DELTA-TOCOPHEROL Assay
Individual Tocopherols in Mixtures Containing Delta- Tocopherol
of
LEONARD WEISLER, CHARLES D. ROBESON, AND JAAIES G . BAXTEK Research Laboratories, Distillation Products, Znc., Rochester, .V. Y .
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A method is described for the estimation of the individual tocopherols in mixtures which may contain a-, y-, and 6-tocopherols. It depends on the estimation of y- and &tocopherols by the difference in the color intensities produced by coupling with diazotized o-dianisidine in sodium carbonate and potassium h j drowide solutions. a-Tocopherol is determined by diflerence. The method is appliHE discovery of &tocopherol (10) has necessitated the T d e v't opment of an improved method for determining the percentage of the individual tocopherols in commercial vitamin E concentrates prepared at Distillation Products, Inc., by the molecular distillation of mixed vegetable oils. These conccntratcs contain, a t the present time, a mixture of a-, y-,and 6tocopherols and are referred to below as distilled concentrates, Type I11 or IT'. -1procedure for the similar assay of vegetable oils has also been needed. \Then y- and a-tocopherols were believed to be the only commonly occurring members of the vitamin E complex, the problem of determining the tn.0 in mixtures seemed relatively simple and procedures based on the dcterniination of y-tocopherol by selective oxidation with silver nitratc ( 2 ) , with nitric acid (4))or by coupling with diazotized p-nitroaniline (6)) were developed. a-Tocopherol was then determined by difference from the total tocopherol concentration as measured by the Emmerie and Engel method. Another procedure n'as ' the direct determination of a-tocopherol in the presence of y-tocopherol by selective oxidation with ferric chloride ( 6 ) .. The advantages and disadvantages of each of these procedures have becn compared ( I ) . The presence of &tocopherol causes considerable error in the published methods. &Tocopherol behaves like y-tocopherol in giving a color Tvith silver nitrate and nitric acid absorbing a t the wave length used t,o determine y-tocopherol in the selective oxidation methods (460 mp). For example, by oxidation with silver nitrate according to ( 2 ) the est,inction coefficients a t 460 mp for y- and &tocopherols mere, respectively, 20 and 6. On oxidation with nitric acid according to (4)the extinction coefficients a t 460 mp were 20 and 10.6, respectively. Thus, these methods for t,he direct estimation of y-tocopherol are inaccurate when &tocopherol is present. A procedure is described here for determining the individual tocopherols in mixtures containing a-, y-, and &tocopherols, which depends on coupling duplicate samples of the preparation with diazotized o-dianisidine in solutions made alkaline with sodium carbonate and pot,assium hydroxide. Both y- and 6tocopherols contain unsubst,ituted positions in the aromatic ring of the chroman nucleus, and couple with thc reagent to give red pigment,s absorbing a t 510 mp (Figure 1). The relative intensity of color formation for the two tocopherols a t the two alkalinities, however, is different and this difference serves as the basis for determining each in mixtures. a-Tocopherol has no unsubstituted, position and hence does not couple. I t can then be measured by difference from the tot,al tocopherol content as determined by the modified Emmerie and Engel procedure of Stern and Baxter (9).
cable directly to tocopherol concentrates prepared from vegetable oils by distillation. Vegetable oils may be assayed after preliminary concentration of tocopherols by saponification and decolorization of pigments by hydrogenation. A series of distilled tocopherol concentrates from mixed vegetable oils and from soybean oil has been assaj-ed as well as soybean, cottonseed, and peanut oils. The diazo method used is b:ised on the procedure developed by Quaife ( 6 ) , ivho showed that y-tocopherol couples n-ith diazotized p-nitroaniline to give a red color absorbing a t 530 n i p while a-tocopherol does not couple. -1 procedure for assaying ytocopherol in mixtures of the two vas developed on this basis. The authors have found diazotized o-dianisidine to be superior to di:izotized p-nitroaniline for this reaction. The use of odianisidinc, first introduced by Talbot et n2. (11) for t'he assay of estrone, has t,lie advantage that the reagent' is more stable. The coupling can be carried out a t room temperature and the diazo reagent can be prepared and stored a t room temperature, without decomposition, for as long as 10 days.
I
.
0.21
300
400 W4VELENGTH
x)O IN
600
YILLIUICROIIS
Figure 1. Absorption Curve for y-Tocopherol Coupled with Diazotized o-Dianisidine in Sodium Carbonate Solution Ell&(510
mp) =
180
-4n advantage of the o-dianisidine method over the selective oxidation procedures is its high sensitivity. The extinction coefficients of the red colors from y- and &tocopherols are over 100 (Table I) and as much as ten times as great in intensity as those produced by oxidation with silver nitrate or nitric acid. BASIS OF PROCEDURE
Absorption Spectra. The absorption curve for the azo dye formed by coupling pure y-tocopherol with diazotized o-dianisidine in sodium carbonate solution is shown in Figure 1. It was determined with a Beckman spectrophotometer. The absorption curves for the dyes formed Jvith y-tocopherol in sodium carbon-
906
907
N O V E M B E R 1947 ate and potassium hydroxide solutions were similar in appearance and had maxima at 398, 515 mp and 395, 510 mp, respectively. The curves for &tocopherol were similar and had maxima a t 395, 510 mp and 385, 490 mp, respectively. The analytical procedure described uses the absorption at the higher wave length because less interference is encountered due to the color of the oil sample.
mined. For example, a concentrate having a total tocopherol concentration of 27.47,, measured as in Equation 3, gave L values of 85.6 and 80.5 by coupling in carbonate and hydroxide solutions. Substitution in Equations 3 and 4 gave 12.1'% 6and 36.97, y- in the mixed tdcopherols. By difference the cytocopherol contcnr was found to be 51.0%. REAGENTS AND APPARATUS
Table I. Tocopherol Y-
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6Ratio 7-16-
Absorption Properties of Dyes L Value with S o . 520 Filter Sa2COa KOH 153 180 213 117 0.63 1.54
E ~ F(510~ ~ , mir)
SapCOa 180 301 0.60
KOH
196 129 1.52
The extinction coefficients at 510 nip (Beckman) and the L values with a S o . 520 filter (determined n-ith the Evelyn colorimeter) of the dyes formed, hoivever, were niarkedly different, as shon-n in Table I. These values viere calculated from the weight of tocopherol used in the coupling reaction and not from the weight, of dye formed. The absorption curves were sufficiently flat so that measuring the extinction coefficients a t 510 my, rather than a t the maximum, altered the value significantly only in the case of &tocopherol in potassium hydroxide solution. This value (129) is 14% lower than the value found a t the maximum. The authors cannot explain, at present, n-hy the intensity of color produced ivith &tocopherol varies so strikingly as the p H of the solution is changed; but it was apparent that these differenccw in color intensities could be used to determine the percentage of y- and 6-tocopherols in mixtures on the assumption that any other tocopherols present ~vouldbehave like a-tocophelol and fail to couplc. Calculations. The use of t h e differences in color formation to assay -/- and &tocopherols by measurements made, for example, n-it h the Evelyn colorimetrr, was based on two equations relating tho intensity of color formed in sodium carbonate and potassium hydroxide solutions for the sample being assaj-ed t o the percentage of y-and 6-torophcrols prcscnt.
Colorimeter. An Evelyn colorimeter equipped x i t h a S o . 520 filter is used for the color measurements. Alternatively and preferably, if it is available, a spectrophotometer such as the Beckman may be employed, using absorption data at 510 mu. Absolute Ethanol. Commercial absolute ethanol is purified by refluxing 5 gallons (19 liters) of ethanol nith 10 gram!: of potassium permanganate and 20 grams of potassium hydroxide pellets, followed by distillation. Potassium Hydroxide. Two grams of potassium hydroxide pellets are dissolved in enough absolute ethanol to make 100 ml., and filtered. Sodium Carbonate. Two grams of sodium carbonate moriohydrate are dissolved in enough distilled water to make 100 ml. Petroleum Ether. Petroleum ether (Skellysolve XI) is obtained from the Skelly Oil Company and used without further purification. o-Dianisidine Dihydrochloride (11). Technical gradc o-tlianisidine (Eastman Hodak Company) is treated by adding to 100 grams of crude powdered dianisidinc (n1.p. 134"-136") 140 nil. of distilled viater and 6 ml. of concentrated hydrochloric acid. The mixture is stirred \vel1 and boiled gent,ly until all t,he solid material is dissolved. Stannous chloride (0.5 gram) is added t,o the dark solution and li,)iliq continued for 5 minutes. To remove the rrmaining coloi,, 2 granis of decolorizing carbou are addcd and the aolution is allo-rcd t o simmer an additional 5 minutes, after which it is ra,)idly filtered by suction through a la?-er of C'elite. To the hot filtrate 50 nil. of concentrated hydrochloric acid are added, whereupon the dihydrochloride innncdiat,ely starts t o precipitate. After the solution is cooled in an ice bat-li, the pre,cipitate is filtered by suction. The c tals arc washed three times with absolut,e ethanol and once 11- ether. and dried in the oven at, 40" for 1 hour. The resulting d isidine dili!:drochlorid; should be colorless and wis found to Iinve a niclting point' of 283 (decomposes). ine. T o 0.200 gram of o-dianisidine d in 60 ml. of distilled xvatcmr canrenml. of jcC aqueous YO mixed thoroughly arid after 5 minutc solution arc added. The reagent ferred to a b r o ~ mbottle or flask, and let stand :tt rooin teiuperature for 24 hours before using. It is stable for ;tilout 10 chys lrhen liept at room temperature. PROCEDCRE
where I& L& Lz, and L t are constants, representing the L values for pure y - and &tocopherols from coupling in sodium carbonate and potassium hydroxidc solutions (Table I), and L;, L;, represrnt the observed L values for the total tocopher01s in the sample being assayed. I, value = 2 - log G 1.9 c '05' (2 - log n-hrre, G is the galvanometer reading, 1.9 .lI C = grams of total toropht~iulspor 100 nil. in the standard volume used (12 m1.1, and 31 = micrograms of total tocopherols per standard volume (3).]
L
By tranqposition and substitution of the L values for the pure tocopherols (Table I ) it can readily be shown that:
Substitution of the observed values of L i and L i then permits the percentage of 6- and y - in the mixed tocopherols to be deter-
I n order to carry out the analysis of a mixture of a-, y - , and &tocoplicrols, tivo coupling reactions are necessar- on duplicate samples of the preparation. Coupling in Sodium Carbonate Solution. A sample containing 50 t o 100 micrograms of y- and 6-tocopherols is macle up to 3 ml. with absolute ethanol in a glass-stoppered cylinder (50 ml.). Aqueous sodium carbonate is addcd (7.5 nil., 2 7 concentration), follon-ed by diazotized o-dianisidine solution (1 ml.). The mixture is shaken and allon-ed t o stand for 5 minutes a t room temperature, t,hen sodium sulfate (0.5 gram) and pet,roleum ether (Skellysolre H, 12 nil.) are added. To extract the dye the.niixture is shaken vigorously three t,imes, the phases being allowed t o separate each time. A blank is prepared in a similar manner except t'hat' no tocopherols are added. %. small interfacial prccipitate forms between the petroleum ether and water layers in the blank as well as the sample but this does not affect the determination. The mixture is transferred t o a 50-ml. separatory funnel and the loner phase \Tit11 the broivn precipitat,e is drawn off and discarded. The petroleum et,her solution is dried by the addition of anhydrous sodium sulfat,e, transferred t o an Evel>-ri colorimeter tube, and L = (2 - log G ) determined witha S o . 520 filter, setting the instrument a t 100 ivith t,he blank. The L t,hen be calculated. If a spectrophotometer is used, E be obtained instead of I, values. Coupling in Potassium Hydroxide Solution. -4 sample containing a t,otal of 50 to 200 micrograms of y - and &tocopherols is made up to 4 ml. n-ith absolute ethanol in a glass-stoppered cylin-
.
V O L U M E 19, NO. 1 1
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der (50 ml.). A solution of potassium hydroxide in absolute ethanol (2 ml., 2% concentration) is added, followed by diazotized o-dianisidine (0.3 ml.). The mixture is shaken and allowed to stand 2 minutes. Water (8 ml.), petroleum ether (12 ml,), and sodium sulfate (0.5 gram) are added and the dye is extracted and treated as described for the coupling reaction in sodium carbonate solution. 4 blank is also prepared in the same manner. Calibration Curves. The calibration curves used as a basis for determining y- and 6-tocopherols in mixtures (Figure 2) were obtained by coupling the two pure tocopherols a t the two alkalinities. It is evident that the color intensities over the range studied are linear functions of the concentration. The high intensity of color produced by coupling 6-tocopherol in eodium carbonate solution contrasts with the lower intensity produced by carrying out the coupling in potassium hydroxide solution. The curves for the dye from y-tocopherol are intermediate. Total Tocopherol Estimation. Calculation of the L values needed for substitution in Equations 3 and 4 requires an estimation of the total mixed tocopherol content of the sample. This may he determined by a method (9) for estimating total tocopherols in mixtures containing 6-tocopherol which makes allowance for the unusual behavior of the latter toward the Emmerie and Engel reagent. RESULTS AND DISCUSSION
Recovery Experiments. The accuracy of the mixed tocopherol estimation method was studied by determining the percentage recovery of pure natural mixed tocopherols alone and after addition to olive oil. For the latter experiments the total tocopherol concentration was set a t 30 and 15%, and the ratios of a-, y-, and &tocopherols were set at 40:45:15 and 10:60:30, respectively. The results are given in Table 11. The error varied from 1 to 1370, depending on the composition of the mixture.
TOCOPHEROL
CONCENTRATION (micragrams/l2 mi.)
Figure 2. Standardization Curves for Diazo Method for y- and 6-Tocopherols in Sodium Carbonate (1,3) and Potassium Hydroxide (2,4) Solutions
When a- and y-tocopherols alone were present the accuracy was increased. This was determined by adding pure a- and ytocopherols to olive oil. When approximately equal amounts of each were added at the low total tocopherol concentration of 1.0% the percentage recovery of y-tocopherol (four determinations) was 99 *2%, In an extreme case where only 6.3% y-tocopherol was pre’sent in the mixed tocopherols and the total vitamin E concentration was 3.8%, there was an average recovery (four determinations) of 94 *5%. The ability of the method to detect small amounts of y-tocopherol with acceptable accuracy has been found useful by Qhaife and Harris (8),who have used the
procedure to estimate the small amounts of y-tocopherol occurring in the mixed tocopherols of blood plasma and foods. No attempt was made in this case to correct for the possible presence of &tocopherol.
Table 11. a-,y;, 8-
in
Mixed Tocopherols
% 40, 45, 15
Total Tocopherol Concentration
% 100 30
15
10, 60, 30
100 30 15
Recovery Experiments Found in Mixed Tocopherols ay6-
% 38.6 41.2 41.1 10.6
%
48.2 41.8 46.5 56.5 10.7 5 7 . 0 10.7 5 7 . 3
Error a-
y-
%
%
%
13.2 17.0 13.4 32.9 32.3 32.0
-3.5 +3 0 +2 8 +6.0 +7.0 +7.0
+7.1 -7.1 +1.1 -5.8 -5.0 -4.5
8-
% -12.0 +13.3 -10.6 +9.7 +7.7 +6.7
Khen only a- and y-tocopherols are present, the calculation of the percentage of each in mixtures is simplified, only a single calibration curve for 7-tocopherol being needed. This is preferably prepared by coupling in potassium hydroxide solution since the estimation can be performed on concentrations of total tocopherols in fats which are as low as 0.1%. Assay of Mixed Tocopherol Concentrates and Vegetable Oils. The diazo procedure was applied to a Type I11 and Type IV distilled concentrate, to two tocopherol concentrates prepared from soybean oil by distillation, and to three vegetable oils. The assays on the vegetable oils were calculated from the values obtained on the unsaponifiable matter after hydrogenation. The data are given in Table 111. The results indicated that the percentage of a- in the tocopherols of the Type I11 concentrate examined was 51%. An assay by the selective adsorption method described below gave the value 48.0%, which was considered satisfactory agreement. The assays were of interest since the concentrate s h o w d a d-atocopherol equivalency of 60 to 65% in the resorption sterility test. A portion of this difference may have been due to the activity of 6- and y-tocopherols and to their antioxidant activity, n-hich served to protect the a-tocopherol from oxidative destruction. The a-tocopherol value for the Type IV concentrate assayed (53%) was similar to that of the Type 111. The distillates from soybean oil, however, had only about 8% a- in the mixed tocopherols. An assay by selective adsorption gave a value of 10.5% on one of these distillates. The percentage of 6- in the tocopherols of Type I11 and I V concentrates n-as about 12% compared to the value of about 33% for the distillates from soybean oil. Assay of the vegetable oils after saponification and hydrogenation indicated that 6-tocopherol is present, not only in soybean oil, but also in cottonseed and peanut oils. The percentage of a- found in the tocopherols of the cottonseed and peanut oils examined was lower than that previously reported ( 5 ) (62 and 63%, respectively). I t is planned to investigate this discrepancy by the selective adsorption procedure. Interfering Substances and Their Removal. The diazo reaction IS not specific for the tocopherols. Known interfering substances include phenols, such as gossypol. High concentrations of fatty acids (acid value = 50 or more) give a color with the reagent for reasons which are not clear. Carotenoids and similar pigments interfere because of their intrinsic color. TVheat germ oil contains a substance reacting with the reagent which distills a t much higher temperatures than the tocopherols; it is found in the residue after molecular distillation of the oil. Such interfering substances usually give colors, after coupling, which are different in tint from those produced by the tocopherols and their spectrophotometric curves shon. only general absorption at 510 to 520 mp. p-Tocopherol does not couple with either the o-dianisidine or
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NOVEMBER 1947
909
p-nitroaniline (6) reagents and if present in the mixture of tocopherols it is determined as a-tocopherol by the diazo methods. This intcrfcrence is minimized by the fact that 8-tocopherol has so far been found only in wheat germ oil. The difference betweeu the total tocopherol concentration and that of y- and 6tocopherols is referred to here as the a-tocopherol concentration. recognizing that in preparations containing @-tocopherol this value will be in error by an amount which can not be determined by this method. Low concentrations of tocopherol during the coupling reaction in sodium carbonate solution arc undesirable. The total tocopherol concentration should be 10% or more to ensure complete reaction. This limitation is less important for the reaction in potassium hydroxide solution, since anhydrous ethanol i j used as a solvent. I n this case concentrations of tocopherol as low as 0.1% can be analyzed. These requirements do not present a problem in the assay of distilled conccntrates, for the preliminary alkali refining of the vegetable oils removes gossypol, low molecular weight phenols, and fatty acids. Substantially all the carotenoids are rejected during distillation. The distillates will also usually be above 10% in tocopherol concentration.
Table 111. Analyses of nlixed Tocopherol Concentrates and Vegetable oils Total Tocopherol in Oil
a-
y-
8-
%
%
%
%
51.0 53.0 8.1 7.7
36.9 35.1 58.4 58.5
12.1 11.9 33.5 33.8
B . Vegetable Oils 8oybean 0.168" 11.9 58.6 Cottonseed 0.086" 47.6 42.2 Peanut 0.0340 37.5 42.2 Calculated from assay on hydrogenated unsaponifiable matter.
29.5 10 2 20.3
Sample
A. Distilled concentrate, Type I11 Distilled concentrate, Type IV Distillates from soybean oil
Found in hlixed Tocopherols
Concentrates
27.4 35.1
15.0 17.7
Direct application of the coupling reaction, hoxever, to vegetable oils containing a-, y-, and &tocopherols is not feasible at present. Preliminary concentration of the vitamin E is neccssarand removal of pigments is desirable. A method of accomplishing this, excluding distillation, is to saponify the oil in an atmosphere of nitrogen (a) and to hydrogenate the unsaponifiable matter as described by Quaife and Harris (?). I n the authors' work this last step was applied to an ethanol solution of the unsaponifiable fraction, using Adams platinum oxide catalyst, I n a Parr hydrogenation apparatus (50 pounds' pressure, room temperature, for 40 minutes). COh-FIRMATION BY SELECTIVE ADSORPTION
It is frequently possible to increase the accuracy of the diazo method, especially on preparations containing small amounts of a-tocopherol, by employing a preliminary adsorption step in order to separate a- and y-tocopherols from 6-tocopherol. In this way only a single calibration curve, preferably prepared by coupling y-tocopherol in potassium hydroxide solution, is needed. The basis for the separation is the observation that the tocopherols increase in the strength of their adsorption on zinc carbonate in the order a-, y-, &. By passing a petroleum ether solution of mixed tocopherols through a column of zinc carbonate-Celite, under controlled conditions, it is possible to collect the a- and a portion of the y-tocopherols in the filtrate, leaving &tocopherol and certain pigmented materials on the column. Application of the diazo reaction to the tocopherols recovered from the filtrate permits calculation of the percentage of CY- in
the tocopherols of the original sample, since t'hc total tocopherol concentration of the latter can be determined and the weight of the sample adsorbed is luiown.
.il cplumn (2.5 X 60 em.) packed with 50 grams of a 70 to 30 misture of zinc carbonate (Baker, precipitated) and diatomaceous earth (Celite, Johns-Manville Corp.) has been found convenient for carrying out the separations. The column must be carefully prepared because t,oo tight, packing results in virtual stoppage of solvent flow and too loose packing gives poorly differentiated bands. A satisfactory column can be prepared by adding t,he adsorbent in four portions to the column, app1,ving suction by R water aspirator through a filter flask after each addit,ion. Since the adsorption method depends on the separation of a-tocopherol from such interfering reducing compounds as 6tocopherol, the ratio of tocopherol concentrate to adsorbent must first be determined which will allow all the a-tocopherol and a portion of the y-tocopherol, but no 6-tocopherol, to pass through the column. The diazo .method is used t o establish the absence of 6-tocopherol in the filtrate from the column. The authors have found a 2-gram sample satisfactory for concentrates having a pot'ency of approximately 30,% mixed tocopherols. For higher potency concentrates (60 to 80%), a 1-gram sample is preferred. The column is first washed with petroleum ether (125 ml.). The sample in petroleum ether (25 ml.) is then added and rinsed into the column -7ith another portion (25 ml.) of solvent. The solution is drawn through the column by gentle suction and washed through with petroleum ether (300 ml.). Care should be taken that the top of the column is covered with solvent or solution at all times after wetting. The washing operation is followed with ultraviolet light. Clean, sharp bands are observed if the packing has been done properly. a-Tocopherol was found to be associated with three bands, fluorescing blue, which passed down the column first and into the filtrate. The liltrate is evaporated on a steam, bath under a stream of nitrogen and the residue weighed. I t is then assayed for total tocopherols as in (9) and for y-tocopherol by the diazo reaction. The percentage of a-tocopherol in the original sample is then readily calculated. For example, a sample (2.00 grams) of a distilled mixed tocopherol concentrate ,which assayed for 27.4% total tocopherols was chromatographed as described and yielded a ftltrate fraction (1.50 grams) which assayed for 27.7y0 total tocopherols, and 36.8Yc -,-tocopherol by the diazo method. Thus,' the Dercentage of CY- in the tocooherols of the i.50 x 27.7 (loo - 36.8) = original sample was 2.00 X 27.4
i8%.
Selective adsorption promises to be a valuable tool in the study of the tocopherols in natural oils and in developing accurate analytical methods for them. LITERATURE CITED
(1) Baxter, J. G., Lehman, 'R. W., Hove,
E. L., Quaife, M. L.,
Weisler, Leonard, and Stern, M. H., Biol. Symposia, 12, 484 (1947). (2) Baxter, J. G., Robeson, C. D., Taylor, J. D., and Lehman, R. W., J. A m . Chem. Soc., 65, 918 (1943). (3) Dann, W. J., and Evelyn, K. A., Biochem. J.,32, 1008 (1938). (4) Fisher, G. S..IND. ENC.CHEM.,ANAL.ED., 17, 224 (1945). (5) Hove. E. L., and Hove, Z., J. Bio2. Chem., 156, 601 (1944). (6) Quaife, M. L.. J. A m . Chem. SOC.,66, 308 (1944). (7) Quaife, M. L., and Harris, P. L., J . Biol. Chem., 156, 499 '(1944). (8) Quaife, M. L., and Harris, P. L., IND. ENO. CHEM.,ANAL.ED., 18, 707 (1946). (9) Stern, M.H.. and Baxter, J. G.. ANAL.CHEM.,19, 902 (1947). (10) Stern, M . H., Robeson, C. D., Weisler, Leonard, and Baxter, J. G., J.Am. Chem. SOC.,69, 869 (1947). (11) Talbot, N. B., Wolfe, J. K., MacLachlan, E. A., Karush, F., and Butler, A. M.,J.Biol. Chem., 134, 319 (1940).
RECEIVED December 6, 1946. Presented before the Division of Analytical and Microchemistry at the 110th Meeting of the ANERICANCHEXICAL
SOCIETY, Chicago, Ill.
Correction I n the article on "Water Analyses by Selective Specific Conductance" by J. W. Polsky [ANAL.CAW., 19, 660 (1947)], Table VII, the value for chloride for water sample 2 should be 52.5 by the standard method and 51.5 by the conductometric method.
