Determination of, Starch in Plant Tissues GEORGE W. PUCHER', CHARLES S. LEAVENWORTH, AND HUBERT BRADFORD VICKERY Connecticut Agricultural Experiment Station, New Haven, Conn. Starch is extracted from a 50- to 250-mg. sample of dried plant tissue with perchloric acid as recommended by Nielsen, precipitated with iodine, and recovered as starch which is hydrolyzed and determined as glucose. The results are independent of the composition of the starch of different species with respect to amylose and amylopectin. Theoretical recoveries of glucose from pure potato starch are obtained; the experimentally determined factor is 0.90 with an uncertainty of =t1.2Yo0. The precision of the method as applied to a variety of plant tissues is approximately 29&
Ac9L
*ORIR.IETRIC method for determining starch in plant tissues mas developed in this laboratory 12 years ago (13) Revision of this method has become necessary because of two recent developments in the chemistry of starch. The first of these is the observation that plant starches are mixtures of t n o main components, usually referred to as amylose and amylopectin (1, 6-8, 15), which are present in more or less constant ratio in the starch from a given tissue but often in quite different ratios in the starches derived from different plant species. The second IS the introduction by Xielsen (10) of perchloric acid as an effective solvent with which to extract starch from the plant tissue as a step preliminary to its analytical determination. The earlier method depended upon measurement with a red tilter of the extinction coefficient of a solution of the starchiodine complex prepared after careful purification of the starch extracted v ith hydrochloric acid from the sample of tissue. This was compared with the extinction coefficient of a standard solution of potato starch; accordingly, the method is subject to error in those cases where the ratio of the amylose to amylopectin in the specific tissue analyzed differs appreciably from that of potato starch. The failure, in many cases, of the color values to agree with the results of titration of the sugar after hydrolysis of the starch is doubtless attributable to this circumstance. illthough correction factors can be determined, as has been suggested by Sielsen and Gleason ( 1 2 ) , it is not certain that such factors are valid for use in studies of metabolism where samples a t different stages of development or of different tissues from the same species are being euamined. Moreover, Schoch and Williams (16) have shown that the absorption of iodine by starch is repressed by the presence of fatty acid in the preparation. If this should result in variation in the intensity of the color, another source of error is possible. The revised method consists of extraction of the starch with perchloric acid, precipitation with iodine under conditions that have been shown to be quantitative (2, 13, 19), decomposition of the iodine complex, and determination of the sugar produced by hydrolysis of the starch. A maximum of 250 mg. of dry tissue is required; proportionately smaller quantities are sufficient if the Once the necessary fundastarch content is greater than mental values for the starch from a given tissue have been determined, in terms of sugar titration and in comparison with a standdrd such as a preparation of potato starch, the more rapid colorimetric procedure previously described can be used if desired in a series of determinations on the same tissue.
lso.
REAGENTS
Perchloric Acid, 72%, 11.2 to 11.7 N , reagent grade. Iodine-Potassium Iodide. Iodine (7.5 grams) and potassium iodide (7.5 grams) are ground with 150 ml. of water diluted to 230 ml., and filtered through No. 3 Whatman paper d i t h suction. 1
Deceased Sovrmhrr 20, 1447.
850
Alcoholic Sodium Chloride. Ethanol (350 ml.), water (80 ml.), and 207, aqueous sodium chloride solution (50 ml.) are diluted to 500 ml. with water. Alcoholic Sodium Hydroxide, 0.25 N . Ethanol (350 nil.), water (100 ml.), and 5 Y sodium hydroxide (25 ml.) are diluted to 500 ml. with mater and filtered as above. Hydrochloric Acid, 0.7 N . Sixty milliliters of concentrated hydrochloric acid are diluted to 1000 ml. Somogyi's Phosphate Sugar Reagent. Prepared as described by Somogyi (28) except that standardized potassium iodate solution is included by dissolving 56 grams of anhydrous disodium phosphate and 80 grams of Rochelle salt in about 1 liter of water and adding 200 ml. of 1.00 S sodium hydroxide; 160 ml. of 10% copper sulfate pentahvdrate are slowly added with stirring and then 360 grams of anhydrous sodium sulfate. When this is dissolved, the solution is transferred to a 2-liter volumetric flask and exactly 200 ml. of 0.1 AVpotassium iodate (3.5760 grams per liter) prepared with quantitative accuracy are added. The mixture is diluted to volume, allowed to stand for several days, and filtered through dry No 3 Whatman paper in a dry funnel into a dry filter flask, the first 50 ml. of filtrate being discarded. The reagent should be stored a t 20" to 25" C.; it is 0.01 AVwith respect to iodate and 5.00 ml. are equivalent to 10 in]. of 0.005 -V sodium thiosulfate. The effective range for the determination of sugar is from 0.05 to 1.0 mg. of glucose. Sodium Thiosulfate, 0.005 N . Sodium thiosulfate pentahydrate (2.73 grams) is dissolved in water and made to 2 liters. The solution is preserved in a dark colored bottle and standardized dailv against 5 ml. of the Somogyi sugar reagent; 1 ml. of 2.5% potassium iodide and 3.0 ml. of 1.5 .V sulfuric acid are added and the mixture is allowed to stand for 5 minutes before titration. Starch Indicator, prepared according to Peters and Van Slyke (12) as follows: one gram of potato or cornstarch is triturated ~ i t cold h rrater and the suspension is poured into 200 nil. of boiling water containing 200 mg. of salicyclic acid. The solution is boiled for a few minutes and allowed to settle and the clear supernatant fluid is decanted for use. In addition, 2.5% potassium iodide stabilized with a little sodium carbonate, 20V0 aqueous sodium chloride, 0.057, phenol red indicator solution, 0.5 .V sodium hydroxide, 0.1 S oxalic acid, and 1.5 S sulfuric acid are required. PROCEDURE
Preparation of Perchloric Acid Extract. It is convenient t o analyze four samples in duplicate simultaneously.
A 50- to 250-mg. sample (chosen according to expected atarch content) of dry poLvdered tissue and 200 mg. of sharp sand are transferred to a 200 x 25 mm. test tube together with 4 ml. of water. The tube is heated in a boiling nater bath for 15 minutes to gelatinize the starch, cooled to room temperature, and placed in a bath a t 22" to 25' C., when.3 ml. of perphloric acid are added rapidly with constant agitatlon. The tissue is then ground against the lower wall of the tube nith a stout stirring rod (a rotary motion is used and firm pressure is exerted) for a minute or so a t a time over a period of 15 to 20 minutes, each tube of the series with its stirring rod being returned to the bath while another is being treated; about 20 ml. of water are then added so as to wash the rod, the solution is mixed and centrifuged, and the clear solution is decanted into a small beaker and set aside. The residue is again treated with 4 ml. of m-ater and 3 ml. of perchloric acid
V O L U M E 20, NO. 9, S E P T E M B E R 1 9 4 8 and ground and extracted as before. The f i s t extract is then returned quantitatively to the tube with the aid of a little water. The rod is rinsed off and the combined extracts are diluted to 50 ml., and are mixed by shaking the stoppered tube vigorously. The tube is finally centrifuged and, if desired, the clear extract is decanted through a plug of dry glass wool in a funnel into another vessel; otherwise aliquots may be removed directly without decantation. Immediate analysis is to be preferred, but the solutions may be stored in the refrigerator for as long as 48 hours if necessary. Precipitation of Starch-Iodine Complex. An aliquot of the. starch extract of from 1 to 10 ml., depending on the starch content, is transferred to a test tube calibrated a t 10, 15, and 20 ml. and diluted to 10 ml.; 5 ml. of 20% sodium chloride and 2 ml. of iodine-potassium iodide reagent are added and the solution is mixed. After being allomd to stand for a t least 20 minutes, the tube is centrifuged and the supernatant fluid is decanted with extreme care to avoid loss of precipitate. The precipitate is then suspended in 5 ml. of alcoholic sodium chloride wash solution by gently tapping the tube, centrifuged, and the fluid is decanted. Decomposition of Starch-Iodine Complex. Two milliliters of alcoholic sodium hydroxide are added to the packed precipitate and the tube is gently shaken and tapped until all the blue color is discharged. A stirring rod must not be used and ample time for decomposition of the complex must be allowed. The liberated starch is then Centrifuged and washed with 5 ml. of alcoholic soIlium chloride as before. Hvdrolvsis of the Starch. TKOmilliliters of 0.7 N hvdrochloEic aGd are added to the precipitate, and the tube is co;ered with a glass bulb, heated for 2.5 hours in a constant-level water bath provided with a cover with holes to accommodate the tubes, and maintained in vigorous ebullition. It is important that holes not occupied bv tubes should be covered. The tube is cooled, a few drops of 0.04% phenol red are added, and the solution is neutralized with 0.5 AT sodium hydroxide: the color is discharged with the necessary amount of 0.1 Y oxalic acid and the solution is diluted to 10, 15, or 20 ml., according to the starch content, and centrifuged. Titration of Sugar. A 5-ml. or smaller aliquot of the hydrolyzed starch solution is transferred to a 200 X 25 mm. test tube (if less is taken, water to make up to 5 inl. is added), exactlv 5 ml. of the Somogyi phosphate reagent are added, and the tube, together with two or, preferably, three blanks that contain 5 ml. of water and 5 ml. of reagent, is covered with a glass bulb and heated in a vigorously boiling water bath for exactly 15 minutes. For this operation, a rack containing all the tubes of a series of determinations is employed, so that each is heated in the same way. The tube is removed nithout disturbing its contents and cooled to 25' to 30 O C., 1 ml. of 2.5.% potassium iodide is carefully added down the wall of the tube without agitation, and then 3 ml. of 1.5 Y sulfuric acid are added rapidlv TTith simultaneous agitation. After all the cuprous oxide has dissolved, the solution is titrated nith 0.005 iV thiosulfate, starch indicator being added when the titration is nearly completed. Thc blank solutions are treated similarlv. Calculation. The percentage of starch in the tissue is calculated from the equation S = 0.90 X 'Oo0 where S is the perWE'A rentage of starch, W is the weight in milligram; of the sample, E is the volume in milliliters of perchloric acid extract taken, V is the volume in milliliters of the starch hydrolyzate, A is the aliquot of the starch hydrolyzate taken (in milliliters), and G is the number of milligrams of glucose in A . G is the difference between the titration of the unknown solution and that of the blank multiplied by 0.129 and by the factor to convert the actual normality of the thiosulfate solution to0.005 A'. The coefficient 0.90 is the theoretical factor to convert glucose t o starch. The factor 0.129 is experimentally determined and depends upon a large number of tests with potato starch. In routine calculations of the results of similar determinations, the constants can be conveniently collected into a single constant, due attention being given to the aliquot ratios should these be different. ~
In carrying out the procedure, special care must be taken in decanting solutions from the precipitates of the iodine complex and from the starch so as to avoid loss of small particles. I t is also important that the boiling water bath used to hydrolyze the starch be covered, so that the contents of the tubes are heated to 100 C. for the necessary times. Open baths are not reliable. After a little experience has been gained with the method, the choice of aliquots to obtain suitable quantities of sugar for thtx h a 1 titration is easily made.
851 * Table I. Comparison of Colorimetric Methods of Nielsen and of Pucher and Vickery for Starch in Various Plant Tissues (Potato starch as standard) Nielsen Alfalfa atem Alfalfa root Tobacco leaf '
Xaize leaf Rhubarb rhizome Beet root
Pucher and Vickery
70
rc
4.06 6.09 1.56 3.10 2.76 33.8 0.20
4.01 5.85 1.53 3.06 2.96 33.1 0.15
EXPERIMENTAL RtiSULT5
Table I shuns a comparison of the starch cwntmt of several plant tissues as determined by the colorimetric methods, rcspectively, of Nielsen ( I O ) and of Pucher and Vickery (13). The data are expressed in terms of pure potato starch as a standard of comparison and serve to demonstrate that perchloric acid, as used in the Nielsen method, may be app1ic.d generally for the extraction of starch. The agreement between the tvio methods is excellent; however, in all save one case, the result by the Sielsen method is slightly higher, possibly evidence of the greater effectiveness of perchloric acid as a solvent for starch as compared with the hydrochloric acid used in the other method. Table I1 shows the quantities of glucose recovered from potato starch hydrolyzed directly, after extraction with perchloric acid, and after extraction and subsequent precipitation as the iodine complex. Within the limitations of the method, the extraction is clearly quantitative and there is no loss during the precipitation and decomposition of the starch-iodine complex. The potato starch was prepared as described by Pucher and Vickery and the weight taken was corrected for moisture and ash on the assumption that the organic residur n-as exclusively starch
Table 11. Recovery of Glucose from Potato Starch Glucose Found Direct hydrolysi-
Extraction with HClO4
45.5
50.2 50.1 49.8
51.0 50.9 51.0
92.2
102.0 103.0 101. A
103.2 103.2 102.0 103.0 103.0 103.2
Starch Taken
Extraction and precipitation with iodinr
.Mg. 49.4 *IO,0 50.7 103.2 103.2 103.2 101.5 103.2 102.0
Glucose Factor for Starch. Although many workers have made use of the theoretical factor 0.90 to convert glucose to starch, this magnitude has only rarely been verified experimentally Sotable among early examples mentioned in Ralton's bibliography (20) are the studies of de Saussure (14, in 1815, lvho obtained the ratio by direct gravimetric analysis, and of Fehlinp (5) in 1849, who used reduction of copper to measure the glucose Factors in the range from 0.92 to 0.95 have frequently been reported and such compromise values are often employed. Etheredge (41, for example, has recently maintained that the theoretical factor 0.90 should be revised upward. Several uncontrolled variable.. may contribute to the slightly high values for the ratio that are usually obtained; incomplete hydrolysis of the starch, the presence of moisture in supposedly dry preparations, or the variability of the moisture content with changes in conditions of storage of the sample of starch may be mentioned as well as the possibility of differences in the behavior
8
ANALYTICAL CHEMISTRY
‘852 Table 111. Factor to Convert Glucose to Starch NO.
of
Detns. Rydrolysis with 0.7 N HC1 6 HC!O4 extract 29 Iodine precipitate 29
Maximum 0.915 0.915 0.929
Minimum 0.900 0.884 0.893
Meana 0 . 9 0 6 =t0 . 7 % 0.900 i 1 . 2 % 0.905 f 1 . 2 %
a Uncertainty expressed as coefficient of variation-Le., tion as a percentage of mean.
standard devia-
Table IV. Analysis of Starches of Different Amylose a d Amylopectin Contents Expressed as Percentage of Weight of Preparations Starch Hydrolysis with 0.7 .\‘ HC1
Precipitation 1%-ith iodine
%
70
7c
5%
9b:3 91 . o 90.0
8?’4 90 4
8814
90.0
87 91
100 36 187 71
88.6 89.5
88.8
90.6
73 67
88.5
90.6 85 4 00.0
73 69 3
00 0
0
Organic Solids Potato starch Waxy maize starcha Amylose6 Amylopectin b Araucaria starche (Brazilian pine) Arrowroot starche Apio starchc (Arracacia es. culenta) Rice starchc Glycogen (animal) Lemon pectin
..
85.7 77.1 29.0
Colorimetric, potato starch standard
show that error from the presence of either of these substances ie entirely avoided by the precipitation with iodine. The analysis of glycogen is of particular importance, inasmuch as Morris and Morris (9) have observed the presence in certain varieties of maize of a substance indistinguishable from animal glycogen. Starch of Plant Tissues. Table 5’ gives the results of determinations of starch in a number of plant tissues. Although Nielsen advocated only a single extraction with perchloric acid, and experiment verified the fact that this is usually sufficient when analyzing preparations of pure starch, the quantitative removal of the starch from dried leaf and root tissues clearly requires two successive extractions. However, a third is not necessary. A few of the samples of Bryophyllum leaf were extracted three times: the second extract gave, as an average 0.73% more starch. I n 8 few cases, a trace of starch representing 0.0401, of the tissue wag found in the third extract. K i t h the root tissues studied, all starch n as removed in two treatments. The f i s t group of experiments with Bryophyllum leaves is presented only in summary the last experiments in the table show the negligible effect of size of sample on the results. The pairs of duplicate analyses illustrate the precision that i p obtained.
Table V.
The authors are indebted t o R. M. Hixon, Iowa State College, for this 3 amp1e, b Prepared according to the method of 1IcCready and Hassid (8). From collection of late T . €3. Osborne. a
of the various sugar reagents employed for the determinations. For example, Somogyi’s modified reagent S o . 50 (17) consistently gave results in this laboratory from 2 to 49&higher than the theoretical and occasionally gave erratic results when applied to the perchloric acid extract. On the other hand, the new Somogyi phosphate reagent was found to give essentially theoretical values, provided moisture determinations were carried out a t the same time as the sugar analyses. Illustrative examples are shoivn in Table 111, in which a large number of experiments on potato starch are summarized. The mean value within each group closely approaches the theoretical and the uncertainty (coefficient of variation) is only slightly over 1y0. ilccordingly, the us? of the theoretical factor 0.90 appears to be justified. Behavior of Starches of Different Amylose and Amylopectin Content. The present method, if it is applied to starches of different origin and different relative amylose and amylopectin content, depends on the assumption that both components are quantitatively precipitated by iodine. Comparison of the data of Clendenning and Wright (S), who examined a variety of starches with respect to specific rotation, n ith those of Steiner and Guthiie (19), who likewise observed the specific rotation but interposed the step of precipitation with iodine, would suggest that both components are equally well precipitated. Xevertheless, it seemed desirable to test the point upon samples of known widely divergent composition. Table IV s h o m the results obtained with several starches as well as n ith preparations of crude amylose and amylopectin. The mean value for starch in the first seven preparations after hydrolysis with hydrochloric acid was 88.6%, whereas the mean value after precipitation with iodine was 88.8Ye and it is obvious that complete precipitation occurs. The last column of Table IT shons the starch content as determined by the colorimetric method of Pucher and T’ickery, using potato starch as standard. The n ide difference betn.ecn the results in this column for amylose and amylopectin, as n ell as the irregular results for the starches of different origin, furnish a demonstration of the fallacy in the earlier colorimetric method, vhen a single kind of starch is used as the standard. The results with glycogen of animal origin and Kith pwtin
Analyses of Plant Tissue for Starch after Extraction with Perchloric Acid One Extraction
Two Extractione
% Bryophyllum leaf (1943) Maximum Minimum Alean Bryophyllum leaf F L
6.61 6.21 6.40 f 10.18 10.18 8.38 8.38 6.00
Bryophyllum leaf W 1 Bryophyllum leaf W2
6.00
Bryophyllum leaf M I
9.36 9.26 36.0 38.0 6.25 6.15 4.80
Rhubarb rhizome Alfalfa root 7639 Alfalfa root 7540
5.00
Alfalfa tops Tobacco leaf E O.l-gram sample 0.2-gram sample 0.5-gram sample Rryophyllum leaf (1943) 0.l-arani sample 0.25-gram sample ’i
b
% 7.24
6.87
3 O
7.05 f 1.7b 11.92 12.00 9.26 9.26 7.14 7.14 9.62 9.72 39.6 40.9 7.52 7.37 5.76 5.76
0.07 0.06
0.08 0.08
2.02 2,lO 2.09
..
6.50 6.30
7.04 7.14
..
27 determinations: uncertainty expressed as coefficient of variation 14 determinations.
Effect of Concentration of Perchloric Acid. Nielsen and Glcason showed that the conccnti ation of perchloric acid best suited for extracting starch is 4.8 -Y. This is attained if 3 ml. of 727, perchloric acid are added to a suspension of the tissue in 4 ml. of water. The alternative procedure of adding 4 ml. of 8.5X arid to 3 ml. of n ater was preferred by Xielsen and Gleason, as It diminishes the effect of momentary high concentrations of the acid. Comparison of the two techniques showed that, with pure starches, identical results Tvere obtained but that, with dried leaf tissues, the use of 72% acid gave consistently higher values. Thue the average of 6 determinations on Bryophyllum leaf with 72% acid was 6.47%, whereas 8.5 acid gave only 5.30%. Effect of Two Precipitations with Iodine. An examination of the possibility that a second precipitation with iodine might contiibute to the purification of the starch indicated that this p r e caution is not necessary. Table VI shom parallel analyses of several tiwues. To obtain the data in the last column, the precipltate 171th iodine TTas suspended in 207@sodium chloride and 1 ml. of 0.16 AI sodium thiosulfate solution was added. After trituration of the precipitate until the color \\as discharged, the r \A
V O L U M E 20, N O . 9, S E P T E M B E R 1 9 4 8 Tahle VI. Effect of One and Two Successive Precipitations with Iodine on Determination of Starch One Precipitation Potato Otarch Bryophyllum leaf 1 Bryophyllum leaf 2 Tobacco leaf Tobacco -tern Alfalfa root 1 Beet tops Beet root Rhubarb leaf
Two
Precipitations
%
70
91 .0 11.74 10.40 0.11
91.0
3.73
6.90 0.09 0.13 0.55
11.74
10,56 0.11 3.76
F.93
0.09
0.10 0.49
aolution was diluted to 10 ml. and 1 ml. of 2.3 S hydrochloric acid and 2 ml. of iodine reagent Tvere added. The precipitate was centrifuged after 30 minutes and treated according to the usual procedure. The agreement of the results with those obtained after a single precipitation is satisfactory. LITERATURE CITED ‘1) B a t e s , F. L.. F r e n c h , D., a n d R u n d l e , 65, 142 (1943).
853 (2) C h i n o y , J. J., Analyst, 63, 876 (1938). (3) C l e n d e n n i n g , K. A,, a n d W r i g h t , D. E., C a n . J . Research, B23. 131 (1945). (4) E t h e r e d g e , M . P., J . Assoc. Oficiald4gr. Chem., 2 7 , 4 0 4 (1944). ( 5 ) Fehling, H., Ann., 72, 1 0 6 (1849). (6) Hassid, W.Z., Quart.Rea. Bid., 18, 311 (1943). (7) H a s s i d , W. Z., a n d h I c C r e a d y , R. M., J . Am. Chem. S O C . ,65, 1157 (1943). (8) h I c C r e a d y , R. M . , a n d H a s s i d , W.Z., I b i d . , 65, 1154 (1943). (9) M o r r i s , D. L., a n d M o r r i s , C. T., J . B i d . Chem., 130, 535 (19391. (10) S i e l s e n , J. P., ISD.EXG.CHmf.,ASAL.E D . , 15, 176 (1943). (11) Kielsen, J. P., a n d Gleason, P. C., I h i d . , 17,131 (1945). (13) P e t e r s , J. P., a n d Van Slyke, D. D., “ Q u a n t i t a t i v e Clinical C h e m i s t r y , ” 1-01. 11, p . 34, B a l t i m o r e , Williams a n d K i l k i n e C o . , 1932. ENG.CHEM., -&SAL. ED.. (13) P u c h e r , G . IT7.,a n d Vickery, H. B., IND. 8, 92 (1936). (14) S a u s s u r e , T. d e , Ann. P h y s i k , 49, 129 (1815). (15) S c h o c h , T. J., J . Am. Chem. SOC., 6 4 , 2 9 5 7 (1942). (16) S c h o c h , T. J., a n d Williams, C. B., Zbid., 66, 1232 (1944). (17) Somogyi, M . , J . B i d . Chem., 117, 771 (1937). (18) Ibid.,160, 61 (1945). (19) Steiner. E. T., a n d G u t h r i e , J. D., ISD.ESG. C H E M . ,.&SAL. ED., 16, 736 (1944). (20) W a l t o n , R . P., “ C o m p r e h e n s i v e S u r v e y of S t a r c h C h e m i s t r y , ” N e w Y o r k , B o o k D e p a r t m e n t of Sugar, 1928.
R. E., J . Am. Chem. Soc., RECEIVEDJanuary 20, 1948.
Separation of Aliphatic Alcohols by Chromatographic Adsorption of Their 3,5=Dinitrobenzoates JONATHiN W-. WHITE, J R . , AND EDWIN C. DRYDEN Eastern Regional Research Laboratory, Philadelphia 18, Pa. Forty pairs of aliphatic 3,5-dinitrobenzoates have been subjected to chromatographic adsorption by Brockmann’s fluorescence technique. In this procedure, ultraviolet radiation shows the adsorbed zones as dark bands on a bright fluorescent background. Of these 40 pairs, involving 12 aliphatic alcohols from methjl to hexyl, 23 >-ieldedtwo zones, 11 gave a single zone of varying composition, and 6 were completely inseparable. The normal primarj alkyl dinitrobenzoates from methyl to amyl were easily separated from one another.
C
HROMATOGRhPHIC methods have proved of great value
in separating small quantities of similar compounds. They have been applied chiefly to naturally occurring colored substances of more or less complex structure. Application to colorless compounds depended on the development of suitable means for observation of the chromatogram. This has been aecomplished in several ways: empirically, by examining successive filtrate fractions or by arbitrary sectioning of the extruded chromatogram; by streaking the extruded column with a color-producing reagent; by pretreating the adsorbent or washing the developed column with a reagent that gives a reversible color reaction with the adsorbate; by examining under ultraviolet radiation if the substances fluoresce, by converting the substances to colored or fluorescent derivatives before adsorption; and by Introducing a colored substance with adsorptive properties similar to those of the colorless materials. I n a new and elegant procedure for the location of adsorbed bands of colorless and nonfluorescent materials, described by Brockmann and Volpers ( I ) , the adsorbents are pretreated with a fluorescent substance so that the entire column becomes fluorescent. Under ultraviolet illumination, bands of adsorbed nonfluorescent material then reveal themselves by a local absence or diminution of fluorescence. By using only radiation absorbed by the substances being separated, such chromatographic procedures
can be extended to many colorless substtnces. Broekmann and Volpers have shown that by using 2537 A. radiation and quartz adsorption tubes it is possible to locate adsorbed substances that show absorption in this region. They sJated that ergosterol and ergosteryl acetate, invisible with 3650 Ad radiation on a fluorescent column, were easily seen when 2537 A. radiation was used. The fluorescent material for treating the adsorbent must be chosen n-ith the following essentials in mind: It must not react with the substances being separated, and it must be strongly adsorbed so that it will not be displaced or leached from the column on development or elution. Brockmann and Volpers used morin on alumina, calcium carbonate, and magnesium oxide, berberine on silicic acid, and diphenylfluorindine sulfonic acid on calcium carbonate. Each of these was adsorbed from methanol solution. The following separations on alumina-morin were described: xylene musk, musk ambrette, and musk ketone; ergosteryl and cholesteryl p-nitrobenxoates; several aromatic aldehydes; phorone-mesityl oxide; and the p-phenylphenacyl esters of acetic and benzoic acids. Sease (4)used fluorescent adsorption columns prepared by mixing fluorescent zinc sulfide with ordinary adsorbents. He separated mixtures of cinnamaldehyde, xanthone, p-nitrobenzyl bromide, salicylaldehyde, azoxybenzene, nitrobenzene, and iodoform on silicic acid by this technique.
