1409
V O L U M E 2 4 , NO. 9, S E P T E M B E R 1 9 5 2 1. Variability in excitation. This appears to be the niost important source of vnriability. 2. Errors due t o the plates. 3. Instrumental errors in measuring t,he plate. 4. ,Uncertainty as to the silica cont,ent of electrodes and hase materials. SUhIRIARY
The technique described provides a convenient means for excit,ation of solid samples by the high voltage alternating current arc. The uniform, thin, adherent sample coatings obtained on t,he electrodes burn in a smooth, reproducible manner. ThiP gives more uniform exposures and better reproducibility in line intensity ratios. The tcchnique therefore permits high voltagcl
alternating current arc excitation, with its superior precision and sensitivity, to be extended to the analysis of nonconducting solid samples. ACKNOWLEDGMENT
It is a pleasure to acknowledge the assistance of Josephine Schulz with the experimental work described here. LITERATURE CITED
(1) Duffziidack, 0. S.,and Wolf? R., 10, 161 (1938). RI.Ct:IT-ED
for review J a n u a r y 18, 1951.
ISD. ENG.CHEM.,A N . ~ LED., .
Accepted J u n e 11. 1952.
Standardization of Adsorbent Mixtures Used in Vitamin A Chromatography J. B. W ILKIE ARD S. F.JONES Division of aVutrition, Food and Drug idministration, Federal S w i r r i t ) igeitcy, Washington 25, D . C . Cnexplained variations in adsorption chromatography have been attributed to many factors. This paper describes a method for evaluating or standardizing one of the more important factors-the strength or adsorptive capacity of the adsorbent. Quantitative determinations of adsorbent strength (as adsorption indexes in terms of grams of F8;D Butter Yellow No. 4 per gram of adsorbent) have been conducted with magnesia as well as w-ith magnesia-Celite mixtures. The study includes the effects of atmospheric constituents upon adsorbents a5 well as correlation of adsorbent strength with separation of vitamin A and carotene in mixtures. The separation of vitamin A and carotene was found possible through a w-ide range of adsorption indexes. Chromatography may be improved and made more dependable by the use of the proposed standardization technique.
C
HEMISTS generally recognize the advantages gained by the application of chromatography in certain quantitative chemical separations, but frequently avoid the use of this procedure, particularly for routine purposes. Unfortunately, poor pwformance of an adsorption column does not give a definite indication of the major cause of a difficulty. The activity of the atlrorbent, which has been found to vary in an uncertain manner, has often been the h s i s for criticism where such difficulties have been observed. The development of an easily applied method for an independent quantitativc estimation of an adsorbent activity was considered of importance for this reason, and it was felt that such a method would permit standardization of the adsorbent and result in a general improvement of the chromatography. Of particular concern in the determination of the vitamin A pot,ency of margarines was the standardizing of adsorbents used in the separation of vitamin A and carotene from impurities and fromeachother ( f , 2j. JIagnesiuni oxide-Celite adsorbent mixtures are in general chromatographic use and are of special interest because they are readily available. flexible in composition, and well adapted to t,he use of ultraviolet light for viewing bands of fluorescent substances on the adsorption column. The activitJ- of magnesium oxide has been evaluated by the use of an iodine number deter-
niination and found by the manufacturer to be satisfactory for control purposes ( 3 ) . It x a s considered important, however, to have a method more convenient for use by the chromatographer, a method that makes use of the common adsorption tools and usual spectrophotometric equipment. Repeated observations have shown t'hat the certified food dye, F&D Butter Yellow 90.4, is strongly adsorbed by magnesium oxide, and information from the Division of Cosmet,ics, Food and Drug *idministration,indicated that this dye is available with a purity of greater than 99%. These facts led to the experimental work reported in this paper. Various techniques viere tried for determining the amount of this dye adsorbable by various mixtures containing the active adsorbent magnesium oxide. The final technique makes use of an original stock solution containing 5 X gram of FBiD Butter Yellow S o . 4 per ml. in a purified petroleuni ctlier. (This petroleum ether must be substantially free from fluorescence arid have a transmittance of greater than 85% a t 300 mp when measured in a 1-cm. quartz cell against a no-cell blank set a t 100% transmittance. A product that neets this specification can he obtained from the J. T. Baker Chemical Co., Phillipsburg, S . ,J.) The authors' purpose w s to correlate the amount of this dye adsorbed viit'h the observed chromatographic behavior, using a petroleum ether solution containing vitamin A and carotene. To do this i t TYas necessary to develop the practical technique that would make the dyestuff adsorbed by a fixed sample, reliably and conveniently measurable. Initially i t was soon found possible to make a rough direct measurement by merely titrating a petroleum ether solution of dye into a sample of adsorbent suspended in petroleum ether until a definite color remained in the supernatant liquid after successive additions with continual stirring. After such an experimental approximation, the means for more precise measurements 1Tere determined experimentally. The method finally chosen depends upon adding under effective adsorption conditions an excess of the dye (as determined by an initial titration approximation above described), and then the amount of unadsorbed dyestuff was determined by spectrophotometric means. From the difference between total added dyestuff and the total excess dyestuff remaining after the adsorption step as calculated from the spectrophotometric adsorptions a t 438 nip, the total dye adsorbed is readily obtained, and this value may be designated as the Butter Yellow KO.4 index of
ANALYTICAL CHEMISTRY
1410 Table I. Adsorption Indexes for Adsorbent Magnesium Oxide Determined by Dye and Iodine Methods Type of
Llixture JIgO 31.0
Index (G. Dye/ G. MIX.,)Talue,
x
lIg0, %
2642
100
21.5 71 7.5
98
2641
IO0
'I!
54
10-0
,
TO.
.I
1 M g O : 1 Celite
2642
.i0
..i Ai' .i i4
1 3 1 ~ 01:Celite
2641
,j0
3 38 7 4.5
a Krstvaoo
Iodine
31goa
Clieinical Division. X e v l-ork. S . 1..
Table 11. Adsorption Indexes for l-arious IIixtrires Containing RIagnesium Oxide (2641) bIixture 3 M g O : 1 Celite 1 MgO: 1 Celite 1 hIgO : 1 Celite 1 M g 0 . 3 Cehte
Index (G. Dye/G. Mix) Values X 10-3 3.63
JIgO, %
Age of Prep.
75 .i0 J0 25
1 yr.
2.45
4 mo. 4 ma.
1 32
2 yr.
1.79
adsorption of activity in terms of grams of dye per grmi of adsorbent. With no further precautions than outlined abovc, check values are easily obtained nithin less than a 5% error. Experimentally certain precautions are necessary, especially in the manner of adsorption and of the thorough removal of the excess nonadsorbed or mechanically held dyestuff. The predetermined excess dyestuff is stirred vigorously with a 2-gram sample of the adsorbent and 25 ml. of the petroleum ether for 2 minutes for intimate adsorption. The adsorbent is then allowed to settle for about 5 minutes and the clear liquid is poured off into a 500-ml. beaker. The adsorbent is then transferred to a 250- or 300-ml. glass-stoppered Erlenmeyer flask and shaken with successive portions of petroleum ether (30 t o 50 ml.) until the supernatant liquid is colorless. Something less than six such rinsings are usually required. The original supernatant liquid and all of the rinsings after filtering through a fritted filter are combined and made to volume and the total dyestuff represented is calculated from the spectrophotometric absorption value a t 438 mu. This subtracted from the total dye added gives the amount adsorbed, and this divided by the grams in the sample gives the adsorption of dye in grams per gram of adsorbent This is designated as Butter Yellow KO. 4 (adsorption index) of the particular adsorbent. Calculation of Adsorption Index. Definitions of terms in formula: = grams of dye per ml. of stock solution actually used 5 X 1.71 X = grams of dye per unit of spectrophotometric absorbency a t 438 mu. (Each analyst should determine this value independently.) V O = volume of stock dye solution added to adsorbent V, = final volume as made for spectrophotometric measurement Valq = ml. aliquot of actual total unadsorbed dye V t = total ml. of unadsorbed dye A = absorbency of spectrophotometric solution at 438 mu in 1-cm. cell Index = (grams of dye added) - (grams of nonadsorbed dye) grams of sample Index = ( g r a m of dye/granis of adsorbent) =
[Vo X ( 5 X lo-')]
-
[1.71 X
X 1X
v Vtj 4
(2 is necessary t o reduce the actual gram sample t o the 1-gram or index basis.) Table I correlates results of the iodine method with those of the adsorption method and shows that the latter is applicable to magnesium oxide either alone or in mixture form. Magnesium oxide 2642 is about twice as strong as magnesium oxide 2641 by the older iodine number method. The nexer adsorption method shows the 2642 magnesium oxide to he ahout three timcs
as strong as the 2641 magnesium oxide. HobTever, as a result of mixing, about one half the strength of the stronger magnesium oxide (2642) was lost, since a 1 t o 1 mixture has only about one fourth the original strength in place of one half strength t o be expected if no loss in strength were incurred. On the other hand, the 2641 mixture, originally weaker, lost very little as the result of the mixing operation. This latter observation seems t o indicate that the originally weaker magnesium is more stable. Table I1 indicates relative strengths of similar miscellaneous mixtures which were currently available for chromatography in the laboratory. A11 these mixtures m r e stored under ordinary (not air-conditioned room) temperature and humidity oonditions, in tight, screw-cap bottles, kept tight except for the few minutes' exposure t o room atmosphere required while loading the chromatographic columns. The 2641 mixtures used in all work reported a t this time, except where the 2642 niivture is specifically mentioned. As might be expected, these mixtures are considerably weaker than the original mixtures of the same composition For example, as can be seen in Table I, mixture 2641 of 1 to 1 composition has an index of 3.5 X 10-3 in comparison with the corresponding values of 2.45 and 1.79 in Table 11. Table I1 might seem to show a discrepancy, as a 1-year sample is shown to havc a greater strength than a 4-month sample of the same composition. Holyever, a check upon the matter indicated that the +month sample was used more frequently than the 1year sample, thus making it appear that the loss in strength might be greater with greater frequency of use or exposure. Table I11 brings out more fully the effect of exposure to atmospheric constituents as well as the practical effects of such exposures upon separating vitamin A and carotene in a particular mixture. The Cmonth 20-hour samples in this table include two old adsorbents of different composition. Each of these compositions was exposed to carbon dioxide plus water, carbon dioxide done, and water plus nitrogen for 20 hours. All these exposure? were made by treating the stated mixtures in a "spread-out" condition to the dry gases or with moisture vapor released by passing nitrogen or carbon dioxide through water and over the adsorbent mixture in a partially closed container. In no case did any gross physical change occur and there xas no noticeable change in packing characteristics when columns were prepared after exposure. From various results of Table I11 it is evident that the combined effect of carbon dioxide and water was the most deteriorative, as evidenced by the approximate decrease of about 30 to 1 0 0 ~ in o the adsorption index, depending upon composition as well as the past history of the mixture. Each mixture reported in Table I11 was used in a conjunctive manner for an actual chromatographic separation of vitamin A and carotene. All performed adequately-that is, straight, well-separated, controllable bands of vitamin A and carotene were obtained except in the cases of mixtures exposed to both carbon dioxide and moisture vapor. In these cases no adequate bands were formed and the chromat,ography was out of control. Thus the limits of useful adsorbent activity can be determined by the Butter Yellow KO. 4 adsorption index. Several other interesting observations are possible based on the figures in Table 111. The freshly prepared mixtures are significantly stronger than the older ones of the same composition -but as the corresponding chromatography was successful in all cases except in carbon dioxide plus water treated mixtures, i t may be generalized that successful chromatography ie possible through a wide rangc of adsorption index but with a rather definite lower limit of about 0.325 X 10-3 gram of dye per gram of adsorbent. Holwver, absolute limits of usefulness cannot b e determined because of other unavoidable factors affecting the characteristics of the chromatography. Lest, the utility of this adsorption index be carried too far, certain other observations from Table I11 must be brought to attention. For example, in the first line, the adsorption index of t'he untreated mixtures not containing sodium sulfate, may be
1411
V O L U M E 24, NO. 9, S E P T E M B E R 1 9 5 2 Table 111. Dye Adsorption Indexes of Vitamin A Chromatographic Adsorption Mixtures (After exposure t o various atmospheric conditions.
x
10-8)
2641 Mixtures. Values
1 M g 0 : l Celite 3 M g 0 : l Celite:6 KazSO, 4-month Freshly 4-month Freshly ?ample prepared sample prepared i\ione 2.92 4.0 1.74 2.91 .. 0.325'' 20 hr. 0.oa COa Hi0 1.44 .. 20 hr. 2 3i Con 0.36 20 hr. 1 45 Ni Hi0 1:9i 2.ii 10 Inin. .. COS HzO 0 .37sn .. 0.oa .. con E80 45 niin. 45 min. .. 1.71 .. Cor HzO 1.28 2.3 .. Room atmos. 4 days ,. a Chromatographic effectiveness for iytamin 4 and carotene lost only in these samples. Treatment
+ ++ ++
.
-~~ _ _ _ _
~~
-
-
compared with those containing sodium sulfate. In both cases the mixtures that do not contain the sodium sulfate have the larger index, but actual chromatographic performance with carotene and vitamin A indicates that the sodium sulfate type is much stronger, as more compact bands are formed in this mixture. It appears that this kind of performance may possibly be accounted for by a mechanical increase of effective surface in the sodium sulfate adsorbent mixture. No doubt such a mechanical picture is oversimplified and could be elaborated upon from the standpoint of the electronic forces involved in such mixtures, but that is beyond the scope of this discussion. The data considered in Table I11 warn one that interpretive comparisons must be largely restricted to compositions of the
the same general type. The relative values given on the last line of Table I11 merely indicate the appreciable effect on adsorption index or activity by ordinary atmospheric exposure. The results of these studies indicate that it is possible to evaluate with precision the strength of a given adsorption mixture and that this evaluation may be correlated with chromatographic performance. Furthermore, it follows that such an evaluation of a magnesium oxide-Celite mixture determines the strength of the magnesium oxide itself from a practical viewpoint, since the Celite is inactive. In fact, a 1 to 1 mixture may be used advantageously for the evaluation of the magnesium oxide, as the distribution of dyestuff and the manipulative technique are thereby facilitated. Some time and material may be saved by adopting this procedure. as the mixture usually is the thing that has practical utility in the laboratory. I t is felt that a new and needed means is offered for more mccessful chromatography by the use of the adsorption index technique just presented in its relationship to the separation of vitamin .\ and carotene. LITERATURE CITED
(1) 11-ilkie, J. B., 1.Assoc. Ofic.Agr. Chemists, 32,455 (1949). (2) TTiikie, J. B., and DeWitt, J. B., Ibid., 28, 176 (1945). (3) Zettlemoyer, \. C., and Walker, W, C., Ind. Eng. Chent., 39, 69 (1947). RECEIVED for review April 15, 1952. Accepted July 3, 1952. Presented before the Division of Biological Chemistry a t the 121st Meeting of the AMERIC A K CHENICAL SOCIETY, Buffalo, N. Y.
Quantitative Paper Chromatography of D=Glucose and Its Oligosaccharides K. .J. DI-MLER, W. C. SCHAEFER, C. S. WISE, A N D C .E. RIST Y o r t h e r n Regional Research Laboratory, Peoria. Ill.
T
HE extension of paper chromatography to the qualitative
separation of mixtures containing di- and oligosaccharides ( I S ) has opened the way to solving the difficult problem of quantitative analysis of such mixtures. The procedure of quantitative paper chromatography presented here was established particularly for the analysis of mixtures of low molecular vieight polymers of D-glucose, such as those obtained in the enzymic hydrolysis of starch fractions ( 3 ) . I t is also directly applicable t o Dfructose and to oligosaccharides containing D-fructose alone or along with D-glucose. The present procedure combines the previously reported chromatographic techniques for oligosaccharides ( I S ) with elution of the resolved spots by an adaptation of the method of Dent ( 2 )and determination of the carbohydrate content by the colorimetric anthrone reaction (16,21). Quantitative paper chromatography of sugars has been described by several authors, usually in application to monosaccharides. Most of' their methods, however are not suitablr for the study of mixtures of glucose polymers of unproved structure and size. Thus the use of methods based on reducing power (6-7, I O , 18, E?),periodate oxidation ( I I ) , or comparison of spot intensity ( I > 8, 17) would be prevented by the lack of samples of the pure oligosaccharides for standardization and comparison. The ant,hrone method overcomes this difficulty, as it essentially determines the amount of monosaccharide which would be obtained on total hydrolysis. It offers advantages of speed and simplicity of operat,ion over other total carbohydrate methods, such as acid hydrolysis followed by determination of reducing power (6. 251, or distillation from phosphoric acid follolr-cd by
ultraviolet spectrophotometric measurement of hydroxymethyl furfural (24). On the basis of sensitivity the anthrone method is well suited to paper chromatography, as a determination ran be made in duplicate on 10 to 100 micrograms of carbohydrate. Since the studies described here were completed, a phenol-sulfuric acid procedure has been reported ( 4 ) for carbohydrate dctcrmination and applied in the quantitative paper chromatography of sugars in wheat flour ( I 4 ) , the known sugars being used for standardization. Evaluation of the merits and limitations of this method must await the publication of further drtails iPPIR4TLS $\I) REIGESTS
Paper chronmtography equipment, as described by Jeanes, Wise, and Dimler ( I S ) , or other suitable arrangements are satisfactory. In the present studies TJ-hatrnan No. 1 filter paper (45.5 X 57 cni. sheets) wab used. A stainless steel tray, 3 x 8 inches and 0.5 inch deep, holds six pairs of 2 X 2 inrh glass slide. in .z 10-inch diameter desiccator (see Figure 1). A spectrophotomet,er, mch as Coleman Universal spectrophotometer Model 11, is used. For the work reported here the cuvette for test, tubes was fitted with brass sleeves to accommodate selected 18 X 150 mm. borosilicate glass test tubes. The dinitrosalicylate spray reagent, contains 0.5 gram of 3,5dinitrosalicylic acid in 100 ml. of 1 S sodium hydroxide ( I S ) . -4mmoniacal silver nitrate spray reagent is prepared by dissolving 5 grams of silver nitrat'e in 95 ml. of water and adding 6 ml. (slight excess) of concentrated ammonium hydroxide ( l e ) . To avoid the danger of explosive silver residues, the solution should be freshly prepared and the unused portion discarded immediately in such a manner that no dr3- residue will form.
