Spectrophotometric determination of molybdenum in biological materials based on flotation of its 3,5-dinitrocatechol complex associated with rhodamine B

MICROCHEMICAL

JOURNAL

42, 197-205 (1990)

Spectrophotometric Determination of Molybdenum in Biological Materials Based on Flotation of Its 3,5-Dinitrocatechol Complex Associated with Rhodamine B RYSZARD Department

LOBINSKI

of Analytical

Chemistry, 00-664

AND ZYGMUNT

MARCZENKO’

Technical University, Warsaw, Poland

ul. Noakowskiego

3,

Received January 5, 1990; accepted February 1, 1990 A flotation-spectrophotometric method for the determination of molybdenum has been developed and its applicability to the analysis of biological materials has been demonstrated. The method is based on the formation of the ion-associate of the anionic 3,Sdinitrocatechol molybdenum complex with rhodamine B. The compound is separated by flotation, washed, and then dissolved in acetone whereupon the absorbance of the resulting solution is measured. Beer’s law is obeyed up to a molybdenum concentration of 0.3 &ml and the molar absorptivity is 2.1 x 10’ liter . molt r cm ’ at 555 nm. The molar ratios of the components in the floated and washed compound have been determined and the formula [(RB’)2][Mo0,(DNC),2-l has been proposed. The method becomes specific after a preliminary separation of molybdenum by its extraction as the o-benzoinoxime complex from 2 M HCl. The accuracy of the method has been demonstrated by determining molybdenum in three certified reference materials. o IWO Academic press, I~C.

INTRODUCTION

The determination of molybdenum in biological materials is of considerable importance as it is an essential trace element for living organisms (1). Molybdenum is involved in various stages of nitrogen metabolism participating in protein synthesis and in a large number of enzymatic reactions (2). In most plant and animal tissues molybdenum is present at the ppm or sub-ppm levels, at which its determination by electrothermal AAS or neutron activation analysis is accurate but requires lengthy separation procedures and expensive instrumentation (3). Spectrophotometry has for a long time been used for the determination of molybdenum in biomaterials (4, 5). The lack of sufftciently sensitive methods, however, necessitates large samples for analysis (even up to 10 g), which are usually not accessible, the use of which complicates the mineralization and the preliminary separation procedure. The most commonly used spectrophotometric methods, e.g., the thiocyanate one and the dithiol one, are relatively insensitive (E = 1,6 x lo4 and 2.1 x 104, respectively) whereas the sensitivity of many other methods rarely exceeds 1 x lo5 liter . mol-’ cm-’ (4, 6). Flotation-spectrophotometric methods have recently given rise to much interest due to the possibility of a considerable preconcentration of elements in trace amounts and the attainment of high molar absorption coefficients (6-9). These ’ To whom correspondence

should be addressed. 197 0026-265X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

198

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methods use ion-associates of multivalent anionic complexes of metals with basic dyes as cationic partners. Such compounds form hydrophobic particles which are separated by flotation and subsequently dissolved in a polar solvent. The intense color of the resulting solution, due to the presence of several dye cations per atom of metal in the multivalent anion, forms the basis of the spectrophotometric determination. In this paper a new flotation-spectrophotometric method for molybdenum based on the ion-associate of its anionic 3,5-dinitrocatechol complex with rhodamine B is developed. Hitherto, the 3,5dinitrocatechol complex/basic dye system has been restricted to the flotation-spectrophotometric determination of tungsten (ZO), vanadium (11), and niobium (12). Attempts to develop an extraction-spectrophotometric method for molybdenum based on such a system have failed (13). EXPERIMENTAL Apparatus

A Specord UV-VIS recording spectrophotometer and a VSUZP spectrophotometer (Zeiss Jena) with lo-mm matched glass cells were used for absorbance measurements. An ELPO Model 1572 pH meter was used for pH measurements. Reagents Molybdenum(VI) stock standard solution (1 mglml). Molybdenum trioxide (1500 g; JMC Puratronic) was dissolved in 25 ml of 2 M NaOH, slightly acidified with hydrochloric acid, and diluted with water to 1 liter in a standard flask. Working solutions were prepared by appropriate dilution of the stock solution with water. The solution was standardized gravimetrically with 8-hydroxyquinoline . 3,SDinitrocatechol

(DNC)

solution,

8

X

lop4 M (ca. 0.01%) in 25% ethanol.

DNC was synthesized from catechol (Aldrich) as described elsewhere (14) and purified by double recrystallization from ethanol. The purity of the reagent was confirmed by the mp measurement, elemental analysis, and IR and NMR spectroscopy. Rhodamine B (RB) solution, 5 X 10p4 M (ca. 0.025%) in water. A commercial preparation of the chloride salt (POCH Gliwice, Poland) was purified by adding 10 vol of anhydrous diethyl ether to 1 vol of ethanol saturated with the dye salt and washing the crystals obtained with water (6). a-Benzoinoxime solution, 0.02% (w/v) in chloroform. Standard solutions of diverse metals for interference studies were prepared according to (4). Analytical reagent grade chemicals and twice-distilled water were used throughout. Procedure

A sample solution containing not more than 3 pg of molybdenum(V1) was placed in a separating funnel. The DNC solution (0.5 ml) and 1 ml of the RB solution were added. The solution was diluted with water to about 20 ml, whereupon its pH was adjusted to about 1.8 with sulfuric acid or Flotation

of molybdenum.

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199

sodium hydroxide solution. The aqueous phase was shaken with 5 ml of cyclohexane for 45 s. After separation of the liquid phases they were both removed, leaving the precipitate accumulated on the wall of the separating funnel. Spectrophotometric determination of molybdenum. Hexane (5 ml) and 2 ml of water were added to the separating funnel, which was thereafter shaken for 30 s. Then both liquid phases were discarded and the precipitate from the wall was dissolved in acetone. The solution was transferred to a lo-ml standard flask, acidified with 0.1 ml of 0.1 M H,SO,, and diluted to the mark with acetone. The absorbance was measured at 555 nm against the blank prepared in the same way. The calibration graph was obtained by applying the above procedure to solutions containing 0.05, 0.2, 0.5, 1.O, 1.5, 2.0, 2.5, and 3.0 p.g of molybdenum. Analysis of biological materials. Samples dried at 110°C or according to the certificate were weighed and ashed in quartz crucibles at 550°C for 24 h. The white residue was dissolved in 1 M HCl, evaporated to dryness with 1 ml of coned HF, and redissolved in 5 ml of hot 2 M HCl with the addition of 0.5 ml of coned H,PO,. The solution was cooled and transferred to a separating funnel whereupon molybdenum was extracted twice by shaking the funnel for 2 min with two 5-ml portions of the a-benzoinoxime solution. After the combined extracts were washed with 5 ml of 2 M HCl molybdenum was reextracted into the aqueous phase by shaking the organic layer with two 5-ml portions of 5 M HCl. The reextracts were evaporated almost to dryness and the determination of molybdenum was carried on as described above. RESULTS

AND DISCUSSION

Choice of the System

Polyphenols are known to form multicharged anionic complexes with molybdenum which are extractable into an organic solvent in the presence of diphenylguanidine or quaternary ammonium salts (6). The possibility that such complexes form ion-associates with basic dyes and their quantitative flotation were investigated. Catechol, tetrabromocatechol, 3,5-dinitrocatechol, pyrogallol, and tribromopyrogallol were tested as polyphenols. The following basic dyes were examined as cationic partners: rhodamine B and rhodamine 6G (xanthene dyes), brilliant green, malachite green, crystal violet, and methyl violet (triphenylmethane dyes), methylene blue, Nile blue A, Capri blue, Meldola’s blue, Safranine T (azine dyes), and Bindschedler’s green (indamine dye). The flotation of the molybdenum-polyphenol-basic dye ion-associates from various media ranging from pH near 0 to pH 4 (HCl or H,SO,) by several weakly polar solvents was then investigated. Extractable or flotable compounds are formed with diphenols but not with pyrogallols. Ion-associates are formed with all the basic dyes examined apart from Safranine T and Bindschedler’s green. They are either extracted into the organic phase (benzene, toluene, xylene, chloroform, isoamyl acetate) or floated (cyclohexane, hexane, diisopropyl or petroleum ethers). However, only in a few cases does quantitative formation and flotation of the ion-associate take place. The greatest preconcentration factor and the highest sensitivity were obtained in the system Mo(VI)-DNC-rhodamine B-cyclohexane, which was investigated further in detail.

200 Optimization

LOBINSKI

of Chemical

AND

MARCZENKO

Variables

Acidity of the aqueous phase and reagent concentrations were optimized in order to find conditions for the quantitative flotation of the molybdenum compound. The effect of the pH of the aqueous phase on the percentage of molybdenum flotation is shown in Fig. 1. The range of the optimum acidity is rather narrow (one pH unit). Quantitative separation of molybdenum takes place at pH about 1.8. It is apparent from Fig. 2, which shows the dependence of the percentage of the molybdenum flotation on the DNC concentration, that a minimum 1Zfold molar excess of DNC (with respect to 3 p.g of MO) is necessary to obtain the quantitative recovery of molybdenum. This excess corresponds to a DNC concentration of 2 x lop5 it4 in the aqueous phase. The necessary excess of rhodamine B is slightly higher (U-fold with respect to 3 p.g of molybdenum), which corresponds to a dye concentration of 2.5 x lop5 M (Fig. 3). In order to obtain the data in Figs. 1-3, molybdenum was determined both in the precipitate after its mineralization and in the postflotation aqueous phase by the developed flotation-spectrophotometric method. The total molybdenum found was 100 + 3%. Shaking 20 ml of the aqueous phase with a 5-ml portion of cyclohexane for 45 s is necessary for the quantitative flotation of molybdenum. Owing to a good adhesiveness to glass the floated precipitate sticks to the wall of the separating funnel and can be separated quantitatively by careful discarding of both liquid phases. The precipitate readily dissolves in polar organic solvents and molybdenum can be determined in the solution obtained by any instrumental technique, e.g., neutron activation analysis or ETA AAS. However, as the solution is intensely colored and its absorbance is a function of the molybdenum concentration in the aqueous phase, the application of spectrophotometry is of advantage with respect both to the sensitivity and to the simplicity of the method. The measurement of the absorbance of the floated precipitate solution may lead to a direct spectrophotometric determination of molybdenum. Its precision, how-

FIG. 1. The effect of pH on the flotation of the molybdenum compound. [MO] = 1.6 X 10V6 M; [DNC] = 2 x 1O-5 A4; [RB] = 2.5 x 1O-5 M.

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1

1.0 1.5 2.0 2.5 [DNCI xl@M FIG. 2. The effect of DNC concentration on the flotation of the molybdenum compound. [MO] = 1.6 x 1O-6 M; pH 1.8; [RB] = 2.5 x lo-’ M. 0.5

ever, is negatively affected by a relatively high blank value (about 0.20) resulting from the fact that the flotation of the analytically interesting molybdenum compound is accompanied by the flotation of the salts of the dye both with sulfate and with DNC. The latter can, however, be washed out. A good system for washing should ensure complete removal of the salts and at the same time should prevent the decomposition of the molybdenum compound. Washing with water fulfills these requirements but the precipitate loses its adhesiveness to glass and remains dispersed in the aqueous phase, which makes its quantitative separation difftcult. Good adhesiveness of the precipitate to the wall can be attained again only after washing it in a two-phase system: water-nonpolar solvent. Particles of the washed molybdenum compound then creep with the solvent up the wall of the separating funnel and remain there after the solvent evaporates. Out of several nonpolar solvents tested hexane was found to be the best. Shaking of both phases for 30 s ”

100

o-

t 80 t

,,I,, , , , , , 0.5

1.0

1.5 2.0 2.5 3.0 IRBI ~16~ M FIG. 3. The effect of rhodamine B concentration on the flotation of the molybdenum compound. [MO] = 1.6 x 1O-6 M; pH 1.8; [DNC] = 2 x IO-’ M.

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at a hexane to water ratio of 5:2 ensured almost total reduction of the blank value. The loss of molybdenum from the precipitate was about 5%. Dissolution of the floated and washed compound in several polar organic solvents, methanol, ethanol, acetone, and dimethylformamide (DMF), was studied. Each of the solvents tested can be used for dissolving the floated molybdenum compound provided that the solution obtained is then acidified. In the absence of acid rhodamine B is partly (in the case of DMF totally) present in the colorless lactone form. Acidifying the solution shifts the equilibrium toward the formation of the colored zwitterion and monoprotonated forms, which allows the maximum sensitivity of the determination of molybdenum to be obtained. Acetone was chosen. The absorbances of the molybdenum compound solutions in this solvent were constant for at least 24 h. Composition

of the Molybdenum

Compound

The molar ratio of DNC to molybdenum in the separated and washed compound was determined by Bent and French’s logarithmic method (curve 1 in Fig. 4) and is equal to 2:l. This result was confirmed by Job’s method. Comparing the absorbances of acetonic solutions of the precipitates from a known amount of molybdenum with those of the acetonic solutions containing the dye in the same amount lead to the conclusion that the molar ratio RB:Mo is 2: 1.

L+&-

0.8 0.6

-0.2 -5.6

-/

I -5.4/

/

I -4.8

-5.0

A 2 1

LOG C

iaZ --0.4 --0.6 --0.8

FIG. 4. Estimation of the components’ molar ratios by Bent and French’s method. Curve 1, DNC:Mo; curve 2, RB:Mo. C, reagent (DNC or RB) concentration; A,, absorbance corresponding to the reagent concentration C; A,, maximal absorbance.

DETERMINATION

OF MO IN BIOLOGICAL

TABLE 1 Statistical Evaluation of the Results of the Molybdenum

203

MATERIALS

Determination

in Standard Solutions

Molybdenum Added 6%) 1.00 2.00 3.00

Found (lx) 1.02 1.99 2.91

Standard deviation” (l-4 0.038 0.040 0.046

Relative standard deviation (%)

Confidence limits (probability level, 0.95)

3.7 2.0 1.5

1.02 k 0.04 1.99 * 0.04 2.97 2 0.05

” For seven determinations.

This result was confirmed both by Bent and French’s method (curve 2 in Fig. 4) and by that of Job. The determined molar ratios of DNC and RB to molybdenum together with the fact that molybdenum is present in chelates as the MoOZ2+ cation allow for the postulation of the following formula for the separated and washed molybdenum compound:

02N

According to Nazarenko et al. (1.5) molybdenum occurs in solution at pH 1.52.5 in the following forms: MoO,OH+, H,MoO,, and HMoO,- . If any of the above forms alone reacted with DNC and rhodamine B the slope of the curve percentage of flotation = ApH) would be an integer and equal to 3, 2, or 1, respectively. The slope is equal to 2, indicating that the form H,MoO, dominates as the reacting form. The reaction of the formation of the molybdenum compound can then be written as H,MoO,

+ 2H2R + 2RB+ -+ [(RB+),][MoO~R~~-]

+ 2H+

TABLE 2 Statistical Evaluation of the Results of the Molybdenum Determination Molybdenum Added 6%)

Found 6%)

Molybdenum recovery (%I

1.00 2.00 3.00

0.96 1.94 2.92

96.0 97.0 97.3

0 For seven determinations.

+ 2H,O,

in Synthetic Samples

Standard deviation” OLP)

Relative standard deviation (%I

Confidence limits (probability level, 0.95)

0.064 0.076 0.082

6.1 3.9 2.8

0.96 k 0.07 1.94 rt_0.08 2.92 -e 0.08

204

LOBINSKI Results of the Molybdenum Material Bowen’s kale Horse kidney H-8 NBS 1577 bovine liver

AND MARCZENKO

TABLE 3 Determination in Certified Reference Biological Materials Molybdenum hk)

found”

2.21 f 0.20 2.05 f 0.19 3.54 f 0.26

Certified value ak) 2.3 f 0.11 2.1 2 0.32 3.4

a For five determinations.

where H,R denotes DNC. Determination

of Molybdenum

The calibration graph obtained under the optimal conditions outlined under Procedure obeys Beer’s law in the molybdenum concentration range 0.005-0.3 pg/ml. The molar absorptivity is 2.1 x lo5 liter * mol-’ cm-’ at 555 nm and the specific absorptivity (a = e/atomic mass x 1000) is 2.29. The detection limit for molybdenum (estimated as 3 SD of the blank value) is 5 &ml. Thus the proposed method is several times more sensitive than the commonly used thiocyanate and dithiol methods and twice as sensitive as the method based on the ion-pair formation by the Mo-SCNcomplex with rhodamine B in the aqueous solution (16). Good precision and accuracy of the method were demonstrated by the determination of molybdenum in standard solutions at three concentration levels. The results are shown in Table 1. The interference with diverse metal ions was examined. Ti, Zr, and V(V) interfere at any level. Equivalent amounts of Fe, Bi, Sb, Ga, and W as well as Al and Cr(II1) in 5-fold excess can be tolerated. Cu, Zn, Cd, Ni, Mn as well as alkali and alkaline earth metals do not interfere even in lO,OOO-fold mass ratio. To avoid the above interferences the determination of molybdenum in biological samples must be preceded by a preliminary separation step. Specificity of the method is reached after extraction of molybdenum with a chloroform solution of a-benzoinoxime from 2 M HCl and its subsequent reextraction from the organic phase into coned hydrochloric acid. To check the molybdenum recovery in the whole procedure synthetic samples containing molybdenum at three concentration levels in addition to 5 pg each of Nb, Ta, and W, 100 pg each of Zr, Ti, Bi, Ga, In, Sb, Sn, Fe, Cu, Ni, and Cu, and 0.5 g of Ba, Mg, Ca, and Sr were analyzed. The results are shown in Table 2. They indicate that a recovery of molybdenum of about 97% and good precision of the method are obtained. The applicability of the method to the determination of molybdenum in biological materials was demonstrated and the accuracy of the method was verified by the analysis of three certified reference materials. Samples of 200 mg were analyzed. The results were in good agreement with the certified values (Table 3). ACKNOWLEDGMENT This work was supported by Research Program CPBP 01.17.

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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il.

12. 13. 14. 15. 16.

Morrison, G. H. CRC Crit. Rev. Anal. Chem., 1979, 8, 287-320. Burgmayer, S. J. N.; Stiefel, E. I. J. Chem. Educ., 1985, 62, 943-953. Marczenko, Z.; Lobinski, R., submitted for publication. Marczenko, Z. Separation and Specrrophotomerric Determination of Elements, Horwood, Chichester, 1986. Schwedt, G.; Dunemann, L. Z. Anal. Chem., 1983, 315, 297-300. Sandell, E. B.; Onishi, H. Photometric Determination of Traces of Metals. General Aspects, Wiley, New York, 1978. Mizuike, A.; Hiraide, M. Pure Appl. Chem., 1982, 54, 1555-1563. Marczenko, Z. Pure Appl. Chem., 1985, 57, 849-854. Marczenko, Z. CRC Crir. Rev. Anal. Chem., 1981, 11, 195-260. Nazarenko, V. A.; Poluektova, E. N.; Shitareva, G. G. Zh. Anal. Khim., 1973, 28, 1966-1969. Lobinski, R.; Marczenko, Z. Anal. Sci., 1988, 4, 629-635. tobinski, R.; Marczenko, Z. Anal. Chim. Acta, 1989, 226, 281-291. Vinarova, L. I.; Malinka, E. V., Stoyanova, I. V. Zh. Anal. Khim., 1983, 38, 2013-2015. Nazarenko, V. A.; Lebedeva, N. V.; Vinarova, L. I. Zh. Anal. Khim., 1972, 27, 128-133. Nazarenko, V. A.; Antonovich, V. P.; Nevskaya, E. M. Metal Ion Hydrolysis in Dilute Solutions, Atomizdat, Moscow, 1979. [In Russian] Haddad, P. R.; Alexander, P. W.; Smythe, L. E. Talanta, 1975, 22, 6169.