquioxide in 250 mg faecal samples collected from rats was oxidised to Cr(V1) as previously described.'.2 Sufficient concentrated H2S04 (=l ml) was added to the fusion mixture to ensure complete oxidation of Cr(II1) to Cr(V1). The fusion aliquot was made to volume (100

366

Techniques for chromium sesquioxide analysis 361

ml) and contained approximately 25 pg ml-' of Cr(V1). Suitable aliquots of this solution were used and chromium content was measured by the four methods. A dilution of 1 in 10 was made before determination by the spectrometric methods.

Subsamples were ground to pass -40 mesh to reduce the variable results previously observed (M. Fisher, unpublished data) because of the inhomogeneous dispersion of Cr203 in the bulk sample.

2.2. Analysis of chromium 2.2.1. Titration Approximately 10 ml of 1 0 ~ H$04 was added to a 20 ml aliquot of sample solution and titrated with freshly prepared, standardised, ferrous ammonium sulphate as described by Fisher et a1.2

2.2.2. Inductively coupled plasma emission spectrometry Chromium in solution was determined using an Applied Research Laboratories 34000 emission spectrometer A two-point calibration procedure was used (0 and 10 pg ml-' Cr) with cadmium as an internal standard for checks on drift and matrix effects.

2.2.3. Atomic absorption spectrometry A Techtron AA4 atomic absorption spectrometer with an AA6 nebuliser was used with the following instrumental conditions: Cr wavelength, 357.87 nm; slit width, 50 pm; single-element Cr hollow-cathode lamp current, 5 mA; nebuliser uptake rate, 4 ml min-'. Chromium was determined using both an air-acetylene flame (single ridge slot Varian Techtron AA6 burner) and a nitrous oxide-acetylene flame (water-cooled single-slot burner as described by Goguel"). In both flames the optimal light path was centred 5 mm above the burner slot. In the air-acetylene flame a stoichiometric (faint yellow coloration) and a luminous flame were investigated (acetylene flows 5.5 and 6.5 respectively on the rotameter gauge). For the nitrous oxide-acetylene flame the height of the red feather was 10 mm. Air and nitrous oxide gas pressures were held at 1.05 kPa. Calibration curves were obtained for each of the flames from standards containing 0, 0.5, 1.0, 3.0, 5.0, 7.5, 10, 12 and 15 pg ml-' of chromium as Cr20$- in the presence of 1000 pg ml-' Na and 20 pg ml-' A1 as their chlorides.

2.3. Interferences Previous s t ~ d i e s ~ , ' ~ have categorised interelement effects in flames as enhancing (Cu, Al, Mg and Ca), depressive (Na, K, Zn and Sr) and selective (Fe). The effects of silicon on chromium absorption have not been well defined. Multi-element analysis of the sample solution by i.c.p.e.s. showed that aluminium, silicon and sodium were present at sufficiently high concentrations potentially to interfere with the chromium by atomic absorption. Standard increments of each of the interferents were therefore added to aliquots of 10 pg ml-' Cr(II1) and Cr(V1) solutions up to 50 pg ml-' or aluminium and silicon and 5000pg ml-' for sodium (all as their chlorides).

3. Results and discussion 3.1. Precision of methods Tables 1 and 2 summarise the results derived from the same samples using the four methods. All methods gave good precision within batches. The titrimetric method was the only technique which did not show any significant variation between batches in the mean chromium concentrations (pooled SD, 66; NS; P<0.50). The i.c.p.e.s. method gave the next lowest variation between batches, although the batch means (five batches) showed significant variation (pooled SD, 207; P<O.OOl ) . An independent calibration for i.c.p.e.s. was made for each batch of analyses which were run over a 2 month period.

Although the variation within batches for both the atomic absorption techniques was small (approximately 1% RSD), the variation between batches was considerable ( ~ 5 % RSD). In

J. Lee et d.

Table 1. Within batch variation (n=11) in chromium content in rat faeces measured by four methods

i.c.p.c.s. Titration AA (N20-C2H2) AA (air-C,Hz)

Mean (,us g-') 10204 9840 9944 10170 SD 96 44 122 108 % RSD 0.94 0.45 1.23 1.10

Min (pg gi') 9995 9774 9760 9867 Max (pg g-') 10330 9912 10163 10226

TableZ. Totalvariation (within batch + between batch) in chromium content in rat faeces measured by four methods; analyses made over a 2 month period on a single set of alkaline

fusions (pg g-') ~~~ ~~

i.c.p.e.s. Titration AA (NzO-C2Hz) AA (air-C,H2)

n 49 44 88 66 Mean @g g') 10025 9805 9939 10272 SD 207 66 461 513 % RSD 2.1 0.67 4.70 4.99

digestibility studies where chromium measurements are compared within a batch, all methods of analyses described here would give acceptable precision. However, in lengthy studies where between-batch chromium analyses are made, the atomic absorption method may give unaccept- able repeatability. This could lead to erroneous digestibility factors, particularly where absolute recovery is assessed. It is apparent that at least part of the observed imprecision in the atomic

al C

n

n a j i

- -0 2 4 6 8 10 12 14 16

Chromium concentrotion (pg mC')

Figure 1. Calibration curves for Cr(V1) for atomic absorption using air-C,Hz flame: -, luminous flame, solutions freshly made; ----, luminous flame, 1 day old; 0-0, stoichiometric flame, flow 5.5, fresh solution; --, stoichiometric flame, flow 5.5 , run next day. Standards contain 20 p g ml-' A1+1000 pg ml-' Na in 2~ HC1 ( Y indicates actual absorbance readings; SD=0.0025 a.u.).

Techniques for chromium sesquioxide analysis 369

0.15-

O . I 0 -

0.05

0 so0

absorption methods is derived from using a large dilution multiplication factor for the dilution (4000). The large dilution is necessary to bring the chromium concentration of the samples into a range that is least affected by interferences and calibration anomalies (Figure 1). Dietary levels of Cr203 are concentrated approximately five times in the faeces as a result of passage through the gut. Lower levels of Cr203 in the diet than those used here result in mixing problems. It is therefore not possible to obtain significantly lower levels of Cr203 in the faeces and hence avoid large dilutions. There were no significant differences in the titration results when either 25 or 2.5 pg ml-' of Cr(V1) was titrated, although the higher level gave a clearer end-point.

Recoveries of chromium from both Specpure Cr203 and Analar Cr203 alone using the spectrometric methods gave good recoveries (i.c.p.e.s., 99.0+0.1%; air-acetylene, 99.2+7%; nitrous oxide-acetylene, 100.9f2.4%). Recoveries using the titration method in earlier work2 gave a mean value of 101.02+0.3%.

3.2. Calibration curves and flame appearance In both the air-acetylene and the nitrous oxide-acetylene flames, conditions were optimised not solely for maximum sensitivity but also for the shape of the calibration curves in response to flame stoichiometry and the presence of interferents (Al, Si and Na).

Characteristic calibration curves for Cr(V1) standard solutions containing 1000 pg ml-' Na and 20 pg ml-' A1 are shown in Figures 1 and 2 for the air-acetylene flame and the nitrous oxide-acetylene flame respectively. These curves were consistently obtained over a 3 month period using several sets of standards. The crosses represent observed absorbance readings (SD, 0.0025 a.u.). All curves were fitted using a cubic spline. Because of the characteristic shape of the curves, the fitting of a linear regression line to the points was inappropriate (Figures 1 and 2 ) . The luminous air-acetylene flame (flow rate, 6.5) although providing better sensitivity gave a convoluted curve which was also more variable than the curves obtained for the leaner

A/ p' 1% -

2 ' I I I I I I I I I 2

0.45 0.501 0.35 O a 4 O I

0-20

8

n a

/* /

370 J. Lee et al.

air-acetylene flame (flow rate, 5.5). These effects have also been observed by Thompson.' The nitrous oxide-acetylene flame (Figure 2) provided a more linear calibration curve and was less sensitive to differences in oxidation state.

The plasma does not suff.er from differences in the rate of atom formation as observed in the air-acetylene flame in particular3s4 and gives a linear calibration curve over a wide concentration range. The emission response from the Cr(I1) 267.72 nm emission line was observed to be independent of oxidation state.

3.3. Interferences The alkaline fusion method2 using disposable porcelain crucibles resulted in approximately 20-40 pg ml-' Al, 5-10 pg ml-' Si and 500 pg ml-' Na in the final analysis solution. These elements are derived from a combination of the indigenous content of the faeces and some leaching from the porcelain crucibles and the sodium peroxide flux. At these levels no interferences from aluminium, silicon or sodium are observed in the inductively coupled plasma or in the titration.' However, aluminium, silicon and to a lesser extent sodium reportedly cause interferences in the air-acetylene flame, particularly in acetylene-rich flame^.^^'^^^^

The effect of increasing concentrations of aluminium and silicon in the presence of 1 mg 8-l Na in 2~ HCI on the chromium absorbance signal are shown in Figures 3 and 4. The presence of aluminium at low levels (4 pg ml-l) was sufficient to enhance the absorbance signal from 10 pg ml-' of chromium markedly. The enhancement was greatest in a luminous air-acetylene flame and least in the nitrous oxide-acetylene flame. The effect of aluminium was greater on Cr(V1) solutions than on Cr(1II) solutions. In the leaner air-acetylene flame and in the nitrous oxide-acetylene flame the plateau region is relatively constant. This enables the addition of aluminium to the standards to match that in the sample. A similar enhancement of titanium spectrometric signals in the nitrous oxide-acetylene flame by aluminium has been observed by Seymour and Boss13 and showed a possible stoichiometric reaction.

-* ,*---------- - - - - - _ _ _ '.*' \ \

0.45 *-.. * _ _ _ _ _ _ - - - - - - *

*-*--*-* *

1 1 I I I I 10 20 30 40 5 0 60

Aluminium concentration (pg ml- ' )

Figure 3. Effect of aluminium on chromium absorbance in the presence of loo0 pg ml-' Na in 2~ HCI: -, Cr(VI), NZ@CZHz, red plume 10 mm; --, Cr(III), air-C,H,, flow 5.5; *-*, Cr(VI), air-CzHz, flow 5.5; ---, Cr(III), air-CzHz, luminous flame; -----, Cr(VI), air-QH,, luminous flame; height above burner slot, 5 mm, nebuliser uptake rate, 4 ml min-'; Cr concentration, 1Opg ml-'.

Techniques for chromium sesquioxide analysis 371

Oe40 r

k-*. .

I Silica concentratlon (pg rnl- )

Figure 4. Effect of silica on chromium absorbance in the presence of loo0 y g ml-' Na in 2M HCI: -, Cr(VI), N,O-&H,, red plume 10 mm; -----, Cr(III), NzO-CzH2, red plume 10 mm; ---, Cr(VI), air-GHz, luminous flame; .--., Cr(II1) and Cr(VI), air-GHZ. flow 5 .5 . Other parameters as in Figure 3.

In the non-luminous air-acetylene flame, silica produced a similar enhancement of absorbance for both Cr(V1) and Cr(II1) solutions (Figure 4). In the luminous flame the effect was less constant with a suppression at low silicon levels followed by an enhancement of the spectrometric signal with further additions of silica. The effect of silica in the nitrous oxide-acetylene flame using the flame conditions described in this study was minimal.

The variable response of the chromium spectrometric signal depending on the oxidation state, flame stoichiometry and the presence of interferents has been discussed e l ~ e w h e r e . ~ ~ ~ ~ ~ ' ~ However, in the context of this applied work it is obvious that proper selection of flame stoichiometry, complete oxidation of Cr(II1) to Cr(V1) and the matching of interferents in standards and samples are necessary to obtain consistent results from Cr203 digests analysed by flame atomic absorption.

4. Conclusions

This comparative study showed that obtaining good precision in the measurement of chromium in Cr203 in biological samples is not simple.

Although excellent recoveries and precision were obtained in the fusion dissolution itself,2 variability in measurement for each of the techniques increased as susceptibility to interferences for each of the respective methods increased. The variability observed in the atomic absorption spectrometric measurements arose from a combination of factors: the high dilution factor needed, the presence of potential interference~,'-~,'~ changes in oxidation state3j5 and the inability to reproduce optimised instrumental operating conditions exactly. In particular consistent settings of gas flows and burner position for control of flame stoichiometry are

372 J. Lee et al.

important. Of the two atomic absorption methods the nitrous oxide-acetylene flame gave the more consistent calibration curves and freedom from interferences.

Chromium in solution must be oxidised to Cr(V1) for the atomic absorption methods (and obviously the titration technique) but this is not a prerequisite for the i.c.p.e.s. determination. In the latter method, however, changes in sulphuric acid concentration result in nebulisation variation due to viscosity changes. However, when digests are acidified with HCI, reduction of Cr(V1) to Cr(II1) occurs slowly with time in samples and standards alike. For this reason it was important to analyse fresh sample and standard solutions.

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spectrometry. Anal. Chem. 1984, 56,2702-2708. 9. Marks, J . Y.; Welcher, G . G. Inter-element interferences in atomic absorption analyses with the nitrous

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performance in the analysis of biological materials. A.B.D. Tech. Rep. No. 3, DSIR, New Zealand, 1981, pp. 1-40. 11. Goguel, R. An improved nitrous oxide burner for atomic absorption spectroscopy. N.Z.J.Sci. 1970, 13, 603-609. 12. Yanagisawa, M.; Suzuki, M.; Takeuchi, T. Cationic interferences in the atomic absorption spectrophotometry of

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