758
ANALYTICAL CHEMISTRY
found was less than 10.0002 nil. Thcsc rcwlts show that the isoamyl alcohol does not iritcrfere aitd a l ~ othat t l i c ovrr-all a(’mracy of measurements throughout this \\ u r l ~is 11rol):thly11 ithiri 10.0002 1111. cONcLusIo6
The results of this work indicate that deductions for adsorbtd or occluded air from air-ignited porous copper oxide are not justified. The practice of deaerating copper oxide by evacuating, saturating with carbon dioxide, and preserving in an atmosphere of carbon dioxide is likewise unnecessary. Upon long air exposure the air adsorbed in a combustion tube attains significant values. I n practice, however, the actual error will be very small, because the time of air exposure while placing the sample and temporary filling is comparatively short. and furthermore a major portion of the adsorbed air is held back by the metallic copper by formation of copper oxide. In an actual drterrnination the legitimate adsorbed air deduction appears to be not over 0.001 ml. It has long been recognized that control of the rate of burning the sample is a critical operation. The authors believe that high nitrogen results have been too frequently attributed to adsorbed air errors, rather than to incomplete combustion, in which case methane and similar products may be formed. The conditions for this combustion are far less favorable t,han in the determination of carbon and hydrogen, where platinum catalyst in presence of excess oxygen assures complete oxidation of suhstnricos that are not easily oxidized by coppcr oxide alonc.
Gonick et al. (3) statc that high nitrogen results can hc causcd release of adsorbed nitrogen on copper oxide when the latter is reduced to the metallic state by burning the sample. They do not state how large such errors are. I n the authors’ work the customary sugar blank was omitted to avoid errors due to the possible formation of methane. Theoretically, a 5 - m ~ . sample of sucrose will reduce about 28 mg. of copper oxide. A(atually, the amount of copper oxide reduced will no doubt be a sniall fraction of the calculated, because a major portion of the' rarbon will remain as charcoal. If the volume of nitrogen released by the reduction of such small amounts of copper oxide i$ significant, the values reported are not conclusive. In that case, however, all blanks of this kind become rather meaningless bt,causc the amount of copper oxide reduced and, in turn, the volume of nitrogen released will depend not only on the fate of thc carbon but also on the size of the samplc and the rcduc-iiig equivnlcnt n cight of the particular substance used.
1)sthe
LITERATURE CITED
J . .4m. Chem. Soc., 52, 2813 (1930). (2) Flaschentriiger, B., Z. nngeu. Chem., 39, i 1 7 (1926). Asar,. ED.,17, 677-82 (1945). (3) Gonick, H., e t a l . , IND. ENG.CHEM., (4) Niederl, J. E., and Niederl, V., “Micromethods of Quantitative Organic Analysis,” 2nd ed.. p. 88, New York. John n’iley 8-
(1) Boughtoil, JV.
A\.,
Sons, 1942. ( 5 ) Ogawa. S . , Sci. Repts. T o k y o I m p . Uniz>., 16, 667 (1927). (6) Pagel, H. A , IND.EXG.CHEM.,ANAL.ED. 16,344 (1944). (7) Trauta, 0. P., hlikrochentie. 9, 300 (1931). ~ ~ E C E I I E for D
rcview .lugtist G , 1031. .iccepted Octoiier 30, 1951
Chemical Assay for Tocopherol in Animal Materials R . W . SWICK A N D C . A. BAUMANN College of Agriculture, Cnicersity of Wisconsin, Madison, Wis.
hlAJOR problem in the estimation of tocopherol in animal
A materials is t,he elimination of interfering substances.
In 1947 Wanntorp and Sordlund ( 1 9 ) used saponification and adsorpt,ion on Floridin for the removal of fat and the carotenoids, but recovered only 92%,pf added a-tocopherol. The more recent assay of Quaife and Dju (13)requires a molecular still and the use of hydrogenation, which is prohibited in certain hospitals (3). The present procedure combines elements of these two methods: i t is easy to perform, requires only readily available equipment, and yields an over-all recovery of 98% of added a-tocopherol. PROCEDURE
Homogenization and Extraction. Ten grams or less of tissuv were cut into small pieces and homogenized with 40 to 50 ml. of cthyl alcohol in the microcup of the Waring Blendor. The homogenate was filtered through an extraction thimble and the cup mashed repeatedly with small amounts of alcohol; the washings also were filtered into a Soxhlet extractor. The thimble was dropped into the extractor, about 100 ml. of alcohol were added to the flask with a boiling chip, and the residur was rrfliised for at least 18 hours. Most tissues were homogenized completely by this procedure, except for small knots of connective tissue which remained intact but offered no hindrance to extraction. The intestine !vas the most resistant to homogenization, although ext’raction appeared to be complete, as shown by comparisons of the amounts of tocopherol in two samples of the same intestine, one of which was homogenized, the other frozen and ground (13j: 285 and 273 micrograms of tocopherol per sample, rePpPctivel>-. Extraction for periods longer than 18 hours f d e d to inrrrape the apparent tocopherol content of the tissuep. Saponification. Because tocopherols w e oxidizat)ln in t h r presence of alkali, ehborate equipment has htwi designed to
provide an oxygen-free atmosphere over saponifi(,ation inixtulcs (Z), and neutralization of the alkali before transfer from this atmosphere has been suggest,ed ( 2 ) . The use of antioxidants such as pyrogallol (18) or p-acetylaminophenol ( 7 ) has also bccn recommended. I n the present study the latter was used. The condenser of the Soxhlet apparatus was removed :inti 10 nil. of an alcoholic solution of 2% potassium hydroxide and 0.5% of p-acetylaminophenol were pipetted down the arm of the ext,ractor. The mixture was heated for 30 minutes, the extract being concentrated t o about 50 ml. during this period. Following saponification, the extract was cooled immediately, an equal volume of water was added t o t.he alcohol, and the solution was saturated with sodium sulfate. The nonsaponifiable matter was extracted into 25 ml. of purified petroleum ether (5) by shaking for 10 minutes in a separat.ory funnel (18). The layers separated rapidly and the pet,roleuni ether was dried over anhydrous sodium sulfate. Under these conditions a second petroleum ether extract of thc aqueous alcohol contained no measurable amount of tocopherol. For oils or very fatty tissues alkali a as added gram for gram of fat (ca. 5 times the theoretical amount required), and in the presence of the extra alkali further extraction into petroleum ether became necessary. Washing the petroleum ether layer Tvith water or dilute acid or alkali did not alter the final values for tocopherol. When 110 micrograms of a-tocopherol were added to tissue extracts, the amounts recovered ranged from 106 to 115 micrograms. p-hcetylaminophenol did not affect recovery in the presence of tissue substances, but in their absence the losses of tocopherol were serious unless a supplementary antioxidant was present. p-Acotyl:iminophenol was used routinely in preference to pyrogallol h x i w the ktttei, gave high recoveries---l04 to 181% (17). Chromatography. .In aliquot of the petroleum ether extract containing 20 to 1000 micrograms of tocopherol was evaporated
V O L U M E 2 4 , NO. 4, A P R I L 1 9 5 2 Table I .
Total Tocopherols in Rat Tissues as Determined by Two Methods Distillation IIydrogenation y / d .
i.i\
759
Saponification Chroniatography extracted
4 95
2,56 ( Intestin? 4 . 1 2 (““,(I l,un&h 3 . 25= Heart 3.53 Spleen 5.01“ Tastes 2.06 Increased with hydrogenation. See text. Sunibers In parrntliebes indicate percentage recovery of 110 tocopherol.
y
of a-
t o dryness with the aid of vacuum and a warm water bath, and i l i e residue was dissolved in 2 ml. of dry redistilled benzene. This solution was adsorbed on a 4- to 5-cm. column in a Hennessy tube containing Floridin XXS ( Floridin Co., Warren, Pa.) Ireviously treated with stannous chloride ( 7 , 8). When the level of the liquid reached the top of the column, 2 ml. more of benzene were added. This was repeated with 6 ml. of benzene. All of the benzene passed through the column was collected in a 10-ml. volumetric flask and made to volume, and an aliquot containing 20 t o 120 micrograms of tocopherol was evaporated to dryness with the aid of suction and warm water. The residue was tiissolved in 8 ml. of purified absolute ethyl alcohol (20).
This procedure completely removed vitamin -4and carotene from the extracts or from solutions of the pure compounds, and the recovery of added tocopherol was 96 to 104%. r-Tocopherol was adsorbed and eluted as readily as a-tocopherol. Traces of water or fat in the ext.racts diminished the flow rates of the columns markedly. Positive pressure (air or nitrogen) was applied to the occasional slow-flowing column. Increased oxygen pressure in this step is undesirable, but probably involves less loss than that due to prolonged evposure of tocopherol to the adsorbent. Freshly prepared adsorbent sometimes resulted in columns with flow rates of 20 ml. or more per hour; and under these conditions a-tocopherol was not always eluted quantitatively. Hence, columns that passed 10 ml. of benzene in less than 45 minutes were discarded. The new adsorbent Florex (Floridin Co.), which is similar to Floridin but with a greater adsorptive capacity, produced columns with rapid flow rates more often than did Floridin of the same mesh. For Florex, therefore, a finer mesh appears desirable. Others have destroyed vitamin .4 and carotene by treatment with concentrated sulfuric acid ( I O ) , but this procedure converted cholesterol into derivatives which interfered with the determination of tocopherol ( 5 ) . Controlled oxidation of carotenoids and tocopherol with subsequent reduction of the tocopherol has also been suggested (6). In the present study hydrogenation over palladium according to the technique of Quaife and Biehler ( I d ) inactivated solutions of carotene or vitamin A, and the recoveries of tocopherol from mixtures ranged from 90 to 99%. This procedure appeared satisfactory when applied to most distillates, but when it was applied to the nonsaponifiable matter of rat tissues, hydrogenation increased the values for tocopherol by 53 to 254%. The apparent tocopherol content of yeast increased 185% on hydrogenation. A4ftermolecular distillation of the nonsaponifiable matter from these materials, hydrogenation increased the apparent tocopherol of the residue but not of the distillate. A curious feature of this increase was that it was roughly proportional to the tocopherol content of the tissue rather than to the amount of tissue analyzed ( 1 7 ) . Conceivably, this effect could be due to easily reduced materials such as quinones or semiquinones, or to a bound form of tocopherol with the rather unlikely properties of being soluble in petroleum ether, and liberated by relatively mild hydrogenolysis but not by saponification. Color Reaction. The concentration of tocopherol in the eluates
was measured with the Emnierie-Engel reagents according to the procedure of Quaife and Harris ( 1 4 ) .
.4blank was prepared with 8 ml. of absolute ethyl alcohol, and 1 ml. of 0.5% of 1,l’-bipyridine in ethyl alcohol was added. The galvanometer of the Evelyn colorimeter was set a t 100 with the reagent blank and the corres onding “center setting” was used when reading the unknown tuies. The blank tube was removed and 1 ml. of 0.2% of ferric chloride in absolute ethyl alcohol was run in from a rapid-delivery pipet. After shaking for about 5 seconds, the per cent transmittance was read a t 515 mp exactly 15 seconds after the addition of the last ferric chloride. This was repeated for each of the tubes containing a sample in 8 ml. of ethyl alcohol, and the amount of tocopherol was calculated from of the sample minus LSlj of the blank. In the formula the Ls,, C = K L , K = 171 where C gives micrograms of a-tocopherol per tube. Reproducibility and Recovery of a-Tocopherol. A sample of beef liver was cut into small pieces and mixed, and five aliquots of about 7 grams each xere analyzed for toropherol. The values obtained were 8.00, 7.88, 7.33, 7.89, and 7.40 micrograms per gram with an average of 7.70’& 0.14. In twelve recovery experiments for the entire analytical procedure, 50 to 500 micrograms of a-tocopherol were added to samples of muscle, liver, or other tissues, and an average of 08 i 0.8% of the added tocopherol was recovered Table 11. Tocopherols in Tissues of Young Adult Male Kats AIaintained on 1 Mg. of a-Tocopherol per Day .\Ii.thod Literature - ~ _ _ Prepent _ Organ -/, 0 1 O J I l -(/gram r/organ Liver ?21 ?= 72a 29.4 & 9.4“ 269 Sinall intestinr 42 0 =t2 8 . 3 8 . 2 zt 4 . 3 171 Muscle 4 . 0 5; 3 . 1 ... Kidneys 24.1’2 7.3 13.0: I3 . 1 24.0 Heart 28.0 i 7 . 1 3 5 . 4 =t 6 . 4 31.0 Testes 2 5 . 4 ?= 1 7 . 1 8.5* 5.8 72.0 Brain 6 . 4 2 2.4 Lung 3;. 9’%.2:, 8 20.1 i l 2 . 3 45:O Y 5; 6 . 2 13.8 I 14 0 Spleen 30.0 1 9 i 1.0 Fat ... ... All but liver, muscle. and i:itc>aiirip are 1,ooIed values. 0 Standard deviation. C).
Value (161 r/mm 25.2 3 . 7 “gut” 13.3
11.8 34.2 22.6
16.2
32.4 51.0 60.0
Comparison with Other Procedures and Results of Others. Several rat organs were analyzed for tocopherol by the chromatographic procedure and by the method of Quaife and Dju (IS). The apparent tocopherol content of the materials examined was usually less when determined by the “saponification-chromatographic” procedure than when measured after distillation and hydrogenat,ion (Table I), although the recoveries of added CYtocopherol were adequate for the procedure giving the smaller valbes.
Table 111. Tocopherol Content of Oils and Meats
Corn oil Cottonseed oil Wheat germ oil Beef liver Beef muscle Pork chop Lamb chop
Total Tocopherol, Mg.:100 Grams Present method Lit. values 86.0,88.8 102 76.Q , 7 7 . 6 262,271 1 . 6 7 , l .88 0 . 2 6 , O . 27 0 . 1 1 . 0 .I? O.(i6,0 68
87
268 1.40 0.33 0.71 0.77
Ref. (11) (11) (11)
8( 41)
(4)
When tissues were examined from male rats maintained on 1 mg. of d-a-tocopherol per day for several months, the values obtained were similar to those reported by Quaife and coworkers ( 1 5 ) for liver, heart, and kidneys, but the amounts of tocopherol in all other tissues, particularly in spleen and abdominal fat, were lower than those reported previously (Table 11). [The diet was composed of glucose monohydrate (Cerclose) 79, extracted casein 12, lard 5, salts 4 with.vitamins added a t the following levels in milligrams per kilogram of diet: thiamine 3, riboflavin 2, pyridoxine 2.5, calcium pantothenate 7.5, and choline 1000. Eaoh rat received 2 drops of haliljut liver oil every 4 weeks.]
ANALYTICAL CHEMISTRY
760 There was no loss when a-tocopherol was added to duplicate samples of spleen or of fat. In three oils and four cuts of meats analyzed in duplicate (Table 111), the amounts of tocopherol found were similar but usually less than those reported by others
( 4 , 9J
ll).
Because so many substances other than tocopherol reduce the Emmerie-Engel reagent, it might be argued that the method giving the lower values for tocopherol is the more accurate. This is almost certainly the case for brewers' yeast, which contains so little tocopherol that it has been used routinely in basal diets for the bioassay of vitamin E. According to the saponificationchromatography method, this material contained 1.03 micrograms of tocopherol per gram, whereas by the distillationhydrogenation method ( I S ) the value was 4.17 micrograms per gram (I?'). Until there is an absolute standard against which the accuracy of tocopherol determinations can be evaluated, Stern's rule of thumb (16) may be helpful. If the apparent tocopherol content of the sample does not increase more than 10% during the interval from 2 to 10 minutes folloiving the addition of ferric chloride to the tubes under his conditions, he assumes the color mearured is due to tocopherols.
( 2 ) Dunford, R. A , , Can. Chem. Process Inds., 3 5 , 4 7 (1951). (3) Farber, hl., hlilhorat, A. T., and Rosenkrantz, H., Federation Proc., 10, 294 (1951). (4) Harris. P. L.. Quaife. M. L.. and Swanson. W.J.. J . 9uLrilion. 40, 367 (1950). ' Hines, L. R., and Mattill, H. A,, J . Biol. Chem., 149,549 (1943). Kachmar, J. F., Boyer, P. D., Gullickson, T. V., Liehe, E., and Porter, R. M., J . Xutrition, 42,319 (1950). Kidlhede, K. T., quoted by Kanntorp, H., and Xordlnind. G., Ark'iv Kemi Mineral. Geol., 25, 1 (1947). Kj$lhede, K. T., Z.Vitaminforsch., 12, 138 (194%). Kofler, M,, Hela. Chim. Acta, 28, 26 (1945). Parker, IT. E., and McFarlane, W. D., Can. J . Rescarch, B18, 405 (1940).
Quaife, hl. L., J . Biol. Chem., 175,605 (1948). Quaife. hI. L.. and Biehler, R., I b i d . , 159,663 (1945). Uuaife, &I. L., and Dju, bII. Y., I b i d . , 180,263 (1949). Quaife, M. L., and Harris, P. L., IND.ENG.CHEM.,ANAL.ED., 18,707 (1946). Quaife, &I. L., Swanson, W.J., Dju, A l . Y., and Harris, P. L., Ann. S . Y . Acad. Sci., 52, 300 (1949). Stern, b l . H., quoted by Baxter, J. G., Biol. Symposia, 12,484 (1947).
Swi'ck, R. IT.,Ph.D. thesis, University of Wisconsin, 1951. Tosic, J., and Moore, T., Biochem. J., 39,498 (1945). Wanntorp, H., and Nordlund, G., Arkia K e m i Mineral. Geol., 25, 1 (1947).
RECEIVED for review August 24, 1951. Accepted October 25, 1951. Pub-
LITERATURE CITED (1) Binnington, D. S., quoted by Chipault, J. R., Lundberg, It-.O., and Burr, G. O., A d i . Uzochem.. 8,321 (1946).
lished with the approval of the director of the Wisconsin Agricultural Experiment Station. Work supported in part h y the Wisconsin hlumni Research Foiindation.
Hydrazones, Semicarbazones, and Other Nitrogenous Substances Requiring a Reductive Pretreatment '4 Semimicro-Kjeldahl Procedure \ E L M E R B. FISH W m . H . Chandler Chemistry Laboratory, Lehigh University, Bethlehem, Pa.
CRING the past few years the micro- and semimicroKjeldahl determination of nitrogen has become increasingly popular in research and control laborat'ories. The Kjeldahl method presents certain advantages over the Dumas method. The time required per analysis is less, because several samples may be analyzed simultaneously. Recent developments in the adaptation of the Kjeldahl method to the micro or semimicro scale and the discovery of very efficient Kjeldahl catalysts have greatly reduced the time required for conversion of organic nitrogen to ammonium salts. There are, however, certain recognized limitations in applicability of the Kjeldahl method which require various modifications of the usual procedure. If the nitrogen atom is attached to either oxygen or another nitrogen atom a special reductive pretreatment is essential before the usual digestion with sulfuric acid in the presence of a catalytic mixture. Certain nitrogen heterocyclic compounds are very resistant to the usual method of acid digestion and require an extended afterboil. Ogg and Willits ( 7 , 1 4 ) and Kirk (6) have brought together a great deal of information regarding the conditions that are most favorable to the application of the Kjeldahl method to refractory compounds. Many of the recommendations cited by these authors are applied in the method of Cole and Parks (3). The catalyst used by Cole and Parks consisted of a mixture of mercuric oxide, selenium, and potassium sulfate, which was satisfactory for use with certain refractory nitrogen compounds if an afterboil of 1 hour was used. The 1-hour afterboil is much less than that recommended bj. Shirley and Becker ( 1 2 ) and others (6, 7 ) . Pat.el and Sreenivasan (8) found that if the afterboil were extended to 6 hours considerable nit>rogen was lost. They found a mercuric oxide-selenium mixture t'o be a more
effective catalyst than either alone m-hen used on refractory nitrogen compounds. A search of the literature for a satisfactory method for use with compounds containing N-S and YO2 groups disclosed that the Friedrich ( 4 ) method, which is highly recommended by Clark ( 2 ) ,is probably the most widely accepted for compounds of this type. Several methods have been used for reducing the nitro group, such as the use of sodium hydrosulfite ( I I ) , zinc dust and acid ( I S ) , sodium thiosulfate (1, 5, 9, IS), and potassium iodide with sulfuric acid ( I O ) . The Friedrich method of reduction with hydriodic acid is satisfactory when correctly applied to derivatives of hydrazine, semicarbaeones, or azo, nitro, and nitroso compounds. However, the elimination of the iodine by steam distillation following the addition of sulfuric acid and water is slow and tedious. The methods described in this paper are designed to eliminate the need for hydriodic acid treatment in the analysis of derivatives of aldehydes, ketones, and other substances requiring a special reduction treatment. The pretreatment is simple and the extra time required is about 30 minutes. The material is dissolved in acetic acid and methanol, reduced by the action of zinc and hydrochloric acid, and digested, and the nitrogen is determined as described by Cole and Parks ( 3 ) . APPARATUS
.i microbalance was used to weigh samples containing 20% or more of nitrogen and others were weighed on a balance adjusted to a precision of 0.03 mg. The samples were weighed in tin-foil cups made by cutting out circles of pure tin foil with a S o . 15 cork borer. The circles nere formed into cups by shaping them over the end of a 0.16-inch plastic rod. The average weight of the sample cups thus prepared x a s about 140 mg.
