chemical attack of the perchlorate I mixture of radical on the solvent. ; products probably results, in view of the observed fact that a fraction of the perchlorate is reduced all the way to chloride. The g-factor and coupling constant reported by 1Iaki and Geske for perchlorate radical agree with those since reported by Cole (3) for chlorine dioxide and the liftetinie of the observed radical, mea,wred in seconds, seems longer than one might espect for perchlorate free radical. Formation of chloride ion esplains the effectiveness with n-hich anodization in acetonitrile .solutions of perchlorates cleans a platinum electrode, since some dissolution of the platinum can be expected. This phenomenon \vas reported by Hoh. llcEwen, and Kleinberg (?) and has been used to advantage by the author in obtaining reproducible electrode surface.. Possibly the net anode reaction in the presence of triethylamine is that proposed by Ptreuli ur ljy Billon. if the mechanism is the follon-ing :
-
(C2H5)aS (,C.H,;JT.-
t
e-
(4)
LITERATURE CITED
(1) Billon, J. P., J . Electround. Chem. 1, 486 (1960).
( 2 ) Billon, J. P., Bull. Soc. Chzrn. Fiance
Cyanomethyl radicals, if formed, could couple head-to-head to form succinonitrile or head-to-tail ( 5 ) to form LVcyanomethylketenimine. Hydroxyl radicals could couple to form hydrogen peroxide and be further oxidized to oxygen. The detailed reaction is currently being studied in this laboratory. A full description of the experiments mentioned in this communication will be published later. Since the s u b m i 4 o n of this communication, a study of amine oxidation a t platinum anode. in dimethylsulfoxide has been published by Dapo and N a n n (4), reporting a reaction sequence which parallel. reaction- 4 and 5 . This reaction sequence can account for Streuli and Hanselman's succr\sful use of the glass electrode as end point indicator and mas suggested by reT-erse-current chronopotentiometric data obtained in this laboratory.
1962, 863. ( 3 ) Cole, T.. Proc. S u t l . A c a d . Sci. C . S. 46, 506 (lb60). (4) Dapo, R. F., Nann, C. K., AKAL. CHEM.3 5 , 6 i i (1963). ( , 5 ) Eberson, L., J . Org. ('hem. 27, 2329 (1962). (6) Hanselman, R . B., Streuli, C. A., ASAL.CHEM.28, 916 (1956). ( 7 ) Hoh, 0.L. IC., McEwen, W. E., Iileinberg, J., J . *1nr. ('hem. Soc. 83, 3949 11961i. ( 8 ) llaki, A . H., Ckske, I). H., J . C'hem. P h y s . 30, 1356 [ 1059). (9) Schmidt, H., Xoack, J., Z. Anorg. d l l g e m . ( ' h e m . 296, 262 (195h). ( l o ) Streuli, C. A , , hs.4~. CHEXI.28, 130 (1956). (11) van Bers, H. C., Feldineyer, J. H., 17eisIciy. Lan(1bouu.A O n d e r z c r k . Rykslandboiiu.p,oejsta 34, 8.3 i 1929 1 . Harvard 1-niversity Cambridge, Mass.
C. 1).R P ~ E L L
RECEIVEDfor revim April 19, 1963. lccepted 3Iay 29, 1963. Work supported by the Killiam F. .\lilton Fund of Harvard I-niversity.
Determination of Nitrate in Water by Ultraviolet Spectrophotometry SIR: rl h. e qtrong ab-oriition of the nitrate ion near 203 nip ( 2 ) ha.; been used for determination or' nitrate in natural water- ( 3 .4 1 . In tlieie methods, the absorbance of the ..ample is measured a t 210 or 220 n i w where nitrate absorbs strongly, and 31.0 a t 275 mp where its absorption i- negligible. By subtracting a multiple of the absorbance a t 2i5 nip, a- a correction for organic matter, from that a t tlie .-liorter wavelength, t,he ab5orption due to nitrate is estimated. This method or' correction i- justifiable if the nonnitratr aliiorption a t 210 or 220 nip is small, h i t thi. i.. not' so for some natural water. or for -ea water, the absorbance of which iiicrea-es st'eeply a t wavelengths shorter than 2% nip (I). The addition of a n equal volume of concentrated sulfuric acid to a solution containing nitrate, wlien chloride is also present. causes 9 change in the nitrate absorption spectrum the niasimum being shifted to around 230 nip. The determination of nitrate liy nieasurement at, this n-ai-elength is ea.ier because the absorption of other -uhstances is a good deal le,ritegive almost I n each of two 150- X 20-mm. identical . C that it i. ni :ro.yl chloride, U-ATERS. Re-Lilt.; for some samples PH~-SICAL EFFI:CT. I n hydrochloric forriicd according to the equation- : acid the wivclength of niaximum abobtained loc.:~lly: without and with knoivn addition. of nitrate. are given sorbance i-: 227 m p , and is unaffected by HSOj 3HC'l = SOC1 f Cl? 2H2O in Table 11. .?ipreenient with a phenoltemimature hetn-een 0" and 40' C. In d f o n i c acid llidl(Jd : 5 ) ic .ati..fact,or\-. sea n-ater it is 233 nip at'0' C. decreasing or to 230 mp a t 40" C. +\t 230 mp, the HSO: f HCl = SOC1 H?O ahsorbance ha- a temperature coefficient of approsimatelJ- - 1% per and that t,lie d f u r i c acid, by removing Table I. Interference with Ultraviolet 10"-C. ri-e in tcmperaturc>. ten- ,5, displace.; the Measurements reaction to the right. :Some confirniaInterference. The method cannot he used for sanipli~sIiaT-ing very high tioii ~ v a - obtained -1, (hstracting holu-4bsoi-bance, tioil3 prepared froni ni.:rate or nitrite ultraviolet alxsorption. Table I gives Cmcn., 230 mp, with carl)nn tetrachloride. and cxaminabsorbances of a feiv substances to Substanw p.p.m. 1 cm. ing thc estract for ehlcrint~. Chlorine indicate the levels a t which inter10 0.6 ference mal- be espcct,etl. Reducing wa- detected (by its ai)-.orlJancr a t 320 10 0.0s nip) onl!. in the nitrate wlution.. substance; are olivioiwly undwirable , 10 n I,; Seitlwr the chlori~knor the d f u r i c but the method n-ill tolerate the Hg-? 10 0.22 Trimethylaniiiir 10 0.16 ence of i o m e organic matter. acid cctncentration- a w ciitical. ChloHumic acids 10 0.11 ride needs to 1w w l l ill c ~ x w of~ , the ~ as indicated abo~.e. -1 trial with 100 Fulvic :icicle 10 0.22 p.p.ni. of g1uco.r h x v e d that about theoreticd aniount. and if it, i- le.*s than Glucose 100 0.15 half the nitrate way reduced on heating about' 0.023.U iii the fir a1 d u t i o n the Glvc.ollic ai.id 100 n.19 G1-c.ine 100 0 0s a t 100" c'. for 30 minute-. S o measfull shift of wavelength i, not ob-erved. Formic. ~ ( i c l 1000 0 5 l'hc -iiliuric arid conteiitration muit urable reduction occurred on cooling ..icetic arid 1000 0 2 quickly after addition of sulfuric acid. rsccwl :ETc I)?. volume; above thiChloroform 1000 0 2 Sitrite i- returned a-' nitrate. arid must levcl tliere is little change either in the SH4C1 1000 0s lie corrected for. if Ire-ent. \vaix,length of niasiniuni ali..or1tt,ion or i n al,-orl)anc~e for a givt n filial conctentration oi iiitratr. !!-it11 ~ r n-atcr, a lesthan 4 5 5 s. v . dfiiric. acid may cauw Table II. Tests with Fresh W a t e r 1JrwilJitatiori of cdciuru sulfate. I t i. convcnient in all case? i.0 U-P 50% hy Sitrwte volume. To destroy nit rat, chloacid 1 6 ) method ride. frrroiii sulfate,. .sulfur diosidc>. Plymouth city water supply 0 0.23 0.22 sodium nzitlc, hydriodic : i d . hydros~-l1 .00 1.% 1.23 amine. d f a n i i c acid i n r l li!-drszine i J . 40 River Tavy, Devonshire 0 0.35 1.4CJ 1.42 1 .OO zulfate were tried. 0111~the 1a.t two w r c ' rffrvAive and without apiireciahle Polluted stream' 0 14.5 14.9 ultraviolet absorption. Eit,her needed 10.0 24 . 0 24.3 to he added before the iiilfuric arid. If .4quariuqa t r o p i d fish 0 32 34 the!. were added afterward, t o tlie cold 50 h *5 83 wlution-, reduction of n,trate ws. onlJa Diluted for analysis. Iinrtisl.
+
+
-+
VOL. 35, NO. 9, AUGUST 1963
1293
LITERATURE CITED
Table 111.
Tests with Sea W a t e r
English Channel, 9/24/62 Kitrate Found, pg. atom S,' liter Hydrazine Sample reduction T.V. no. Added (6) method 2 5 1 9 2 0 11 5 11 7 10 4 0 2 2 2 6 10 13 3 12 8 6 0 3 5 4 3 8
10 0 10
14 4
31 14 0
14 3 3 9 13 6
SEA ~I'ATER. Results for samples from the English Channel with salinity approximately 35°/00 are given in Table 111. S o completely satisfactory alter-
native method for nitrat'e in sea water is a t present available; the Tvidely used reduction m-ith hydrazine to nitrite (6) !vas employed. Since it gave less consistent results than the ultraviolet method, the agreement between the methods can hardly be expected to be very close. Reproducibility. I n the range 0.03 to 3 p.p.m. of SOa-S, (1-cm. cuvette) the standard deviation was =0.05. I n the range 0.25 to 25 pg. atom of K03-IK per liter (9-em. curette), the 0.4. standard deviation was
*
Kote: The sulfuric acid solutions on which absorbance meawwiients are made are viscous: highly refractil-e. and saturated with hydrochloric acid. Care must be taken. in transfer to cur-ett'es. to avoid "schlieren" clue to imperfect mixing, and bime may be niwled to allonsmall bubbles to clear.
(1) .%rmstrong, F. -4.J., Boalch, G. T., J . Slorine Bzol. dssoc. U.K. 41, 591-7
(1961).
( 2 ) Bastin, R., Keberling, T., Palilla, F., AN.41.. CHEM. 29, 1795-7 (1957). ( 3 ) Goldman, E., Jacobs, R . J , J . - 4 ~ 1 . W a t e i W o r k s Assoc. 53,187-91 (1961). 1 3 ) HoathPr. R. C . Rackham. R . F.. d n ~ ( u s84, t 548-51 (1959)
( 5 ) Scott, IT I T , "Standard Method8 of Chemical .%nalvsis,'' 5th ed., Vol. 2 , I) 2076, Tan Sostrand, Nen- Yorli, 1944.
(6) Strickland, J. D. H., Parsons. T. R., Bull. Fisheries Res. Board Can. 125, 61 11960).
F. A . J. ARMSTROXG
3Iarine Biologicla1 ;Issociation of the United Kingdom Citadel Hill Plymouth, England RECEIT E D fc!r revim December 26, 1962. .-lccepted IIal-13. 1963
2-Methylalkanoic Acids as Internal Standards in Gas Liquid Chromatographic Assay of Fatty Acids SIR: Gas liquid chromatography (GLC) has proved to be a rapid technique to determine the relative composition of fatty acid components. as well as their radioisotope levels, in lipid mixtures often weighing lesq than 100 pg. It is frequently of interest. however, to calculate absolute levels of fatty acids in mixtures, but standard titrimetric or gravimetric procedures are difficult Then assaying microgram quantities. Through the addition of a known amount of an internal standard to mixtures of fatty acid e*ters prior to GLC, we have found it relatively simple to determine microgram levels of fatty acid components. The advantages of an internal standard obviate volatilization of solvent or loss of sample a t the injection ports, both of nhich are frequent critical events when using microsyringes to inject samples quantitatively. Thus. calibration of recorder response to known amounts of fatty acid esters iniected is not necessary. I n the course of studies of the properties and metabolism of 2-alkylalkanoic esters synthesized in our laboratory (f), we have found that the nonnaturally occurring 2-methyl homologs of dc decanoic, tetradecanoic, and hexadecanoic acid have retention times that are distinct from common, naturally occurring fatty acids when assayed by GLC with 17% polyethylene glycol succinate (EGS) columns. Methyl substitution a t the a- or 2-position results in a relative retention time (0.879) t h a t is distinctly less than that of the unsub1294
ANALYTICAL CHEMISTRY
stituted straight chain acid (Figure 1). This would be in contrast t o naturally occurring fatty acids having methyl substitution at the ultimate (iso) or penultimate (anteiso) positiontraight chain acids (2, 5 ) . The decreavd relative retention times on polar polyester liquid phases resulting from subditution of alkyl groups a t the 2-position wggebt that diminished polarit\- j-teric hindrance) of the ester grouping outn eigli. the increase in molecular n eight or number of carbon atom< 1. elpected. honever, a plot of the logarithm of the retention time 2's. the number of carbon atoms for the 2-methyl fatty acid esters is linear. K h e n EGS columns are employed under conditions noted in Figure 1, the separation factor (5) hetneen the
2-iiiethj-1 substituted acids and their uii.;ub.tituted analogs-e.g., retent'ion time of laurate to retention time of dl2-methyllaurate-was found to be 1.14 for the three derivatives studied, as compared. for example, to a separation factor of 1.20 betiveen oleabe and Aearat'e. Figure 2 illustrates GLC of microgram level* of fatty acids obtained from 34 mg. (wet weight) of a human intestinal biopsy (courtesy of Dr. Robert J. Bolt,). The lipids were extracted (3) and the fatty acid methyl esters prepared for GLC by tranqesterification with methanolic-HC1 ( 6 ) . The dried (in va.cwo) fatty acid esteri were dissolved in 50 pl. of hexane containing dl-2-methylmyristate (100 mg. per 100 ml.), t'hen injected into the GLC apparatus. The relative composition of the fatty acids was then
Figure 1 . GLC of the methyl esters of dl-2-methyl substituted laurate, myristate, and palmitate as compared to the unsubstituted normal homologs. in., 1 7y0 E G S on 80-1 00 mesh Chromosorb-W; tritium ionization Column conditions: 185', 8 ft. X detector (220'C.) a n d argon inlet pressure 26 p.s.i.
