V O L U M E 23, NO. 12, D E C E M B E R 1 9 5 1 periment, with the value of 8.73 calculated from an equation given by Reicher (9) and with the value of 8.68 obtained by graphical interpolation of Warder’s data (16). From Figure 2 it may be observed t h a t t h e value of ko obtained with the titrator appears consistent within k0.2 liter per mole-minute, as shown by the dotted lines. Greater scattering of points and deviations in the value of k, was obtained using the usual titration method (4). Investigations are already under way in this laboratory to use the titrator in the study of other saponification reaction rates a t different conditions with refinements in the equipment used. Efforts are also being made to develop a more linear and more stable titrator. The method herein outlined may prove useful in the kinetic study of saponification reactions which proceed a t rates too rapid to follow by the ordinary method (4). The limiting factor is the response lag of the titrator used. I n this investigation the lag was not determined, as the titrator response was adequate to follow the reaction studied. LITERATURE CITED
(1) Anderson, Bettis, and Revinson, ANAL.CHEM.,22, 743 (1950). (2) Anderson and Revinson, Ibid., 22, 1272 (1950).
1773 (3) Blaedel and Malmstadt, Ibid., 22, 734 (1950). (4) Daniels, F., Matthens, J., Williams, J., and Staff, “Experimental Physical Chemistry,” pp. 167-9, New York, McGraw-Hill Book Co., 1941. (5) Jensen and Parrack, Texasii. & M.College, Eng. E s p t . Sta., Bull. 9 2 (1946); ISD.ENG.CHEM.,ANAL.ED.,18, 595 (1946). (6) Jensen, F. W., Watson, G. XI., and Vela, G., ANAL.CHEM.,23, 1327 (1951). (7) MacInnes, “Principles of Electrochemistry,” pp. 378-80, New York, Reinhold Publishing Corp., 1939. (8) Nichols and Kindt, ANAL.CHEM.,22, 781, 785 (1950). (9) Reicher, Ann., 228, 257 (1885); 232, 103 (1885); 238, 276 (1887). (10) Rieman, IT., Seuss, J. D., and Kaiman, B., “Quantitative Analysis,” pp. 363-70, Xew York, McGraw-Hill Book Co., 1942. (1 1) Scarborough, “Numerical Mathematical Analysis,” pp. 363-70, Baltimore, Johns Hopkins Press, 1930. (12) Stead, B., Page, F. M., and Denbigh, K. G., Discussiona, Faraday Soc., 2 , 2 6 3 (1947). (13) Terry, E., and Stieglitz, J., J . Am. Chem. SOC.,49, 2216 (1927). (14) Walker, J., Proc. R o y . SOC.(London),A78, 157 (1906). (15) Warder, R. B., Ber., 14, 1361 (1881); J . Am. Chem. SOC.,3 , 203 (1881). (16) Kest, Burkhalter, and Broussard, A N ~ LCHEM., . 22, 469 (1950). RECEIVED February 16, 1961. Presented a t the Sixth Southwest Regional CHEJ~ICAL SOCIETY, San Antonio, Tex., December Meeting, AMERICAN a, 1960.
Separation and Determination of Crystalline Vitamin B,, Synthetic Vitamin Mixtures 1
AIAX 31. hIARSH AND NORBERT R . KUZEL Eli Lilly and Co., Indianapolis, Znd. Recent interest in the value of vitamin BIZa3 a nutritional supplement has led to the preparation of mixtures of this substance with one or more of the other vitamins. One of the important factors involved in the therapeutic evaluation of these mixtures is the question of the stability of vitamin B 1 > in the presence of such compounds as thiamine, riboflavin, ascorbic acid, folic acid, pyridoxine, pantothenates, and the oil-soluble vitamins. A method has been devised, utilizing a mixed cation-and anion-exchange resin column, whereby substances
I
SVESTIGATIOSS of t,he possible therapeutic value of vitamin B,, as a nutritional supplement (8) have led to the preparation of various mixtures cont,aining this subst,ance with one or several of the other vitamins. Thus the problem of determining vitamin B1?in the presence of such substances as ascorbic acid, the B complex vitamins, and certain of the oil-soluble vitamins li~tsevolved. As t8hestability of vitamin BIZ in such mistmurepis a primary factor in the evaluation of the usefulness of the various preparations from t,he therapeutic viewpoint, i t \vas necessary that, the methods of determination devised also reflect with reasonable accuracy the amount of vitamin BIZ present during the course of deterioration of the samples under rather severe condit,ions. The probleni of analysis Tvas considerably simplified when i t was decided to initiate the stabilit,y studies utilizing pure cryst’alline vitamin Bl2 rather than fermentation concentrates or liver extract concent,rates. Under these circumstances the separation of vitamin BIZ was required only from mixtures, the original composition of which was knoxn.
in these mixtures w-hich interfere in the spectrophotometric determination of vitamin B I ~ can be satisfactorily removed. Removal of interferences by this technique is also observed following photodecomposition and heat decomposition of the mixtures. The procedure is useful for the routine control of synthetic vitamin mixtures containing crystalline vitamin B,?in concentrations of 5 micrograms per ml. or more. Stability studies have been carried out using this technique; correlation with results of microbiological assays is reasonably good.
Although the structure of vitamin BIZhas not been completely elucidated, certain physical constants of the pure, crystalline substance have been adequately described (3, 6, 7 ) . Among these are the values for aqueous solutions of the pure material. I n these studies, the authors have used the light absorption of the vitamin BIZa t 550 mp (E;?& = 66) as a means of meaeuring the concentration of the vitamin after separation from interfering constituents. Other chemical methods of analysis have been described (1, 2, d ) , some of which could conceivably have been applied directly to the samples without attempting a preliminary separation; however, the separation procedure developed is relatively rapid and simple, whereas the chemical assay procedures so far described are somewhat complex and require considerable time. It was observed t h a t vitamin BIZ in aqueous solution, being essentially nonionic in its behavior toward some ion exchange resins, would pass through coluniris of these resins nithout significant exchange o r adsorption on the column. Such substances
1774
ANALYTICAL CHEMISTRY
as thiamine hydrochloride, riboflavin, nicotinamide, calcium pantothenate, pyridoxine hydrochloride, and ascorbic acid, however, are ionic in their activity toward these ion exchangers and can be removed from solution by passage of aqueous mixtures containing these ingredients over a n ion exchange column. Interfering degradation products of most of these, as well as those of vitamin B12itself, are also removed by the resins.
acids and bases a t different concentration levels to establish optimum conditions for maximum vitamin BIZ recovery without loss of retentive power for interfering materials. The resins were prepared for use by standing overnight in contact with an aqueous solution of the regenerant; excess regenerating solution n a s decanted and the resins were washed with distilled water until the washings were neutral (to bromocresol green for acid-regenerated resins and to phenolphthalein for alkali-regenerated resins). All exchange studies were made in columns prepared in shortr ened 50-ml. burets fitted with either a stopcock or a rubber tube and pinchclamp to control column flow. A glass wool plug was inserted a t the base of the column and an a ueous slurry of the washed resin was poured into the column. TCfie resin was packed by tamping under a head of water until a measured column height was reached. A pledget of glass wool then was laced on the top of the column to hold it in place duriAg the aidition of sample and washing solvent. The columns were washed thoroughly - . with water before use. The eluates were collected in 25- or 50-ml. volumetric flasks containing approximately 1 ml. of pH 5.1 buffer (Coleman buffer tablets, Coleman Electric Co., Maywood, Ill.). This amount of buffer was found adequate to prevent the eluate from becoming alkaline as a result of displacement of hydroxyl ion by the anions of salts in solution in most of the mixtures so far encountered. Preparations having high concentrations of alkali metal salts require larger quantities of buffer solution. The variation of absorption characteristics of vitamin BIZin the pH range 5 to 7 does not appear to be appreciable; however, alkaline solutions of vitamin BI2 deteriorate rather rapidly with resulting low recoveries.
Table I.
WAVE LENGTH IN
Figure 1. 1. 2. 3.
4.
Mp
Effect of Variables
Reference curve of crystalline vitamin BIZin water at 15y/ml. Same material after 16 hours' exposure to ultraviolet radiation and 6 hours' heating at 100' C. Sample represented by 2 after passage over 10% sodium hydroxide-regenerated Amberlite IRA 400 Sample represented by 2 after passage over mixed 10% sodium hydroxide-regenerated Amberlite IRA 400 and 2.5 % sulfuric acid-regenerated Amberlite IR 120
I n the case of mixtures of the oil-soluble vitamins (A, D, and
E) with vitamin BIZ,dispersions of the oil-soluble vitamins were made in propylene glycol-water mixtures with the aid of dispersing agents. The direct application of these samples to the exchange columns did not provide a satisfactory eluate for the determination of vitamin Biz. Apparently, the solvent and/or the dispersing agent in these preparations functions to prevent exchange of some of the interfering components of the mixture, thus allowing the interferences to pass through the column and appear in the eluate. It was found that a preliminary extraction of the samples containing the oil dispersions with a suitable mixed solvent would permit the aqueous phase remaining after extraction to be passed over a n ion exchanger with satisfactory removal of the interfering components. REAGENTS AND APPARATUS
All spectrophotometric measurements were made with the Beckman Model DU spectrophotometer using matched 1-, 5-, and IO-cm. Corex cells. A tungsten filament lamp provided the incident beam used for the 550 mp readings; the same light source with the proper filter inserted in the beam was also used for 361 mp measurements. Measurements below 330 mp were made in matched 1-cm. quartz cells, using a hydrogen discharge lam as a source of the incident beam. T t e resins studied mere primarily those of the Amberlite series (Rohm & Haas Co., Philadelphia) and included a nuclear substituted sulfonic acid resin ( I R 120), methylene sulfonic acid resins ( I R 100 and I R 105), and carboxylic acid resins ( I R C 50 and XE-66); the anion exchangers studied were I R 400,. I R 410, XE-75, and XE-67. These were regenerated with various
Recovery of Vitamin Bi2 from Various Types of Resins
(10-ml. aliquot8 containing 15 micrograms of vitamin Bn per ml. pawed over columns and eluted with water into 25-ml. volumetric flasks) Column Regenerant Height. Cm. Recovery, % Resin 1. XE-67 1.5 10% N a O H 99.0 10% S a O H 2 . XE-75 81,4 10% NaOH 100.0 3. 4. I R 410 120 79.7 10% HzSOP 92.0 5. I R 120 5% H ~ S O I 94.7 6. I R 120 2 5% HzS04 78.6 10% HCI 7. I O N - X 100.5 8. I R A 4 0 0 5% (CHa)rNOH 99.5 9. I R A 400 10% S a O H 100.5 10. I R A 400 5% KOH 5'70 N H I O H 100.5 11. IR.4400 5% NaOH 99.0 12. I R A 4 0 0 13. I R 105 Essentially none 10% HC1 14. I R 100 Essentially none 10% HCI 5 % HOAC 33 15. I R C 50 Essentially none 16. XE-66 5% HQAC 90.8 17. IR.4 400 10% P a O H ] I R 120}1:1\ 10% HZ904 I R A 400 10% NaOH 18. I R 4 4 0 0 (1 part) 10% S a O H } 90.8 I R A 120 (1 p a r t ) } 10% HtSOa I R A 400 10% % a O H 95.1 19. I R A 400 120}':'\ 10% 2.5% N aH,SO,] OH I R A 400 J 20. Same mixtures as 19 with other 21. lots of resin
};
}
}; };
11
10% S a O H 93.1 97.1
For the preliminary extraction of the mixtures of vitamin B12 with the oil-soluble vitamins in propylene glycol-water solution a 1 to 1 mixture of tetrahydrofuran and chloroform was found to extract materials that interfered with the exchange of ions on the column as well as t o extract the oils themselves. Chloroform alone did not remove the desired components adequately, nor did petroleum ether prove to be satisfactory for this extraction. EFFECT OF VARIABLES
Effect of Various Resin Types and Regeneration Concentrations. As interfering substances encountered in the separation of vitamin Blz in synthetic vitamin mixtures consist of both cations and anions, a mixed resin column was desirable. The comparison of absorption curves of crystalline vitamin BIZin Figure 1, before and after passage over various resin columns, indicates the extent of removal of degradation products
V O L U M E 2 3 , NO. 1 2 , D E C E M B E R 1 9 5 1 of vitamin HI, liv t h c iinioti resin :inti cation-anion wiiii rrinil)ination. The nece;sity For using l)oth types of resin is evidrnt froin
177s for t,he small, ccnsistent I ( $ r OF vitamin B,, o n the c~olunin. Thus N general c:ilrul:ition would involve the follon-ing equation:
the iniprovetl cwrve ahitpe olwerved after treat,ment x i t h t8hcI R 120-IRA 400 combination :is ronipared to the rurve o f t h r rlutite from IR.1 400 alone. T:ihle I illustrates t h c i'cc'overy o f crystalline vit:tniin t ' i , l l l l i v:irious individual uvins :itid niised resin colunini.
83 iJ
i ~ ~ n r e n t ~ . a t iofo ivitrrnin i H,, in saniplt. x h e r e .4 Sdl,il,~e is the a1)sorliiinry of t h e s:iniple eluate to 1 ('in, if 5-cm. or 10-cni. cell:: are used) and . l Q t d . is theoretical :ibsorlianc~,of a specified concentration of 3t:indard vit,nniin B12-e.g., a solution of 10 inirrogrania per i d . of vitamin B,, in 1-mi. ahsorption cells hap :I the( retical alisorhancy of 0.066. The per cent reroverg for the resin misture is determined o n :I column ronstructed frcni t,he same lots and regeneration batches and of the same approsim:itc. dimensions as ai'e used for the s a n i p l r ~ . The value is estal~lishetitiy passing :I sample of standard cryst:illine vitamin €3, lution OW^ the rolunin :inti coniparing the absorbancy of the eluate (after correction for volume changes :ind differences in rell tiimensions) n-ith t h t ( i f the origin:il wnipl(>, Thur
ASSAYS OF SYYTHETIC VIT4MIY \IIXTURES
involved in wbsequeiit Thc, c~oluninoeltvtetl 1'01. t h r sqiarat studies consisted of an 8-cni. bed of sodium hydrosiciP regwerated IRA 400 overlaid with a i-cm. bed of a 1 t o 1 misture of 1 0 5 sodium h!.tirositle-rrgenerated IRA 400 and 2.5% ( v , /v.) sulfuric aciti-rc.g~,iierated IR 120. Some studies were also conducted using 10yo sulfuric acid-regenerated IR 120 in place of t h c s 2.5('c-regeneratetl material. The lower layer of I R A 400 ( ( ) € I -) was desirable twrause preliminary studies indicated that in niistures containing rilmflavin 15 ith high concentrations of other a n i o n , the riboflavin hand n as not so well retained in the niised rc,sin XL: i i i the anion c~srhangeralone. Effect of Column Height on Recovery. Table I1 s h o w thr recovery of vitnmin HI? :is :i function of column height when the same concentration and diquat, volume are applied to each column. The results indicate th:it minor (0 t o 1 cm.) variations from :I selerted column height do iiot, appreciably affect, recovery; hon.ever, larger h(Jight rhanges would require :t nen- recovery f:ict,or evaluatioii. Effect of Aliquot Concentration and Eluate Volume on Recovery. The results of application of different concentrations of vitaniin E312 in the s m i e snniple volume are shon-n in Table 111. T:ihlc IV includes ii study of the recoverp of vitamin B I as ~ a fuuction of voluint~of eluate. Thus it is seen that. volunie of eluat,e and concentxition level :iw not critical f:ic,tors in t,he range studied. Flow Rate. ;ilthough the time rcquired for the aliquot to pass tliwugh the colunin arid for the proper volume of eluate to be collected was not studied in detail as a variable, it vas satisf:ic$orily established that small variations from a flow rate of 0.8 nil. per minute Ivould riot :Iffcct recoveries appreciably. Pollo\ving t h e w stuclirs, it n - : ~established that t h e application of 10-nil. :iliquutq to the c~oluninand their elution into 25-nil, volunirtric fln.sk.4 (wishing c~oluninsivith water and diluting t,o mark with washings) \\-oulti >-ic,ld satisf:tctory recoveries. The light al)rorption of the eluates after mixing \-,-as measured in 5cni. :~I,sorption cells. T Mriity-milliliter samples, when eluted iiito 50-nil. volunietrie flinsks, \rere occasionally used and found to yield satisfactory recoveries. I n the latter case. tiiisothncies roultl I)e nieasured in 10-rni. ahsorption cells. C'alcul:ttions of vitaniin BI2 concentrations from :ilixort)anry v:ilues o f t h e eluates will c*ont:iiri it correction term t,o nrrount
Several vitamin combinations have lieen studied and the assays obtained are recorded in Table . ' 1
Table 111. Effect of Variation of Concentration Level on Recovery .\liquet Volume
.ipplied, All.
Vitamin B M Concentration, ",/HI,
Voluine of
Eluate, 1\11.
Recovery, 'I
1.
10
I
25
91.5 91. ,5
2.
10
10
25
89.: 90.0
3.
10
15
25
91.0 89.9
4.
10
30
23
91.4
89.5 89. .j
Column components: 7 cn:. of 1:l 10% H?RO~-repenerated I R 120 a n d 10% XaOH-regenerated IRA 400 overlying 8 e m . of I O ? S a 0 1 1 rc'pt'nerated I R h 4CO alone
They indicate that there is no bignificant interferenre in any of the mixtures studied unless extensive decomposition of the components has orcurred. The limitations of precision imposed ~ ~ readings o n these samples also influence by verv 1 0 absorbancy the assays reported. DISCUSSION
It is apparent from the comparative absorbancy readiugs in Table VI that the precision of recovery of vitamin B,, alone or i n
Table IV.
Effect of Yariation of Eluate Volunie on Recovery
Aliquot Volume
Vitamin Bln Conrcntration. -,/1\Il.
10 10 10
13 15 1.j
r\pplierl, 311.
1. 2. 3.
Volriine of
Eluate, LII. 20 25
50
Rccovrry.
L;
83 1 513, 1 92.5
Column components: 7 cm. of 1:l 2.5%1, HzSOi-regenerated I R 120 and 10% NaOH-regenerated I R A 400 overlying 8 e m . of 10% NaOH-regenerated I R A 400 alone
1776
ANALYTICAL CHEMISTRY
Table V. Comparison of Chemical Assay for Vitamin BIZwith Theoretical Concentration and with Microbiological Assays T- h.e.l n_.
______
Sample Vitamin
I.
Blt
Hours
3.
4.
Same as 3
5.
Same as 3 + ascorbic acid 50 mg./nil.
6.
Vitamin BIZcrystalline+ ascorbic acid 50 ma./ml.
33.7
33.7 33.7 33.7 33.7
33.7 14.1 32.0 29.5 32.2 30.9
30.8
1
33.7 33.7 33.7 33.2 33.,
33.2 13.7 2.4 29.0 9.5
35.7 14.1 0.9 30.0 9 0
27.2 27.2 27.2 27.2 27.2
26.9 8.3 6.1 20.0 7.9
None
15.0
14.9
15 5
None
15.0
14.6
14 3
33.7 33 7 33.7 33.7 33.7
29 0 22.1 8.6 3.0 0.8
Iione
14.1
14.7
14.6
Kone
14.1
14.4
15.6
ACKNOWLEDGMENT
14.1
14.3
15.8
T h e authors gratefully acknowledge the aid of J. T. Stephenson and his associates in provid-
1 1
Kone 100” c. 100° C. UVb
UV
Kone 100’ C. 100’ C. UV UV
hone 1000 c. 1000 c.
YV LV 7. 8.
Same as 6 Vitamin BIScrystalline folic acid 3.5 mg./ml. Vitamin BI? cryntallinef vitamin A 6000 U/ml., vitamin D 5000 U/ml., vitamin E 5 mg./ml., Equiv. of a-toropherolin?O% propylene glyrol-water
+
9.
[
b
y/hl!.
Microbiological Assaya, -f/Jll.
Chemical Assay, -,/All.
..
crystalline in w a t w None (controll 1000 c. 1000 C. YVb UV
Vitamin Bn crystalline t thiamine IICl 3 mg./ml.. riboflavin 3 mg./ml., pyridoxine HCI 5 mg./ml. Ca pantothenate 5 mg./lni., nicotinamide 75 mg./ml., Vitamin Bu crystalline+ thiamine HCI 5 mg./ml., rihoflavin 2 nig./ml., nicotinamide 75 mg./ml,, ?;a p a n t o t h m a t e 2.5 mg./ml.. iiyridoxin? HC1 5 mg./ml.,
2.
retical Concentration,
1
None
.. 4 1 4
1 4 1 4
I 4 1 4
1
4 1 4
14.1
15.6
27.9 27.7
35.4
33,2
29 . 3 ~. .
.
19.8 7.3 2.7 < 0.1
Jlodification of method of Skeggs el al. (6). Exposure t o ultraviolet radiation from Hanovia mercury arc lamp a t distance of 1 foot.
_combinations with other vitamins is within a level of f 2 % . Partially decomposed samples exhitiit Pomewhat less precision of recovery, partly because of the lower absorbancy level of the eluate and partly because of the tendency for the decomposition products of some samples to break up portions of the column and cause small “channeled” areas. The comparisons with microbiological assays indicate a good approximation of potency evaluation to the extent that the precision of microbiological results will permit. eucept a t the lower
Table VI.
tion are encountered which in turn limit the quantity of sample that can be applied to the column. A practical limiting concentration mag be considered to be approximately 5 micrograms per m1.-a 20-ml. aliquot of which when eluted to a volume of 50 ml. and measured in 10-em. cells will have a theoretical absorbancy of 0.132. Lower concentrations of vitamin B,, can, of course, be measured with less precision. The conditions for separation and determination of vitamin BI2 which have been described here in detail are in a sense a special case of a rather flexible procedure. T h e technique of separation of vitamin B12from mixtures in which the other components are ionic in their behavior toward ion euchangers can be modified to fit a large number of combinations and concentrations, prsvided the proper changes are made in resin types and amounts and in the amount of buffer used to control the pH of the eluate. The extension of the procedure to vitamin BIZ concentration ranges below those indicated will depend to a certain extent on the utilization of more sensitive light absorption-measuring devices o r the use of lowvolume absorption cells having a relatively long light path.
Reproducibility of Hecovery of Vitamin R I iti ~ Synthetic Vitamin Mixtures
Average Absorbancy Sample KO.of rlnsays (550 mp) Rsnge Crystalline vitamin Bin aolution (control) 8 0.173 0.172-0.17.5 Vitamin BIZ with vitamin R complex 8 0.183 0.181-0.181 Vitamin Biz with vitamin B complex and ascorbic acid 4 0 179 0 . 1 7 8 M . 180 Vitamin B I with ~ vitamins .4, D, and E in propylene glycol-water mixture 4 0.176 0.1744.177 Vitamin B I with ~ folic acid 4 0.178 0.177-0.170 Vitamin BIZ with ascorbic acid 4 0.181 0.180-0.182 Column components: 7 om. of 1:l mixture of 2,5Y0 ISnSOi-regenerated I R 120 and 10% NaOII-regenerated I R A 400 overlying 8 cm. of 10% NaOHregenerated I R A 400
vitamin Bl2 potency levels. T h e capacities of the 5- and 10-cm. cells used in absorption memurements are approximately 14 and 34 ml., respectively; for this remon, minimum volumes for elu-
ing the microbiological assay results on the samples tested; the suggestions of W. H. Rix and S. W, Arnett in regard to the preparation of the various vit,amin mixtures are also greatly appreciated. LITERATURE CITED
(1) Boxer, G. E., and Rickards. J. C., Arch. Biochem., 29, 75-84 (1950). ( 2 ) Boxer, G. E., and Rickards, J. C., presented a t AAAS meeting, Cleveland, Ohio, December 1950. ( 3 ) Brink, N. G., Wolf, D. E., Kaczka, E., Rickes, E. L., Koniussy, F. R., Wood, T. R., and Folkers, K.. J . Am. Chem. Soc.. 71, 1854-6 (1949). (4) Fantes, K . H., Ireland, D. M., and Green, N., Biochem. J.. Proc., 46, xxxiv-xxxv (May 1950). (5) Jackson, W. G., Whitfield, G. B., and De Vries, W. H., J . Ant. C h m . SOC.,73,337-41 (1951). (6) Skeggs, H. R., Huff, J. W., Wright, L. D., and Bosshardt, D. K., J . Bid. Chem., 176, 1459 (1948). (7) U. S.Pharmacopoeia XIV, pp. G61-2. (8) Wetzel, N. C., Fargo, W.C . , Smith, I. H., and Helikson, J., Science. 110, 651-3 (1949). RECEIIE D April 18, 1951. Presented before the Division of Analytical Chemistry a t t h e 119th Meeting of the AMERICAN CHEMICALSocI?.‘rI. Clevcland. Ohio.
Correction In the article on “Lithium Aluminum Hydride as a Qualitative Test Reagent for Aromatic Nitro Compounds” [Nelson, L. S., and Laskowski, D. E., AKAL.CHEM.,23, 1495 (1951)], the last column of Table I should have been labeled “Limit of Detection, G./MI. x 104.”
