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A rapid spectrophotometric method for determination of zinc in milk
Tulunru. Pergamon Press Ltd
Printed in Great Britain
A RAPID SPECTROPHOTOMETRIC METHOD DETERMINATION OF ZINC IN MILK 0.
S. CHAUHAN,
Department
of Chemistry,
Department
of Chemistry,
B. S.
GARG,
University
R. P.
FOR
SINGH
of Delhi, Delhi 110007. India
ISHWAR SINGH
M.D. University,
Rohtak-124001,
India
(Receioed 29 August 1979. Revised 21 November 1980. Accepted 5 January 1981) Summary-l-(2’,3’-Dihydroxypyridyl-4’-azo)benzene~-sulphonic acid and the corresponding S-chlorosubstituted acid are proposed as sensitive reagents for zinc. Cyanide masking and selective demasking of zinc can be used to deal with the interference of many metal ions, cadmium can be masked with semithiocarbazide, and thiosulphate can be used for masking Hg(II), Pd(I1) and Os(VII1). The molar i. The method has been used for determinating zinc in absorptivities are about 1.3 x lo5 I.mole-‘.cmmilk.
Zinc is essential for growth of micro-organisms, plants and animals, but its essentiality for human beings was recognized only recently. It is now evident that not only nutritional but also conditioned zinc deficiency may cause complications in many diseases. It plays an important role in the metabolism of proteins and nucleic acids and is apparently essential for synthesis of DNA and ribosomal RNA.’ A number of papers have been published giving values for zinc in individual foodstuffs and in diets, but little attention has been paid to zinc in various kinds of milk. The present communication introduces two new water-soluble 1-(2’,3’-dihydroxypyridyL4’heterocyclic azo-dyes, azo)benzene&sulphonic acid (DHP&, I) and 1-(5’-chloro-2’3’-dihydroxypyridyl-4’-azo)benzene-4sulphonic acid (CPD-4S, II) as spectrophotometric reagents for zinc.
EXPERIMENTAL Solutions Stock solutions. Standard zinc solution was prepared by dissolving appropriate amounts of analytical-reagent grade zinc oxide in perchloric acid and standardized complexometrically with EDTA. CPD-4S and DHP4S were synthesized’ by coupling diazotized p-sulphanilic acid with %chloro-2,3-dihydroxypyridine and 2,3-dihydroxypyridine respectively, at -0 The product was kept overnight in a freezer, filtered off and dried. The purity was checked by TLC, and 1 x 10m3M solutions were prepared by dissolving 0.330 and 0.295 g respectively in doubly distilled water and making up to 1 litre. A 1% chloral hydrate solution was prepared for demasking zinc from its cyanide complex. A 5% ascorbic acid solution was prepared and kept in amber glass bottles; it was discarded after two days. Fresh samples of cow, goat, buffalo and camel milk were procured from Bawana village, Delhi. Other samples were collected from government supply milk booths and milkmen in the university area. All other reagents used were of analytical grade. Procedure for zinc To a suitable aliquot (in a lo-ml standard flask) containing 1-5 peg of zinc per ml, add sufficient I or II (Table 1) followed by 2.5-4 ml of 0.02M sodium hydroxide. Dilute to volume and measure the absorbance at i.,,, against a reagent blank. Prepare a calibration curve similarly.
I, DHP-4S, X=H II, CPD4S, X=Cl The sensitivities of the reagents for zinc are superior to those of the well known heterocyclic azo-dye reagents [molar absorptivities (l.mole- l .cm-t): zincPAN,’ 5.8 x lo4 at 550nm; zinc-PAR,3 6.34 x lo4 at 495 nm; zinc-PAC4 2.8 x lo4 at 580 nm; zinc--CPD4S, 1.25 x 10’ at 550 nm; zinc-DHP4S, 1.32 x 10’ at 540 nm]. The proposed method has been made selective for the determination of zinc by use of the differential demasking technique of Platte and Marcy’ and a modified method proposed by Watkins et aL6
RESULTS AND DISCUSSION
Addition of aqueous CPD-4S or DHP-4S solution to a zinc solution gives a water-soluble magenta complex at pH above 10. The solubility of the reagents and their zinc complexes in water at any pH gives an advantage over PAR and PAN.* However, coloured complexes are also formed with Cd(H). Hg(Il), Fe(II), Co(lI), Ni(II), Cu(II), Pd(l1) and Os(Vll1) at room temperature. The interference of 399
SHORT COMMUNICATlONS
400
Table 1. Physico-chemical and analytical characteristics of the complexes
550 1.5-4.0
A,,,, nm pH range (0.02M NaOH, ml per /O ml) Moles of reagent required per mole of metal ion for complete complexation Beer’s law validity range, ppm Optimum concentration range, ppm Composition (M:L) determined by Job’s method
Hg(II), Pd(I1) and Os(VII1) can be removed by masking with thiosulphate, and Cd(I1) can be masked with 0.5% semithiocarbazide solution. Interference by other transition metals can be dealt with by the methods given by Platte and Marcy’ and Watkins et a1.6 Preliminary investigations showed that ascorbic acid or chloral hydrate did not affect the zinc-CPD4S or zinc-DHP-4S complex. Hence 2% potassium cyanide solution is added to form the cyanide complexes of the interfering metals and zinc. and the zinc complex is then prercrentlally destroyed by excess of 1% chloral hydrate solution in presence of 5% ascorbic acid (a few drops), leaving the other stable cyanide complexes in solution. The zinc ions thus released are determined spectrophotometrically with CPD4S or DHP4S. The role of ascorbic acid6 is to reduce Cu(I1) or Fe(III), if any, to Cu(I) or Fe(II), the cyanide complexes of which are more stable towards chloral hydrate. Other base metals either do not form complexes with I or II, or else form very weak complexes or those which decompose on standing or boiling. Various physico-chemical characteristics of the complexes are summarized in Table 1. offoreign ions
E&t
Zinc-DHP4S complex
Zinc-CPD-QS complex
Characteristic
In the analysis of a 0.33~pg/ml zinc solution with CPD-4S or DHP4S, the ions F-, Cl-, Br-, I-, NO;, NO;, SCN-, S,O:-, SO:-, SO:-, SzO;-,
20
,I
,
phate, Al(III), In(III), Sn(Ir), Sb(III), La(III), Ti(IV), Cr(III), Mn(II), Fe(III), UO:+, Mo(VI), W(VI),
25 0.55 0.10.48
W.58 O.W.50
1:2
1:2
Zr(IV), Hf(IV), Th(IV), alkaline-earth and platinummetals [except Os(VII1) and Pd(II)] do not interfere at room temperature when present in up to at least lOO-fold w/w ratio to zinc. When masking agents are used the maximum limits for other ions (in ppm) which do not cause a deviation of more than 2% in the absorbance are: Cd(I1) (10, masked with 0.5% STC); Hg(II), Os(VII1) and Pd(I1) (20, masked with S#or thiourea); Pb(I1) (25, masked with 0.5% STC); Cu(II), Co(II), Ni(I1) and Fe(I1) (10, masked as CN- complexes in presence of chloral hydrate). Determination
of zinc in milk samples
Dry-ashing’ is used in determining the zinc contents of milk. Add 25.0 ml of milk dropwise to a heated crucible to evaporate without frothing. After the moisture has been removed, heat strongly to 45&500”. Cool and add l.Oml of concentrated nitric acid. Evaporate to dryness and ignite again at 45&500” for - 1 hr. Utmost care should be taken to avoid loss by sputtering. Dissolve the resulting white ash in the minimum of dilute nitric acid and make up the volume to 25.0 ml in a standard flask. Pipette 1 or 2 ml of the solution, mask the interfering metals as indicated below and determine zinc by the procedure just given. Recovery
oxalate, thiourea, citrate, tartrate, semithiocarbazide (STCI. diethvldithiocarbamate (DTCI. borate. Dhos~~
540 2.5-5.0
of zinc from
milk samples
The standard milk samples were spiked by adding 1.60 and 3.25 pg of zinc and the total zinc was determined with both reagents. Table 2 reports the recovery data obtained with DHP-4S and confirms the precision of the method. It was noted that the zinc
Table 2. Results and recovery data for zinc, in various milk samples, with DHP-4S as reagent Milk sample cow Buffalo Goat Camel Govt. supply: Mother Dairy Delhi Milk Scheme * In milk sample.
No. of analyses
Zn(I1) found*, pg/lO ml average range
Zn(lI) added pg/lO ml A B
Zn(I1) recovery, 1,:
A
B
8 8 5 3
3.65-4.00 2.9c3.25 3.8W.10 2.5c2.80
3.80 3.10 3.95 2.60
1.60 1.60 1.60 1.60
3.25 3.25 3.25 3.25
96 95 103 102
102.0 99 91 91
10 8
3.8W.20 3.65-4.00
4.05 3.85
1.60 1.60
3.25 3.25
91 96
104 98
SHORT
content of various milk samples collected from booths or of milk kept overnight in galvanized sits was higher than that of fresh samples. This be attributed to contamination with zinc during teurization. etc.
COMMUNICATIONS
milk utenmay pas-
is thankful Acknowledgement-One of^ the authors (O.S.C.) totheUr’ . . __ _ uversrty Grants Commtsston, New Delhi, for the award of a Teacher Fellowship. REFERENCES 1. F. Fernandez-Madrid, A. S. Prashad and D. Oberleas, J. Lab. Clin. Med., 1973, 82, 951.
401
2. W. Burger and H. Elvers, 2. Anal. Chem., 1959, 171, 225. 3. M. Hnilifkova and L. Sommer, Collrrrion ~rech. Chem. Commun., 1961, 2.6, 2189. 4. G. Nakagawa and H. Wada, Nippon Kagaku Zasshi, 1962, s3, 1098. 5. J. A. Platte and V. M. Marcy, Anal. Chem., 1959, 31, 1226. 6. R. Watkins, L. M. Weiner and B. Zak, Microchem. J., 1971, 16, 14. 7. J. P. Gupta, Ph.D. Thesis, Universitv of Delhi. 1977. 8. S. Shibata, in Chelates in Analytica; Chemistry. H. A. Flaschka and A. J. Barnard, Jr. (eds.). Vol. 4. Dekker. New York. 1972. 9. A. A. Schilt and P. 1. Taylor, Anal. Chem., 1970, 42, 220.
Printed in Great Britain
A RAPID SPECTROPHOTOMETRIC METHOD DETERMINATION OF ZINC IN MILK 0.
S. CHAUHAN,
Department
of Chemistry,
Department
of Chemistry,
B. S.
GARG,
University
R. P.
FOR
SINGH
of Delhi, Delhi 110007. India
ISHWAR SINGH
M.D. University,
Rohtak-124001,
India
(Receioed 29 August 1979. Revised 21 November 1980. Accepted 5 January 1981) Summary-l-(2’,3’-Dihydroxypyridyl-4’-azo)benzene~-sulphonic acid and the corresponding S-chlorosubstituted acid are proposed as sensitive reagents for zinc. Cyanide masking and selective demasking of zinc can be used to deal with the interference of many metal ions, cadmium can be masked with semithiocarbazide, and thiosulphate can be used for masking Hg(II), Pd(I1) and Os(VII1). The molar i. The method has been used for determinating zinc in absorptivities are about 1.3 x lo5 I.mole-‘.cmmilk.
Zinc is essential for growth of micro-organisms, plants and animals, but its essentiality for human beings was recognized only recently. It is now evident that not only nutritional but also conditioned zinc deficiency may cause complications in many diseases. It plays an important role in the metabolism of proteins and nucleic acids and is apparently essential for synthesis of DNA and ribosomal RNA.’ A number of papers have been published giving values for zinc in individual foodstuffs and in diets, but little attention has been paid to zinc in various kinds of milk. The present communication introduces two new water-soluble 1-(2’,3’-dihydroxypyridyL4’heterocyclic azo-dyes, azo)benzene&sulphonic acid (DHP&, I) and 1-(5’-chloro-2’3’-dihydroxypyridyl-4’-azo)benzene-4sulphonic acid (CPD-4S, II) as spectrophotometric reagents for zinc.
EXPERIMENTAL Solutions Stock solutions. Standard zinc solution was prepared by dissolving appropriate amounts of analytical-reagent grade zinc oxide in perchloric acid and standardized complexometrically with EDTA. CPD-4S and DHP4S were synthesized’ by coupling diazotized p-sulphanilic acid with %chloro-2,3-dihydroxypyridine and 2,3-dihydroxypyridine respectively, at -0 The product was kept overnight in a freezer, filtered off and dried. The purity was checked by TLC, and 1 x 10m3M solutions were prepared by dissolving 0.330 and 0.295 g respectively in doubly distilled water and making up to 1 litre. A 1% chloral hydrate solution was prepared for demasking zinc from its cyanide complex. A 5% ascorbic acid solution was prepared and kept in amber glass bottles; it was discarded after two days. Fresh samples of cow, goat, buffalo and camel milk were procured from Bawana village, Delhi. Other samples were collected from government supply milk booths and milkmen in the university area. All other reagents used were of analytical grade. Procedure for zinc To a suitable aliquot (in a lo-ml standard flask) containing 1-5 peg of zinc per ml, add sufficient I or II (Table 1) followed by 2.5-4 ml of 0.02M sodium hydroxide. Dilute to volume and measure the absorbance at i.,,, against a reagent blank. Prepare a calibration curve similarly.
I, DHP-4S, X=H II, CPD4S, X=Cl The sensitivities of the reagents for zinc are superior to those of the well known heterocyclic azo-dye reagents [molar absorptivities (l.mole- l .cm-t): zincPAN,’ 5.8 x lo4 at 550nm; zinc-PAR,3 6.34 x lo4 at 495 nm; zinc-PAC4 2.8 x lo4 at 580 nm; zinc--CPD4S, 1.25 x 10’ at 550 nm; zinc-DHP4S, 1.32 x 10’ at 540 nm]. The proposed method has been made selective for the determination of zinc by use of the differential demasking technique of Platte and Marcy’ and a modified method proposed by Watkins et aL6
RESULTS AND DISCUSSION
Addition of aqueous CPD-4S or DHP-4S solution to a zinc solution gives a water-soluble magenta complex at pH above 10. The solubility of the reagents and their zinc complexes in water at any pH gives an advantage over PAR and PAN.* However, coloured complexes are also formed with Cd(H). Hg(Il), Fe(II), Co(lI), Ni(II), Cu(II), Pd(l1) and Os(Vll1) at room temperature. The interference of 399
SHORT COMMUNICATlONS
400
Table 1. Physico-chemical and analytical characteristics of the complexes
550 1.5-4.0
A,,,, nm pH range (0.02M NaOH, ml per /O ml) Moles of reagent required per mole of metal ion for complete complexation Beer’s law validity range, ppm Optimum concentration range, ppm Composition (M:L) determined by Job’s method
Hg(II), Pd(I1) and Os(VII1) can be removed by masking with thiosulphate, and Cd(I1) can be masked with 0.5% semithiocarbazide solution. Interference by other transition metals can be dealt with by the methods given by Platte and Marcy’ and Watkins et a1.6 Preliminary investigations showed that ascorbic acid or chloral hydrate did not affect the zinc-CPD4S or zinc-DHP-4S complex. Hence 2% potassium cyanide solution is added to form the cyanide complexes of the interfering metals and zinc. and the zinc complex is then prercrentlally destroyed by excess of 1% chloral hydrate solution in presence of 5% ascorbic acid (a few drops), leaving the other stable cyanide complexes in solution. The zinc ions thus released are determined spectrophotometrically with CPD4S or DHP4S. The role of ascorbic acid6 is to reduce Cu(I1) or Fe(III), if any, to Cu(I) or Fe(II), the cyanide complexes of which are more stable towards chloral hydrate. Other base metals either do not form complexes with I or II, or else form very weak complexes or those which decompose on standing or boiling. Various physico-chemical characteristics of the complexes are summarized in Table 1. offoreign ions
E&t
Zinc-DHP4S complex
Zinc-CPD-QS complex
Characteristic
In the analysis of a 0.33~pg/ml zinc solution with CPD-4S or DHP4S, the ions F-, Cl-, Br-, I-, NO;, NO;, SCN-, S,O:-, SO:-, SO:-, SzO;-,
20
,I
,
phate, Al(III), In(III), Sn(Ir), Sb(III), La(III), Ti(IV), Cr(III), Mn(II), Fe(III), UO:+, Mo(VI), W(VI),
25 0.55 0.10.48
W.58 O.W.50
1:2
1:2
Zr(IV), Hf(IV), Th(IV), alkaline-earth and platinummetals [except Os(VII1) and Pd(II)] do not interfere at room temperature when present in up to at least lOO-fold w/w ratio to zinc. When masking agents are used the maximum limits for other ions (in ppm) which do not cause a deviation of more than 2% in the absorbance are: Cd(I1) (10, masked with 0.5% STC); Hg(II), Os(VII1) and Pd(I1) (20, masked with S#or thiourea); Pb(I1) (25, masked with 0.5% STC); Cu(II), Co(II), Ni(I1) and Fe(I1) (10, masked as CN- complexes in presence of chloral hydrate). Determination
of zinc in milk samples
Dry-ashing’ is used in determining the zinc contents of milk. Add 25.0 ml of milk dropwise to a heated crucible to evaporate without frothing. After the moisture has been removed, heat strongly to 45&500”. Cool and add l.Oml of concentrated nitric acid. Evaporate to dryness and ignite again at 45&500” for - 1 hr. Utmost care should be taken to avoid loss by sputtering. Dissolve the resulting white ash in the minimum of dilute nitric acid and make up the volume to 25.0 ml in a standard flask. Pipette 1 or 2 ml of the solution, mask the interfering metals as indicated below and determine zinc by the procedure just given. Recovery
oxalate, thiourea, citrate, tartrate, semithiocarbazide (STCI. diethvldithiocarbamate (DTCI. borate. Dhos~~
540 2.5-5.0
of zinc from
milk samples
The standard milk samples were spiked by adding 1.60 and 3.25 pg of zinc and the total zinc was determined with both reagents. Table 2 reports the recovery data obtained with DHP-4S and confirms the precision of the method. It was noted that the zinc
Table 2. Results and recovery data for zinc, in various milk samples, with DHP-4S as reagent Milk sample cow Buffalo Goat Camel Govt. supply: Mother Dairy Delhi Milk Scheme * In milk sample.
No. of analyses
Zn(I1) found*, pg/lO ml average range
Zn(lI) added pg/lO ml A B
Zn(I1) recovery, 1,:
A
B
8 8 5 3
3.65-4.00 2.9c3.25 3.8W.10 2.5c2.80
3.80 3.10 3.95 2.60
1.60 1.60 1.60 1.60
3.25 3.25 3.25 3.25
96 95 103 102
102.0 99 91 91
10 8
3.8W.20 3.65-4.00
4.05 3.85
1.60 1.60
3.25 3.25
91 96
104 98
SHORT
content of various milk samples collected from booths or of milk kept overnight in galvanized sits was higher than that of fresh samples. This be attributed to contamination with zinc during teurization. etc.
COMMUNICATIONS
milk utenmay pas-
is thankful Acknowledgement-One of^ the authors (O.S.C.) totheUr’ . . __ _ uversrty Grants Commtsston, New Delhi, for the award of a Teacher Fellowship. REFERENCES 1. F. Fernandez-Madrid, A. S. Prashad and D. Oberleas, J. Lab. Clin. Med., 1973, 82, 951.
401
2. W. Burger and H. Elvers, 2. Anal. Chem., 1959, 171, 225. 3. M. Hnilifkova and L. Sommer, Collrrrion ~rech. Chem. Commun., 1961, 2.6, 2189. 4. G. Nakagawa and H. Wada, Nippon Kagaku Zasshi, 1962, s3, 1098. 5. J. A. Platte and V. M. Marcy, Anal. Chem., 1959, 31, 1226. 6. R. Watkins, L. M. Weiner and B. Zak, Microchem. J., 1971, 16, 14. 7. J. P. Gupta, Ph.D. Thesis, Universitv of Delhi. 1977. 8. S. Shibata, in Chelates in Analytica; Chemistry. H. A. Flaschka and A. J. Barnard, Jr. (eds.). Vol. 4. Dekker. New York. 1972. 9. A. A. Schilt and P. 1. Taylor, Anal. Chem., 1970, 42, 220.