24 hours, pulled thrwgh the solution of ninhydrin, and again left in darkness at room temperature for 12 hours. The colored spots are cut out, weighed to the nearest milligram (9), cut in strips, and placed in test tubes. For lysine and arginine 10 ml. of 50% ethyl alcohol are added and for histidine 5 ml. of 50% ethyl alcohol are added. The tubes are shaken for 30 minutes, the contents are filtered, and the color intensity is read at 570 mp. Absorbance for hydrolyzate is divided by the absorbance for standard, and the factor obtained is multiplied by the amount of standard amino acids applied on paper. From these data the percentage of individual basic amino acids in the hydrolyzate is calculated (3).
Table I. lysine, Arginine, and Histidine in Casein as Determined by Horizontal Paper Chromatography
(Results expressed against 16 grams of nitrogen) Chromatogram Lysine Arginine Histidine 1 2 3 4 5 6 7 8 9
Av. Std. dev. (6)
8.72 8.74 8.66 8.40 8.63 8.69 8.55 8.83 8.55
8.67
3.69 3.70 3.98 3.82 3.80 3.81 3.78 3.73 3.75 3.78
3.10 3.04 3.30 3.04 3.04 3.04 3.08 3.09 3.20 3.10
0.139
0.087
0.089
n
.
on caseinic hydrolyzate are shown in Table I. The standard deviation for these amino acids shows that these determinations might be repeated with good accuracy. These values are in good agreement with literature values (1, 41
LITERATURE CITED
(1) Bergdoll, ?*I. S., Doty, D. M., IND. EKC.CHEM.,A K A L . ED. 18, 600 (1946). (2) Block, R. J., “Paper Chroniatog-
raphy,” Academic Press, New York,
1952. (3) Fischer, F. G., Dorfel, H., Biochem. 2. 324, 544 (1953). 14) Guirard. B. hl.. Snell. E. E.. Williams. R.J., Proc. Soc.‘ Exptl. Biol: Med. 61; 158 (1946). (5) Himes, J. B., Metcalfe, L. D., ANAL. CHEM.31, 1192 (1959). (6) Roberts, H. R., Kolor, 111. G., Ibid., 29, 1800 (1957). (7) Ibid.. 31. 565 (1959). ? S i Rob&,”. R:, Kolor, M. G., Nature 183, 460 (1959). (9) Thompson, F. J., Morris, J. C., Gering, k‘. R., ANAL.CHEM.,31, 1028, 1031 (1959). (10) U.S.Dept. Agr., Washington, D. C., “Amino Acid Content of Foods,” 1957. \
RESULTS AND DISCUSSIONS
In Figure 1 is shown a chromatograni obtained after the solution of basic amino acids (standard solution and casein hydrolyzate after elution from Dowex 50-X4 resin) was chromatographed for 3l/* hours at 40’ C. by paper and phenol, both buffered at p H 6,8. The results obtained from the determination of the three amino acids
hydrin was much better if carried out at room temperature than a t any other increased temperature. Blanks were not taken into consideration, as the determinations of standard amino acids on the same paper were available. Only the size and weight of the spots should be taken into consideration, to be as accurate as possible.
lo).
The development of color with nin-
,
\ - I
RECEIVEDfor review May 11, 1960. Accepted February 27, 1961.
n
bpurious Kecovery Tests in Tocopherol Determinations V. H. BOOTH Dunn Nutritional laboratory, Milton Road, Cambridge, England
b Fat solvents contained small amounts of reducing substances that simulated tocopherols by reacting with the ferric chloride-bipyridyl reagent. In proving new methods these substances enable 100% recovery to be reported, even though some tocopherol has really been lost. The substances were separable from tocopherols by paper chromatography, but were not always separable by column chromatography. Reducing substances were also found in solvents after they had percolated through columns of various adsorbents.
I
N DISCUSSIONS on the determination of tocopherol, while attention is given to removing interfering substances from the material to be analyzed, little has been written about artifacts introduced into the system by the reagents. Writings on determination include a comprehensive review by Dicks (4), general articles by Lehman (11) and by the Analytical Methods Committee ( I ) , and paragraphs on the Emmerie-Engel test or other particular aspects by Kjolhede @), Edis-
1224
ANALYTICAL CHEMISTRY
bury, Gillow, and Taylor (6), and Diplock, Green, Edwin, and Bunyan (6). .Most authors give directions for purifying ethyl or alcohol diethyl ether but not always other solvents. For example, Lehman (11) says the absorbance in the control can be reduced by distilling ethyl alcohol. Quaife and Dju ( 1 2 ) say “petroleum ether must be purified owing to impurities which give an Emmerie and Engel reaction,” and refer to Werner: The paper by Werner ( 1 4 ) , however, is on etherpresumably diethyl-not petroleum. Hobson-Frohock (8) observed a n effect of water on the color developed in the Emmerie-Engel reaction. Rindi (IS) reduced error by making a complete blank run in every test. Some authors circumvent reducing reactions, and measure absorbance a t 292 m,u; Lambertsen and Braekkan (10) did this and corrected for irrelevant absorbance by a geometric procedure. These references provide scanty information on reducing substances present in solvents, and on tocopherol simulators eluted from ndsorbents. In this paper experiments are de-
scribed that show how such artifacts may interfere with accuracy, so that apparently complete recoveries have little meaning unless the recovered tocopherol is properly characterized.
BlPYRlDYL COLOR TEST
In most methods for the determination of tocopherols the lipides are extracted, saponified, and chromatographed, and a modified EmmerieEngel test is done on the residue. In this test tocopherol reduces ferric salt to ferrous, which reacts with 2,2’bipyridyl (2,2’-bipyridine) to give a red color. The modification used in the present work was that of the Analytical Methods Committee (1). Standard procedure included a blank test for subtracting from each reading. With 3.5 ml. of 0.072% bipyridyl 0.5 ml. of 0.2% FeCI,.6H20, both in ethyl alcohol (f), and optical path of 1 cm., this blank absorbance \$as usually about 0.060 after 2 minutes in the dark. With more dilute reagents the blank was lower.
+
UNSPECIFICITY OF COLOR TEST
The bipyridyl test is sensitive but unspecific. Any ethyl alcohol-soluble substance that reduces ferric chloride to ferrous, that escapes purging, or that is introduced during the procedure, may be recorded as tocopherol. ARTIFACTS IN SOLVENTS
I n the penultimate stage of most methods for their determination, the tocopherols are extracted or eluted into a polar solvent, which is then evaporated before the bipyridyl color test is applied. Benzene is popular; acetone or diethyl ether in light petroleum, and other solvents, are also used. After 25 ml. of analytical grade benzene was evaporated and the residue tested with bipyridyl and ferric chloride, it simulated atocopherol contents of 5, 5, and 9 pg. on different occasions. The unknown reducing substance may consist of one or many compounds. It, or they, have been found in all solvents tested. The amount found varied from bottle to bottle and was least in light petroleum and in recently distilled solvents. There was, for example, only little in freshly distilled benzene (Table I, test 5). Because the variation was considrrable, the values observed may not be typical and are not presented here. Diethyl ether may contain a reducing substance purposely added to inhibit the formation of peroxide. TOCOPHEROL SIMULATORS FROM ADSORBENTS
In many methods crude tocopherolcontaining extracts are purified chromatographically. Of several proposed adsorbents, the most popular are based on magnesium silicate-e.g., Floridin and Florisil-although alumina (IO), Decalso (6), magnesium phosphate (0, and others are also in use. When freshly distilled solvent was passed through columns of these materials, evaporated, and tested, the bipyridyl color sometimes exceeded that due to solvent alone. As a column was washed with successive amounts of solvent, the bipyridyl test progressively lessened. The removal of SnClz from Floridin columns (9) by solvents accounted for only part of the bipyridyl color. The almost ubiquitous occurrence of reducing substances means that if a tocopherol solution is evaporated, a bipyridyl test on the residue may produce a false reading. Such falsity is exemplified in the following experiments. OVERCOMPLETE RECOVERY
Light petroleum (40-60') was used to dissolve dl-a-tocopherol to a concentration of 14.8 pg. per ml. Chromatographic columns of Decalso F (5) 1.3
Table 1.
False Gain in Tocopherol Content by Passage through Chromatographic Column of Decalso Test No. 1 2 3 4 5 6 a-Tocopherol solution, m1.G 0 1 1 0 0 1
+
0 0 Decalso column 0 0 25 25 0 0 25 25-k Benzene, ml. Gross absorbanceb at 520 mp 0.060 0.211 0.315 0.167 0.064 0.216 Minus blank from test 1 -0.060 -0.060 -0.060 --0.060 - - _ _-0.060 _Net absorbance 0.151 0.255 0.107 0.004 0.156 Uncorrected tocopherol, pg. 25.0 15.3 Recovery, % 169 103 Net absorbance, as above 0.255 Minus net absorbance, test 4 -0.107 Corrected net absorbance 0.148 Tocopherol, pg. 14.8 14.5 Net recovery, % 100 98 14.8 pg./ml. b Of residue after evaporation of solvent and treatment with bipyridyl-FeC13 reagent.
cm. in diameter by 2 cm. high were washed with light petroleum. Tocopherol solution (1 ml.) was measured onto a column and eluted with 25 ml. of freshly distilled benzene. The solvent was evaporated and the residue tested with bipyridyl reagent. The results are shown in Table I. The net absorbance in the no-column control (test 2) of 0.151 multiplied by the conversion factor 98 (1) gives 14.8 pg. of tocopherol found, as expected. The recovery of 169% after passage through Decalso (test 3) is beyond expected error and patently absurd. However, when the complete blank (test 4) is subtracted, the recovery is reduced to (0.255 - 0.107) X 98 X 100/14.8 = 98%. PAPER CHROMATOGRAPHY
Whatman No. 4 filter paper was treated with zinc carbonate (7), and chromatographic runs without tocopherol were made in two dimensions (1). A piece of the paper the size of a tocopherol spot was cut out and tested with bipyridyl-FeC13 reagent. The paper increased the blank absorbance slightly: typically from 0.060 to 0.063 (8). Benzene was passed through a Decalso column as in test 4 (Table I), concentrated to small volume, and applied to paper. After running the chromatogram ( I ) , a piece of the paper was cut from the position where atocopherol ran in a parallel test, and treated with bipyridyl reagent. The absorbance was 0.063, the same as the paper blank: The reducing substances had been eliminated. Moreover, no spot was seen during examination of the fluorescein-treated paper under ultraviolet light. The eluate from a duplicate of test 3 was also run on paper in two dimensions. The a-tocopherol spot produced a gross absorbance of 0.208. Less blank 0.063 this represents (0.208 - 0.063) X 98 = 14.2 pg. The results are means of duplicates. The precision is indicated by the relative standard deviation for one ob-
servation: This was 7.4%, calculated from the readings for 16 tocopherol spots after running each paper in two dimensions. The unknown reducing substances from columns of other adsorbents or from solvents-even the considerable amounts in diethyl ethers purchased from two different suppliers-were always removable from tocopherol by twodimensional paper chromatography. CHROMATOGRAPHY ON FLORIDIN EARTH XS
An experiment with Floridin, formerly the most favored adsorbent for purifying tocopherols, illustrates another example of false recovery. Floridin was boiled with SnC12 in HC1 (9),made into columns 1 cm. in diameter and 3 cm. high, and washed (1). A solution of a-tocopherol in light petroleum (10 pg. per test) was applied to the columns, which were developed with 20 ml. of 2y0 ethyl alcohol in 30-40' light petroleum. The eluate was concentrated in a stream of nitrogen while being gently warmed, applied to paper impregnated with zinc carbonate ( 7 ) and fluorescein (f), and run in two dimensions. Controls showed no loss during the evaporation, yet only 6.4 pg. per paper was recovered. When five columns were washed with a further 40 ml. of 2% ethyl alcohol, the combined eluates contained negligible tocopherol, showing t h a t recovery was not much improved by using more eluent. TJnder ultraviolet light having a maximum near 250 mp, a new spot was seen on each paper close to a-tocopherol, though it was invisible under light having a maximum near 360 mp. The substance was evidently produced during adsorption of atocopherol on Floridin. The substance ran closely below a-tocopherol in the first dimension, and not far behind tocopherol in the second-that is, it was less polar-and it gave a reading with the bipyridyl-FeC13 reagent. If the VOL. 33, NO. 9, AUGUST 1961
0
1225
second-dimension chromatographic stage were omitted, this compound would be included with a-tocopherol. The transformation may be comparable with the conversion of ubiquinone to ubichromenol by Florex XXS reported by Crider, Alaupovic, and Johnson (3). Tests were done with other batches of Floridin and with other eluting solvents, and complete blanks. The loss of tocopherol was variable, of the order of 3 pg. or more. On the other hand benzene, or mixtures of light petroleum with ethyl alcohol, acetone, methylene chloride, or other solvents, when passed through columns of Floridin, extracted reducing substances approximately equivalent to 3 pg. of tocopherol. Hence 100% recovery could be simulated, even though part of the tocopherol had been lost. Although in this example the gain balanced the loss, both are variable and not always similar. The loss of tocopherol increased only slightly with the amount applied to a column; in other words, the percentage recovery improved with the amount used.
I n methods such as the Analytical Methods Committee's (1) in which 1000 pg. or other large amounts of tocopherol are chromatographed, and only a small aliquot is used for later stages, a loss of a few micrograms might pass unnoticed. With other materials than seed oils or concentrates, much smaller amounts of tocopherol may have to be taken through all stages. In such cases small losses represent large percentages, which would be detected if other reducing substances were eliminated.
LITERATURE CITED
(1) Analytical
Society
for
Methods Committee, Analytical Chemistry,
Analyst 84,356 (1959). (2) Bro-Rasmussen, F., Hjarde, W., Acta Chem. Scand. 11,34 (1957). (3) Crider, Q. E., Alaupovic, P., Johnson, B. C., J . Nutrition 73,64 (1961). (4) Djcks, M., Feedstufls 1960,92. (5) Diplock, A. T., Green, J., Edwin, E. E., Bunyan, J., Biochem. J . 76, 563 (1960). (6) Edisbury, J. R., Gillow, J., Taylor, R. J., Analyst 79, 617 (1954). (7) Green, J., Marcinkiewicz, S., Watt, P. R., J . Sn'. Food Agr. 6,274 (1955). (8) Hobson-Frohock, A., Analyst 84, 567 (1959). (9) Kjolhede, K. Th., 2. Vitaminfwsch. 12,138 (1942). (10) Lambertsen, G., Braekkan, 0. R., Analyst 84, 706 (1959). (11) Lehman, R. W., Methods of Biochent. Anal. 2, 153 (1955). (12) Quaife, M. L., Dju, M. Y., J . Biol. Chem. 180, 263 (1949). (13) Rindi, G., Intern. Reu. Vitamin Research 28,225 (1958). (14) Werner, E. A,, Analyst 58, 335 (1933). RECEIVEDfor review July 11, 1960. Resubmitted April 19, 1961. Accepted May 3, 1961.
CONCLUSION
Two-dimensional paper chromatography has advantages over other methods of purification, in that the tocopherol can be more certainly identified. The results above emphasize another advantage-that artifacts introduced during determinations can be eliminated. The possibility exists that alternative methods are not as good as recovery tests suggest.
Flame Photometry Using Oxycyanogen Flame J. W. ROBINSON Esso Research laboratories, Baton Rouge Refinery, Humble Oil & Refining Co., Baton Rouge, La.
b Many more metals exhibit line spectra in oxycyanogen flames than in oxyhydrogen flames. Of 40 metals examined, 29 emitted useful line spectra. These metals were contained in Groups I and II of the Periodic Table and the transition elements. The composition of the flame--.e., either oxycyanogen or oxyhydrogen- determined whether a metal exhibited line spectra or a continuum. However, the type of solvent determined the intensity of this excitation, whether it was line spectra or continuum. Important variables noted were feed rate, fuel-oxygen ratio, relative position of the flame in the inlet slit of the monochromator, burner design, and a steady flow of fuel-oxygen mixture to the burner. Use of this type of flame should considerably broaden the application of flame photometry and result in cheaper and faster trace metal analyses in samples for which the more expensive spectrographic equipment is now used.
I
1955 Baker and Vallee (1) demonstrated that a flame made from oxygen and cyanogen could be used as a source in flame photometry. The advantage of this flame was the temperatures reached-of the order of
4500' C., whereas the temperatures
reached in conventional oxyhydrogen or oxyacetylene flames are of the order of 3000" C. Further excellent work by Gilbert (4) has illustrated the potential of this high temperature flame. The object of this study was to define how much these developments have extended the usefulness of flame photometry and to obtain information on instrumental and fundamental advantages and limitations involved in this system. The principles involved are exactly the same as in conventional flame photometry. The variable which affects the intensity of emission is the number of excited atoms in the system. This is dependent on a number of variables, such as rate of feed, type of solvent used, and the combustion pattern in the flame. These factors are discussed later. Recent work (6) has shown that ambient
ultraviolet light in a flame appears to have a major effect on the intensity of emission spectra. It is probable that the ultraviolet light increases the number of excited atoms in a given atom population over and above those excited thermally. EQUIPMENT
The equipment used was a DK-1 spectrophotometer, 1P28 detector, a burner designed for use with this fuel, and an all-metal fuel line. Metal Fuel Line. Cyanogen is usually obtained as a liquid contained under pressure in metal cylinders. Unfortunately, it does not flow in a steady rate and this seriously affects the type and stability of the flame. It was necessary to control this irregular flow by inserting a constant pressure valve between the cylinder and the burner. Another difficulty
To Pilot Flame
Cyonogen
N
1226
ANALYTICAL CHEMISTRY
TO Main
Oxyprn
Flame 20' i8/8 Steel 0.085'l.D.
0.125. O.D. Coiled 2-1/2 Inches
i" Stainless 1.0 Steel I - 5ie'o.o.
Figure 1.
Metal fuel line
