As many of the transition r l e n i ~ n t s absorb strongly in the ultratiolet ( 6 ) , even sniall amounts present 11-ith terbium or terbium-lanthanon-yttrium mixtures should be removed prior t o tlie spectrophotometric determination. The \later used should be free of copper. Some difficulty in thc 200- to 240-mg range was traced to copper contaniinatioii in the distilled natcr. With a tloulh-beam instrument such a b the C a r ) , there will be coinpcnsation for impurities if the same solrciit water is u s d In both beams. Hoirever, it is brtttsr to use conductivity IT ater, to avoid the difficulty of incrcasrd slit n idth due to the impurity absorption. There are so many ahsorptions beh c e n 210 and 380 mp, and thry are so close together (Table 11), that there arc deviations from B e d s lan if the absorptions are figured onlj- by a general background correction. Compensation for adjacent absorptions gives good correlation n i t h Beer’s la\\ ~ i t h a f c w exceptions. The absorptions a t 3i5 and 281 mp have a lower value of E than expected at high conccntrations of terbium perchloratc; a t low concentrations, the value of e a t 284 ~
ACKNOWLEDGMENT
Table 111.
Data for 263- to 2 6 5 4 , ~ Absorptions E
Pcrcliloratc Solutions 0 610, 0 640 263.2, 264 8 1 010 0 765, 0 781 262,8, 265 0 0 0778 0 133, 0 163 262.4, 264 9 0 0099 Chloride Solutions 1 80, 1 81 265 0 0 279 262.3, 1 1 7 , 1 19 262,8, 264 6 0 0088
nip is larger than the average value a t intermediate concentrations. The absorptions a t 263 to 265 nip do not follow Beer’s law (Table 111) ; they are broader than the remaining absorptions, except a t 219.8 mp, hence probably are not due to f electron transitions in the siniple hydrated terbium ion. Complex ion formation may influence the intcnsity of the absorptions. Table I11 shows that chloride ion, a stronger complexing ligand than perchlorate ion, enhances the absorption more than does perchlorate ion.
The spectrographic analysis: was done by 0. R. Simi. T. W.Newton loaned the second spectrophotometer. LITERATURE CITED
(1) Ahrendt, >I. E., private comniunica-
tion. (2) Banks, C. Y., Klingman, .)I W., A i d . C h h . Acta 15. 356 11950). ( 3 ) Holleck, L., Hartingbr, L’., Angeui. Chenz. 67, 648 (1965). (4) lloeller, T., Moss, F. d. J., J . A m . Chem. Soc. 73, 3149 (1951). ( 5 ) Prandtl, IT.,Scheiner, K Z. anory. u. allgem. Cheni. 220, 105 (1934). (6) liosenbaum, E. J., A N ~ L . CHEM. 26,20 (1954). (7) Stewart, I). C., Argonne Xational Laboratory, Doc. ANL-5624 (October 1956). (8) Steaart, D, C., University of California Radiation Laboratory, Doc. UCRL-182 (Sentember 1938). Kato, Dorothy, ( 9 ) Stewart, I).‘ ,ZAAL.CHEII.30, 104 (1958).
e.,
RECEIVEDfor review May 28, 1957. Akccepted Koveniber 12, 1957. Work done under the auspices of the Atomic Energy Commission.
Colorimetric Method for Determining Dialdehyde Conte nt of Perio date - Oxidize d St a rc h C. S. WISE and C. 1. MEHLTRETTER Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture, Peoria, 111.
b
Micro quantities of periodate oxystarch have been determined colorimetrically. This rapid method i s particularly suited to the precise estimation of low percentages of carbonyl groups in periodate-oxidized starches. A p-nitrophenylhydrazone of oxystarch i s precipitated from aqueous solution, dissolved in ethyl alcohol, and determined spectrophotometrically.
D
an investigation on the industrial applications of periodate ox!-starches, it became necessary to deterniine very sinal1 amounts of periodate oxystarch having rarious extents of oxidation. A marc preciv procrdure than either the sodium borohydride (3) or the alkali consumption ( I ) method was desired which could also be used for the analysis of oxystarchcs of low dialdeh ~ d ccontent. Kcuberg and Strauss (b) showed that dicarbonyl compounds in niicro quantitivs can b(1 determined by convrrting
174
URISG
ANALYTICAL CHEMISTRY
them t o insoluble nitrophenylhydrazones, which in ethanolic sodium hydroxide solution produce a violet color capable of spectrophotometric evaluation. Applying this procedure t o periodate-oxidized starches produced results of low precision because of color instability. Improved precision was obtained by eliminating the ethanolic sodium hydroxide treatment and by measuring direetly the yrllon- color of ethyl alcohol solutions of the p-nitrophenylhydrazones of the 0x1 starches. The absorbance nieasurd a t 445 mg nas as intense as the maximum blue lriolet color obtained with ethanol alkali a t 575 inw and was constant for a t least 3 days. An\- foreign substance present that can form an insoluble p-nitrophenylhydrazone n ill interfcre n ith the method. APPARATUS A N D REAGENTS
Spectrophotonieter, Coleman Junior Xodel G A .
Fritted glass filter funnel, 3.5-cm. inside diameter, medium porosity. Borosilicate glass tubes, 18 X 150 mm., selected for uniforniity in spwtrophotometric measurements. p-Sitrophenylhydrazine (East man). Stock solutions are made by dissolving 0.25 gram (Procedure A) or 0.50 gram (Procedure B) in 15 ml. of glacial acetic acid. C(.lite hltcr aid, analytical grade. PROCEDURE
Procedure A. Analysis of Oxystarches Containing Less Than One Dialdehyde Unit per Hundred Repeating Units. Weigh out oxystarch samples approximately equivalent t o 0.25 nig. of 1007, osystarch. For example. a 25-mg. sample of 1% oxystarch \vas taken and a 250-nig. sample (dry \wight basis) of 0.17, oxystarch was used. After placing the weighed sample in a trst tube, add 20 nil. water and heat in the &am bath for one-half hour. Occasionally agitate the mixt’ure TYith a
tliiii
glass stirring rod left in thc test
tubr .
Dissolve 250 mg. of p-nitrophenylhytlrazine in 15 nil. of glacial acetic acid. T o the tcst tube add 1.5 nil. of the solution (105 moles of reagent per dialdeIi~-tleunit of o y s t a r c h ) . Heat for 1 lionr 11I t h occasional agitation t o cornp k t c the forniation of the deep red p-nitrophcnylhytlrazoii(~. This period of reaction is not critical, brcausc 98% mnuiniuni-hydrazone formation occur5 in 15 minutes and reaches 99.87c rvithiii aii hour. Cool the tub(>.add 0.4 gralii of filtr,r aid. and vacuum filter t h r p-iiitrophc~nylhydrazone onto a fritted glass fuiinc.1. Kash thc rcd precipitatr with tn-o 5-nil. uortions of i% acetic acid, fol~on-ec~ i,y two s-nil. 'portions of Gater. I n washing. first rinsc t h r test tube and tlicii pour the rinsc watcr into the funnP1. Discard the washings and roplacc the receiver with a clean, dry, 500-ml. filter flask. R(~peatedl\-wash tlw t,est tube aiitl funnel with hot 95% ('thy1 alcohol until all of the p-nitroplic~nylhydrazone has becn dissolved :in(l collccted in the rcceiving flask. Transfer the p-nitrophenylliydrazoni. solut,ion to R 25O-ni1. voluniet'ric flask :iiitl dilute to the mark n-ith ethyl al(,oliol. Plac~approxiniatrly 15 nil. of thci solution in a test' tube (matched tuhw should bc uscd) and measur(' the :ihsorhance at a wave lcngth of 445 nip against a blank, made as described, hiit using starch instead of osystarch. Thc blank need not be prepared daily, a s it is stable for a t least 3 days. Procedure B. Analysis of Oxystarches Containing More Than One Dialdehyde Unit per Hundred Repeating Units. Weigh out a sample npprosimately equivalent t o 5 nip. of lOOyo osystarch on a dry basis. For a IO%, oxysbnrch this \rould be 50 m g . To dissolve highly oxidized s:iitiplrs (80 t o 100 dialdehydc units Iwi~ 100 repeating units), i t is atlrisahlc t o place 62 t o 50 nig. in a 200nil. volumetric flask with 180 ml. of tlistillcd water and heat n-ith occasional agitation for 2 t o 3 hours in a s t m m bath a t 90 t o 103" C. Cool, dilute t o 200 ml., and reniore a 201111. aliquot t o obt,ain t h e 5 nig. of osystai,c-li rcquired. arch sample in a test 11. of witer, or use the 20-1111. nliquot, d(~wribec1. Proceed ivith tlir annlysis as tlcscribrd in Procedure -4. Hon-cvrr, in this caw prcpaSc a ~ t o r ksolution of 0.50 gram of p-nitro~ilic~iiylIi~-tlrazillc in 15 nil. of glacial :icc>tir :icid arid usc 1.5 nil. in the Lirialysis. Altcwiatc~ly. 3 nil. of the prcl-iolis stork solution may he used. This qu:mtity of rc3qynt is cquiralent olvs p r dialdchydc nnit of
TIicl (thy1 alcohol solution of p-nitro-
Table
Sample so. 1 2 3 1 5 ii
I.
Analysis of Periodate Oxystarches by Different Methods
Periodate consumed in preparation 0.05 0.10 0.20 1.00
Dialdehvde Units wer 100 Reueatinc! Units Periodate Sodium oxidation of Alkali borohydride Colorimetric osystarch consumption reduction method 0 051 .. , . .. . ,
.. ..
...
..
. .
..
1O h
10h
. . ..
..
..
..
29
98 98 9T
95 95 9T
3
0 102
.. ..
0 198 0 98 2 99 29 2
..
96 5 95 0 100 G
11 100 97 A 50-mg. sample heated in 200 ml. water for 3 hours, and 20-ml. aliquot reacted vith p-nitrophen? lhydrazine. b Used same oxrstarch as in lo", but weighed 5-mg. samples for direct reaction with p-nitrophenylhydrazine. a
phenylhydrazone is rathrr concentrated,
so that a 5-nil. aliquot from the 250ml. volumetric flask is diluted t o 100 nil. for almrhanrc measurements. CALCULATIONS
The color is compared with that produced by a known standard. Absorbance of the ethyl alcohol solutions deviates slightly from Beer's l a y particularly above a value of 0.7. Therefore, for maximum precision the absorbance of the standard and of the unknown should not differ appreciably. Because different dilutions and different n eights are used, it is convenimt to calculate to a basis of absorbance per milligram of oxystarch per millitcr. For esample, 13.96 mg. of 35.3070 o\\-starch (determined by sodium horohydride analysis), when diluted as dcscribed, had an absorbance of 0.461. This equals an absorbance of 165.1 per nig. per ml. Because this saniplc contained 35.30% dialdehyde units. a 1007, ouystarch would give 365.1,0.3530. or an absorbance of 467.7 per ing. per i d . Example. A IO-mg. sample of an unknolj-n ouystarch n as converted t o t h e p-nitrophenvlhydrazone and dissolved in 250 nil of ethyl alcohol. A 5-ml. aliquot of this solution, diluted t o 100 nil. n i t h ethyl alcohol, had an absoibancc of 0.470, IT hich is equivalent t o a n ahsoibance of 235.0 per mg. pel m l . Since a 1007, ouystaich n or:ld h a w an absorbance of -167.7 per mg. pci ml.. thc u n k n o n n is 235.0 467.7. 01 50.257c oxystaich. RESULTS
A comparison is nladc. in Table I of the dialdehyclc coiitc,nts of a n-idc range
of oxystarchcs xs determined by spcetrophotometry and other proeedurm. The results agree within 0.002% for ouystarches of ntremely low carbonyl For periodate oxystarchcs content. having more than 10 dialdcJhyde units per 100 rqicating units, the results obtained are n-ithin 2% of the average of the wlucs found by alternative nnalytical nirthods. Standard deviations w r r deterniined for replicates on oxystarchcs a t three different Icv(~1sof oaidation. Ten separate determinations on 0.1% oxystarch and on 1 % o y starch gave standard deviations of 0.0019 and 0.030%, rvspoctivrly. Eight replicates on a 3% oy.starch gavr a standard dcviation of 0.089%. ACKNOWLEDGMENT
The authors nish t o thank ,John C. Rankin for some of the determinations for dialdchyde content of o\j-qtnrches h\- nicaiis othcr than colorimctry. LITERATURE CITED
(1) Hofreiter, B. T., Alexander, B. H., \I-Olff, I. A , , A X . 4 L . C H E l f , 27, 1930 i19551. (2) Xeuberg, C., Straws, E., Biochevi. 7, 211 (1945).
Arch.
(3) Rankin, J. C., Mehltretter, C. I,., ASAL. CHEX 28, 1012 (1956).
RECEIVEDfor review June 20, 1957. Accepted September 20, 1957, llention of firm names or trade products does not implv that they are endorsed or recommended by the Department of Agriculture over other firms or similar produrts not mentioned.
VOL. 30, NO. 2, FEBRUARY 1958
175
