V O L U M E 28, NS). 5, M A Y 1 9 5 6 the reagents used in this work. Indeed, from a n inspection of the contour of biguanide, i t appeared that a compound was present between cyanourea and biguanide. With the exception of dicyandiamide, urea, and thiourea, the other compounds were acids and bases and it is possible that a compound could be located in more than one spot in order to preserve electrical neutrality of the spots. Compounds H, I, and J were characterized in part as the guanidinium ion, but the identity of the anions was not determined with certainty. The nature of compound B, which appeared from its color reactions to contain sulfur and which \Vas not thiourea, guanylthiourea, or thiocyanate, is open t o speculation. An ultraviolet absorption spectrum of a n aqueous eluate of this zone gave a band at 236 mp which is the wave length maximum reported for thiourea ( 9 ) , indicating the presence of a closely related molecule. Only the areas corresponding t o the guanidinium salts underwent the Sullivan reaction. However, a n orange color was formed b - cyanamide when an aqueous solution of 1,2-naphthaquinone-4sodium sulfonate and alkali was sprayed on the chromatogram and this color disappeared in successive stages of the Sullivan reaction. These two reactions suggest specific methods of analysis for cyanamide and guanidine in miltures of related compounds.
849 ACKNOWLEDGMENT
The authors wish t o acknowledge the assistance of S. C. Blodgett. This work was part of the development program of North American Cyanamid, Ltd. LITERATURE CITED
(1) Adachi, S., Kagaku 23, 582 (1953). (2) Berry, H. K., Biochemical Institute Studies IV, pp. 88-92, Univ. Texas Publ. 5109, Austin, Tex., May 1, 1951. (3) Berry, H. K., Sutton, H. E., Cain, L., Berry, J. S.,I b i d . , pp. 22-55. (4) Bertrand, AI., Myers, J. L., Can. J . Chem. 31, 1252 (1953). ( 5 ) Bode, F., Ludwig, E. M., Schweiz. med. Wochschr. 84,629 (1955). (6) Bourjol, S., Teindas, LIrs., Mbm. poudres 31, 51 (1949). (7) Hiibener, H. J., Bode, F., Mollat, H. J., Wehner, M., H o p p e Seyler’s 2. physiol. C h e m . 290, 136 (1952). (8) Kennerly, G. W., unpublished data. (9) Alaaon, S. F., J . Chem. SOC.(London) 1954, 2071. (10) Roche, J., Van Thioai, S . ,Hatt, J. L., Btochem. et B i o p h y s . Acta 14,1,71 (1954). (11) Sullivan, 11.X., Proc. Soc. Exptl.B i d . M e d . 33, 106 (1935). (12) Tuppy, H., Monatsh. Chem. 84,342 (1953). Chrisp. J. D., -$SAL. CHEM.26, 452 (1954). (13) Watt, G. W., (14) Williams, H. E . , “Cyanogen Compounds,” Edward Arnold & Co., London, 1948. RECEIVED for review July 14, 1955.
Accepted February 17, 1956.
Rapid Paper Chromatography of Carbohydrates and Related Compounds H. T. GORDON, WAYNE THQRNBURG, and L. N. WERUM Department of Entomology and Parasitology, University of California, Berkeley 4, Calif-, and California Packing Corp., €meryville, Calif.
This method is useful for the rapid separation and tentative identification of carbohydrates and related compounds in biological fluids and extracts. No desalting or other purification is required. Interference by inorganic salts is prevented by a simple “overspotting” technique using pyridinium sulfate. Ascending onedimensional chromatograms are completed in 2 hours. Spots of carbohydrates, polyhydric alcohols, aldonic and uronic acids, nucleosides, phosphate esters, and other derivatives are semiquantitatively detected by improved specific color reagents. R j values are highly reproducible; this makes possible an analysis of some of the causes of variation in R j , especially the systematic variations due to interference by inorganic ions and to varying loads. The method is not suitable for the complete separation of structurally similar carbohydrates or of a large number of carbohydrates and closely related compounds on one chromatogram; it is designed for the rapid identification of a small number of carbohydratesin complex biological systems containing considerable quantities of many other materials.
T
HE basic objective of the work reported in this paper has been to develop a rapid method of separation and tentative
identification of carbohydrates and related compounds in biological fluids without preliminary purification. This research was instigated by the discovery t h a t a n isopropyl alcoholpyridine-water-acetic acid solvent, used previously for paper chromatography of alkali and alkaline earth cations ( 5 ) ,has the useful property of moving sugars, polyols, uronic acids, purine,
and pyrimidine ribosides, and their phosphate esters all within the Rj range of 0.1 to 0 9. The solvent tends to separate carbohydrates and their derivatives into groups of similar structure, pentoses have a n R! all moving within a narrow Rj range-e.g. of 0.70 to 0.77, and hexuronic acids have a n R j of 0.33 to 0.38. This is useful for a preliminary classification of unknowns; more specialized solvents, with higher resolving power, can then be used to separate and identify individual compounds. The solvent has other useful properties. Inorganic salts normally do not interfere with the movement of organic compounds on the chromatogram, and desalting is usually unnecessary. The low viscosity of the solvent allows i t to ascend rapidly, and an ascending one-dimensional chromatogram can be completed in 2 hours. The high volatility of the solvent makes possible complete drying of the chromatogram in 15 minutes. The solvent power is high, and loads of several hundred micrograms of most carbohydrates move as compact spots, without streaking. The commonly occurring inorganic ions (potassium, sodium, calcium, and magnesium) present in the unknown solution are well resolved and can be easily identified. A second objective has been to study the systematic variation of spot size and R/ with the quantity of substance spotted on the chromatogram. If paper chromatography is done with great care, R, values are reproducible to f 0 . 0 1 ; the variation of R f with load can then be clearly demonstrated, especially for ions (6). Such “load effects,” together Kith interference effects due t o other substances, are important causes of the R/ fluctuations which have led many workers to run known standards simultaneously with unknowns, and to replace RJ by R, (the ratio of the distance traveled by the substance to the distance traveled by glucose).
ANA
850 -1third objective has been to determilie which of’ the many spray reagents described in the literahre are most specific and sensitive, and to apply these reagents to a large number of carbohydrates and related compounds, on developed chromatograms, and over a hundredfold range of concentration. REAGENTS
Paper-Washing Solvent. Distilled water, pyridine (epwtroscopic grade) and glacial acetic acid (ACS reagent grade) are mixed in the volume ratio 80 to 15 to 5. Developing Solvent. Isopropyl alcohol (98 to 99y0, Eastman 212), pyridine (spectroscopic grade), glacial acetic acid (ACS reagent grade), and distilled water are mixed in the volume ratio 8 to 8 to 1 to 4. Solution CD-1 (0.1M 2-aminobiphenyl hydrogen osalate). Dissolve 1.69 grams of 2-aminobiphenyl (Eastman 2523) and 0.9 gram of anhydrous oxalic acid (or 1.26 grams of oxalic acid dihydrate) in a mixture of 5 ml. of glycerol, 10 ml. of distilled water, and 84 ml. of C.P. acetone. This solution keeps indefinitely. It is a reagent for all carbohydrates that can be pyrolyzed to furfural derivatives. Solution CD-2 [O. 1M LY-(1-naphthyl)-ethylenediamine dihydrochloride]. Dissolve 1.3 grams of S-(1-naphthyl)-ethylenediamine dihydrochloride (Eastman 4835) in a mixture of 2.5 ml. of glycerol, 5 nil. of distilled water, and 42 ml. of C.P. acc,tone. The solution keeps indefinitely a t 0” C. It is a fairly specific reagent for ketoses. Solution CD-3-A (0.1.M periodic acid). Dissolve 228 mg. of periodic acid (H510e, G. F. Smith Chemical Co.) in 10 ml. of distilled water. This stock solution is relatively stable, and may he kept for several weeks at room temperature, i7ith only slight loss of periodic acid. It keeps for months at 0 ” C. Solution CD-3-B (0,005M periodic acid). Dilute 1 nil. of solution CD-3-il with 19 ml. of C.P. acetone. This solution is unstable, and should not be used for more than 3 hours after being prepared. It is a fairly specific oxidant for 1,2-glycols. Solution CD-3-C (0.01M benzidine). Dissolve 184 nig. of benzidine in a mixture of 0.6 mi. of glacial acetic acid, 4.4 ml. of distilled water, and 95 ml. of C.P. acetone. The solution is yellow and keeps indefinitely. It detects unreacted periodic acid on paper chromatograms. Solution CD-4-A (0.3N potassium permanganate). Dissolve 4 8 grams of potassium permanganate in water to a volume of 100 nil. This stock solution keeps indefinitely. Solution CD-4-B (0.01M potassium permanganate). Dilute 1 nil. of solution CD-4-A with 29 ml. of C.P. acetone. The solution is stable for about 1 hour. Pyridine, lo%, in Water. This is a useful solvent for extraction or dilution of carbohydrates and derivatives, especially acids. Pyridinium Sulfate, 1M. Mix 20 ml. of pyridine, 50 ml. of water, and 10 ml. of 10M sulfuric acid; cool to room temperature and dilute to 100 ml. with water. This is a useful additive to eliminate interference on chromatograms by divalent cations such as calcium. Pyridinium (Ethylenedinitrilo)tetraacetate, 0.65M. Dissolve 2.92 grams of (ethylenedinitri1o)ttraacetic acid (ethylenediaminetetraacetic acid) in 4 ml. of pyridine plus 10 ml. of water: cool, and adjust volume to 15.4 ml. with water. This is a good solvent for insoluble barium and calcium salts, and an additive useful to eliminate divalent cation interference on chromatogranis. Barium Acetate, 1.OM. Dissolve 2.73 grams of barium acetate monohydrate in water plus a few drops of glacial acetic arid to a volume of 10 ml. This is a n additive to retain acidic suhstances near the starting spot on chromatograms. APPARATUS
The chromatographic apparatus is that previously described for one-dimensional ascending chromatography using I-inchwide strips ( b ) , except for one useful modification. The suspension of the paper strips in the earlier design IYLW by attachment to a paper clip inserted in the upper No. 26 cork on the glass chromatographic tube, This has been replaced by a glass hook, made by drawing out one end of a 12-em. length of 5mm.-diameter glass rod and bending it into a hook. The straight shaft of this hook is inserted in a small hole drilled through th: center of a No. 26 cork; the fit should be loose enough to permit the rod to be pushed up and down easily. Whatman KO.4 paper rolls, 1 inch wide, are cut into strips 580 mm. long, TThich are folded in the middle to form “double strips” 290 mm. long; on each side, a pencil line is drawn 38 mm. from the fold (these are the “starting lines,” to the center of which spots of solution are applied). The free ends of the double strip are then clipped to-
L Y T I C ,A L C H E M I S T R Y
gether 17ith a paper clip, and the etrip is hung on the glass hook by the paper clip. The glass hook should be close to the lower end of the cork. A 1-inch length of glass rod is inserted in the fold a t the loner end of the double strip, to act as a separator. T h e cork and double strip hanging from its hook are then placed in the chromatographic tube. The strip hangs from 2 to 4 em. ahove the level of solvent in the solvent container a t the base of the chromatographic tube. When all the strips that are to be run have been hung in the tubes, each strip is lowered into the solvent by pushing down the glass rod to which the hook is attached, until the strip is dipping about 10 mm. beloT\ the level of the solvent. This modified suspension system gives results identical to those of the earlier system, but is much more convenient and easy to adjust. Many spray reagents require heating the chromatograms t o 100’ C. in an oven; the temperature and time are fairl) critical, especially x h e n minute quantities are to he detected. Many ovens fail t o heat the paper uniformly, and give erratic resiilts. Gravity-convection ovens n ith heaters in the lower section are unsatisfactory for rapid uniform heating; forced-circulation ovens tend to dry the paper too rapidly, iinless the spraj reagent contains glycerol as a humectant The folloa-ing special heating svsteni, however, has given satisfactory reproducibility: -4600n d t t Forma-Vac vacuum oven (Forma Scientific Co , Marietta, Ohio) is operated on a 750-matt, 0- to 130-volt variable autotransformer The thermoregulator of the oven is set a t its maxiniiiiii or short-circuited so t h a t the oven is on continuously. T h e oven is warmed up quickly by setting the voltage a t 130 for 15 to 20 minutes; it is desirable to have this warm-up controlled by a time snitch to safeguard the oven from overheating. K h e n the temperature, indicated by a thermometer hanging inside the oven and visible through the glass nindoiT-, is 100” to 110’ C., the transformer is reset to 50 volts. This maintains a constant internal temperature of 110’ t o 115’ C. The heater in this oven is tliqtributed throughout the n all, so that there are no hot spots, and the heat flow is very uniform. This system provides constant infrared radiation and is superior to the interrupted heating ohtained by the action of a thermoregulator. PROCEDURE
Preparation and Development of Paper Strips. The only change from the method previously described is in the length of the paper strips, which are 40 mm. shorter because of the modified strip-suspension system used in the present work. The 290-mm.long double strips are n-ashed by ascending chromatography in the “paper-washing” solvent, to remove divalent cations, which might form complexes with and alter the mobility of carbohydrates or their derivatives. Washed strips should be handled carefully to avoid contamination by fingerprints, etc. I n preparing solutions or extracts for spotting strips, looo pyridine in water is a useful solvent. Such solutions can be kept in a refrigerator for a week or tTvo v.-ithout danger of Obacterial contamination; they must not be heated above 50 C., because hot pyridine can epimerize sugars. Strong acids, baseq, or salts should not be added. For hot acid hydrolyses of polvwccharides or phosphate esters, a measured volume of 101.1 sulfuric acid should be added; this is neutralized and precipitated by adding an equivalent quantity of barium acetate. Insoluble compleues containing calcium, iron, and the like can often be diesolved in 0.65M pyridinium (ethylenedinitri1o)tetraacetate.
If the solution to be chromatographed contains a considerable amount of free, nonvolatile acid or base, it must be neutralized n ith pyridine or acetic acid before development of the chromatogiaiii. Otherwise, as the solvent ascends over the initial spot, its composition will be altered by removal of pyridine or acetic arid to balance the excess acid or base. This will cause small b u t significant changes in the R, of the spots. The presence of excess acid or base is easily checked by applying a 1-pl. spot of the solution to a test strip and treating n ith bromocresol purple solution ( 5 ) . If addition of pyridine or acetic acid to the original solution is inconvenient, exposuie of spots on paper strips to pyiidine vapor or acetic acid vapor n ill effect the neutralization. It may be desirable t o add a precipitating or complexing agent to a solution before spotting, in order to eliminate mutual interference by incompatible substances, or to eliminate some of the compounds from a solution giving a too complex and full chromatogram, or t o obtain additional information on the chemical nature of the spots revealed on a chromatogram of the untreated solution. -4 1M pyridinium sulfate solution can be used to
V O L U M E 2 8 , NO. 5, M A Y 1 9 5 6 pi,ecipitate large excesses of ralciuni, and also t o minimize interfcrencae by most cations. .4 0.65.1f pyridinium (et,hylenediiiitri1o)trtraacetate solution is .a solubilizer and clarifier for solutions, and also mininiizes cation interference; b u t because it is a zxitterion it may interfere with the movement of small itmounts of aiiions. A 1.1' barium acetatc solution will prccipitate sulfate, phosphate, and many organic. acids, o r greatly x t a r d their niovenient on the chromatogram; lead acetnte has Ileen used for this purpose (Z), hut escess lend forms a spot a t IZ TO on the chromatogram, n-hilc 1)wiiini remains close t o the startiiig line, below R 20, antl is less likely to iiitcrf('i,c with detertion of carbohydrate spots. If the addition of a precipitant or cwiiiplcsiiig solution to the p t t i i i g tec-, especially at I r i ~ l s below 10 y . Sensitive reagents are nreded to detec,t, a m i d l quantity of one substance mixcd n-ith large qunntitics i i f other sul)stances, lircause the total lond of all substance? 011 tlic paper must not exceed a fe\v hundred micrograms in order to yield small qxits and good separations. Two reagents proved to be cswptioilally good: the aniline hydrogen osiilate rragent of P u t ridge ( 0 ) and the periodate-berizidirie reagent. of Cifonclli :inti Smith ( 2 ) . However, these reagents have a high water c o i i t r n t and must be applied by spraying, which is an incoiiveiiieiit o p r a t i o n requiring great care to ensure uniform coverage and to pi,event smearing of t'he spots. T h e reagents were thrrefore modified b y using acetone as the major component PO t h a t they ( . o d d be simply poured onto the paper strips. The pouring technique is simple, clean, and quick and gives sharp1)- defined spots. 111 modifying Partridge's aniline reagcnt, the aniline \vas replaced k)y 2-nminobiphenyl because aniline hydrogen oxalate is iiisolitble i i i acrt,one. Application of a pouring reagent can be done neatly by filling a I-nil. srrological pipet, applying the tip t o the papcr strip a t the solvent front, and allowing the reagent, t o flow out quickly so t h a t the strip is saturated in a fe\v seconds. T h e acetonr evapor:ites Tithin 30 seconds, leaving a iiniform deposit of reagent. One milliliter saturates a length of about 200 mm. of a 25-mm.\vide paper strip. Each of t h e spot-detector solutions used in this n.ork has an idrntifying symbol-e.g., 0 - 1 is carbohydrate detector number I-which is marked on eatah c*hroniatograni t o n-hich it has been applied.
85 1 Solution CI)-1 (0.1.U 2-:~miriobipiicii~-I hydrogen osalate) is poured on a strip, which is allowed t o dry in air for 10 to I5 minutes. The strip is placed in an oven at' 110" C. for 5 minutes. The hackground is pale yellou.. Spots of pentoses are red, hesoses grecnieh brown, uronic acids purple. After several days the background darkens slightly, some spots become more intense, and colors change t o shades of brown. It is desirable t o outline the spots in pencil and note t h e intensity and color while they are fresh. The sensitivity of CD-1 is approsiniat'ely t h e same as that of the aniline reagent from nhich it is derived, and is of the order of 0.01 pniole in a spot area of about 1sq. cni. on a developed rhromatogram. Like all other primary aromatic amine reagents, CD-1 is relatively specific for compounds t h a t readily form furfural derivatives on acid pyrolysis. 1Ionosaccharides and disacchad e s react strongly, trisaccharides and tetrasaccharides react ive:ikly, and the higher polysaccharides give no color. -4ldoses react more rapidly than ketoses, and Partridge ( 9 ) claims t,hat glurose and sorbose can be differentiated hy this means. It is true that, when equal amoiinte of sorbose and glucose are present, some rolor is developed by glucose before any color is developed by sorbose: but the glucose color intensity a t this stage is much less than the ni:isininm that can develop on fiirther heating. If heating is continued until tlie glucose spot attains maximum intensity, the sorbose spot will also have developed color. Solution CD-2 ( 0 , l X -V-(I-naplitliyl jethylenediamirle dihydroc,hloride) is poured on a strip, which is allowed to dry in air for 10 t o 15 minutes and placed in an oven a t 110" C. for 4 minutes. Xt levels of I pmole, ketoses give reddish spots on a pale tan background, \vliile aldoses and uronic acids give fainter yellow, red, or bro\vii spots. At lower ketose levels tlie rolor is a characteristic golden yellow. T h e limit of se:isitivity is of the order of 0.02 pmole per square centimeter. CD-2 is not markedly superior t o other ketose-specific reagents described in the literature (6, 8); it is sometvhat less specific but more sensitive and gives chlear, well-defined ketose spots. Solution CD-3-B (0.005.lP periodic acid) is poured on a strip, wliich is allowed to dry in air a t room temperature for 3 to 4 minutes. Solution CD-3-C (0.OIJP benzidine) is then poured on and allowed to dry. -4s i t dries, t h e background becomes deep blur with n-hite or yellon- spots. I n 5 to 10 minutes, when t h e spots shon- maximum contrast, they are outlined in pencil or with i~ ball-point pen and the intensit'y is noted. T h e background color slo\vly fades t o gray and the spots to grayish white. The limit of detect'ion for niaiiy polyols is of the order of 0.005 kinole per square centimeter, b u t may be a3 high as 0.5 pmole prr Equare centimeter for many compounds, especially polyrsccharides, which react very slowly with periodic acid. CD-3 is much less specific than CD-1 but i t is useful for many important rompounds, such as glycerol, difficult to detect by any ot>herreagent, escept the unstable lead tetraacetate in benzene ( I ) or the extremely uiispecific potassium permanganate. Although the periodic acid in CD-3 is not a powerful general oxidant, i t reacts Tvith 2-aniinoethanol, serine, threonine, and st'rong reducing agents, and gives faint spots with amines and amino acids when they are present a t levels above 0.2 pmole. Spots obtained with this reagent ~hoiildtherefore be interpreted x i t h caution, and a variety of other tests (such as ninhydrin) applied if possible. Solution CD-4-B (0.01M potassium permanganate) is poured on a strip, which is allowed t o dry in air, and continuously obw v e d for spot development during the first 10 minutes. T h e liackground is a t first bluish purple, gradually fading t o magenta, and finally t o rose. Spots develop a t markedly different rates, according t o the quantity and the reducing power of the substance in the spot; the initial color is greenish yellow, and may change l o yellow (manganese dioxide) and then t o white (manganous salt). -1s each spot appears, the time in minutes after application of CD-4-B is marked on t h e paper near the spot; this time is a useful indication of both reducing power and quantity of the
ANALYTICAL CHEMISTRY
852 Table I.
Movement and Detection by Reagents of Carbohydrates in Isopropyl Alcohol-Pyridine-WaterAcetic Acid (8:8:4:1) (2 hours, 30° C.)
Rf X 100
Substance
Micromole" Spotted
(Ethylene1.0 dinitriloltetraacetic 0 . 2 acid Rlucic acid
1
...
0.02 Calcium 0 . 5 ~ saccharate 0.1C
a-D-Galacturonic acid
...
.. ..
...
,
.
...
..
...
.. .. ,,
,..
...
0.2c
Calcium lactobionate
..
fW
0.2
Potassium 0.25 glucose-1- 0 . 2 5 c phosphate 0 . 1 0.05c
Detectionb by C D 2 3
+BR +BR
+B'L
:$$" 2++FV
8 (12-3) 20 (22-18) 8 (11-4)
++T
3++P
17 (23-90) 18 (21-15) 17 124-9) 16 17 (22-10) (21-13)
iBL
+W
+ -I7 ++Y
kBR zk
IBL fBL
..
' '
..
(+I
..
1.Od O.1d
.,. ... ...
.,
++\'
,,
++Y
0.02d O.OO5d
,.. ,..
..
1.0 0.1 0.02 0.005
++P
+P +P
+P (i)
meso-Inositol 0 . 5 ... 0.1 ... Barium 0.25d + B R fructose-6- 0 . 1 0 d + B R phosphate 0.0%' ,, .
3+BL-W 3fBL 3*BL ...
10 (15-7) 18 (25-10)
3++T 4 i T
23 (37-12) 20 (32-21-
++%
.,
tW
4 i Y
+w
.
...
...
0,5 0.1
T a r t a r i c acid 1 .O 0.1 0.02 0.005 Scylloinosose
D-GIUCOSamine hydrochloride Gluconic acid
++I+W
+$5'
0.5
+BR
IY-R
0.1 0.02 0,005
+BR
.. ....
1.0
+BR fBR
0.1 0.02 0.005
... ... ...
... ... ... ... ...
1 0 0.1
f R , .
..
..
.. , .
.. ..
0.5 0 . 3 0.5C
0 . Id 0.lC
Sodium 0.25 glycero0 25C phosphate 0 . 1 0.02
+W
T+Y
+F%:
+ r\ ++Y CY
+ -Y
++P-BR +P-BR ++P-BR
+
.. .,
+P +P
.. ,,
,.
., ,. ,, ..
.
l + + W - Y 53 (62-45) 2+Y 70 (78-62) 38 (44-32) 37 (43-32) .. 40 (44-36)
.
fv-
2 t l W - Y 38 (48-26) iY (82-48) 4+\89 (94-82) 2+T 38 (46-33) 1(78-46) 3+Y 39 (44-33) ., . 39 (43-353
$$';-I+'
2+ fW-Y (46-17) 2 + +W-l' 39 (48-31)
+W
2++Y
+Y +Y +Y
++
+Y
... ... ... ...
+G-BR +G-BR +BR
+I7 iY ..
+BR +BR
iY
+W
0.25c
++P-BR
tP-BR
+tK
0".l0C 2c 0.5
" i
.,
++W
L-~uinic acid
j-+W r + Y
Melezitose ~ ~
llaltose ~
l + + W - Y 39 (48-30) 40 (46-33) 41 (47-35)
0~. 5 0.1 0.02 0.5 0.1
~ i ~ 0,jd i ~ glycerate O . I d 0.02d
Guanosine
0.1
D-Galactose
1.0 0.1 0.02
Sucrose
0 5 0 1 0 02
n-Glucose
0.1 0.02
1.0 0.1 0.02 1.0
4- +W-Y 4 + B L - W
Floridoside
+W
L-Malic acid 0 . 2 0 02
+ +W-Y +I-
20 (28-12) 3iBL 36 (46-27) 6 i B L - W 40 (49-31) ,,, 37 (41-33)
.
,
.
,
,, , ,
. .
...
..
..
...
.
,
. ... , ,
++W
4+Y
47 (55-38)
+W iW iM-
iY
47 (55-38) 46 (52-38) 46 (50-40)
++R +\V +E
8 i Y 8 i T
+w k~ +R
++G-BR +BR
iY
++W
+~ B R IBR
+I--BR
iK
-
+Y +BR
..
i~ , , ,,
, . .
,.
,..
.
,
,..
, ,
t + G - B R iY -G-BR .. iBR ..
,.
...
... +BR iBR
.,
+R-BR +Y-R +Y
..
, . . , . .
,.
... ...
..
..
...
I
... ...
... ...
++G-BR +G-BR +BR
... ...
...
.
.. ..
, . .
fY
+R
iP IT
+BR , , . .~
1 0 0.1 0.02 0,005
0.5
.
iT
...
0.005
Turanose
,
,.
, . .
Dulcitol 0 5 (galac t i t 01) 0 . 1 0.02 D-Sorbitol
r T
i t y
Quebrachitol 1 . 0 0.1
+
a Where calcium salts are used, calculations are based on one half the molecular weight. T h e number of micromoles is therefore t h a t of the acid, not the salt. b Reagents used for detection are: CD-1, 0.1.M 2-aminobiphenyl hydroen oxalate: CD-2, 0.1.M A'-(1-naphthyl)-ethylenediamine dihydrochloride; D-3, 0,005A\fperiodic acid, followed b y 0.01.W benzidine; and CD-4, 0.01M potassium permanganate. Colors of spots are abbreviated as follows: B L , blue BL-W, blue-white B R , brown G-BR, greenish brown P, purple P-BR, brownish purple fading t o brown R , red R-BR, reddish brown R-Y, reddish yellow W, white
8
0.5 0.1 0.02 0.5 0.1 0.02 0.5 0.1
ii $ ~ ~Cellobiose ~ ~0 , 5 { 0.1
l + + J T - Y 35 (43-27 20) 38 (43-33) 3 8 (42-32) ,, 38 (43-33)
2-+Y 8+T
$kY $2; +w +T
0.02 0,005 Calcium 2-ketogluconate
Lactose
Calcium
...
46 (54-3821) 44 (50-38) 43 (50-36)
++G-BR
Rlalto triose
3 1 +W-Y 30*($7-23) 3fT 28 (34-23) 29 (31-27)
4+Y
+K
0 5
31 (37-25) 32 (36-27)
1 fi)
+IT
Isomaltose
D-Trehalose
++Y
+BR
i T
i B R IBR
31(37-24)
,,
40 ( 5 7 - 3 2 ) 46 (53-38)
++G-BR
3+-T
2+BL-W fBL-W 3++T
7iY ...
0.1 0.02
++W
..
kJT
0 5
'-lieto gluconate
+BL
+\I-
Raffinose
31 31 (36-23) (35-26)
..
6kY .,.
+BR
3iY 3+Y
..
... ...
4+Y
+w
0.2 0.05
i: fz::iE{
33,~ cf. l onolactone, Rf0.84 33 (43-22) 29 (36-21) 36 (45-24-
++W +W
iY
RIaltotetraose
+W (+)
2%
4
cf. Gulonolactone, Rf 0.75 45 (53-36) 46 (52-39) 44 (50-39)
++BR +G-BR kBR
,
::
Detectionb by C D 2 3
1
0.5 0.1 0.02
(+)
,
Micromole" Spotted
R j X 100 Values of Spot Center and Limits
Gulonic acid ( + lactone)
,.
D-Glucuronic acid ( + lactone) 3-4denylic acid
(27-17)
++P
,,
+Y
3+T 2++IV-P 2++W-Y 2+Y
,.
++P
+W
Substance
D-Rlelibiose
12) lo(+) 22 (28-17) ,. 28 (32-23) 2++Y 30(41-21) 2i+Y + ~ ~ - y
.. +P
4
F'alues of s p o t Center and Limits
.,
+T +Y +Y
.. ., .. ., .,
..
++w
+W ++I'
++Y
+W
.,. ...
50 (61-39) 50 (56-43) 50 (52-47) l+W 50 (59-40) ... 5 1 (57-42) 2 + + W - Y 51 (60-4337) 2 i + W - Y 53 (60-46) 3*y 53 (60-48) 8 i Y 52 (58-47) 7fY 53 (62-43) ... 5 2 (58-46) SAY 54 (64-43) .,, 54 (61-47) ,,. 54 (60-47) ,
.
.
3i.y 1OiY
57 (65-49) 58 (64-52)
2 + + W - Y 58 (66-48) 2++Y G O (65-55) 3+Y 5 8 (62-5'2)
+W
3 i Y
BO (66-54)
++T-JV ++W +TT
4+Y 6+Y
60 (69-50) 63 (67-59) 63 (87-57)
++R
4 i T
6 2 (69-54) 63 (67-59)
fW
+ W. .
8=kI7
63 (70-j5) 62 (68-95) 61 (66-55)
++Y
++w +w
3 + i T 3+T
++w ++R +w in-
3++T 3++Y 4 i T
++Y
6i;T
in-
iW
fY +\T
... ...
. . .. ...
...
... ...
61 (71-52) 64 (69-56) 65 (69-60)
++W ++W
3++Y
+R
4+T
i R
2 + +W-Y 6 3 173-56) G+T 61 (01-57)
in. ,
.
... ... 05 (73-58)
W-T, white center, yellow peripher)
? ' , "v.Pllnw . ....
Y-R, yellowish red Y-JT, yellow center, white periphery 3-0color symbol, color too faint t o identify Spot intensity is coded as follows: maximum intensity distinct spot, of less t h a n maximum intensity faint spot, near or a t limit of detection For spots detected by CD-4, t h e intensity symbol is preceded b y a number from 0 t o 10. This is the number of minutes elapsed between the time of application of t h e reagent and the time of appearance of the spot. C Solution or spot treated with pyridinium (ethylenedinitrilojtetraacetate t o eliminate interference b y cations. d Solution or spot treated with pyridinium sulfate t o eliminate interference b y cations.
+ ++ +
V O L U M E 2 8 , NO. 5, M A Y 1 9 5 6 Table I.
853
Movement and Detection by Reagents of Carbohydrates in Isopropyl Alcohol-Pyridine-WaterAcetic Acid (8 :8 :4: 1) (Continued) ( 2 hours, 30' C.)
R/ X 100 Micromoles
Substance Mannitol
Citric acid
Spotted
1
1.0 0.1 0.02 0,005
... ... ... ...
0.1 0.02 0.5 0.1 0.02
Pinitol
1.0 0.1
Inosine
0.5 0.1 0.02
Adenosine
++R +R I R
L-Sorbose D-Fructose
1.0 0.1
i R I R
L-Arabitol
Adonitol (D-ribitol) D-Xylose
66 (76-55)
67 (74-59) 64 (69-59)
++W
+iI?
3 I Y 81Y
..
++JV+w
67 (75-60) 67 (74-61) 68 (71-65)
8 i Y
,.
t PV
3 +Y 41Y
.,.
69 (78-60) 70 (76-64) 70 (74-66)
4- +w + IT iI?-
2++Y 5&Y 6 i Y
66 (76-56) 69 (74-54) 69 (73-65)
++W ++W
2++W-Y 5 i Y . ,. 69 (74-64)
...
..
...
..
...
+BR IBR
f R
++R-Y
w
7-
..
+R-Y
+Y
+Y
Iw
&Y
+w
++G-BR +G-BR =tG-BR
i R
++IT
..
+W
,.
It\V
..
+fwTT ++ ++ YY
... , . .
... ...
.. ..
++ R+ R
i R
i R
..
..
0.02
f R
1.0
++BR
+Y
L-Fucose
0.1 1.0 0.1 0.02
++G-BR iG-BR IBR
i R
1.0
++R -rR
i R
.. .. ,.
.. ..
.,.
I
...
iR
2++Y 2+Y 6 i Y
.. ..
1.0
0.1 0.02
2 + +W-Y 65 (75.55) 62 (66-57) 62 (66-58)
3+W-Y 41Y
67 (74-60) 67 (72-62)
DL-Glyceraldehyde
D-LyxOse
..
(72-44) (72-58) (69-61) (69-61)
4 + +\T lOiY
IY iY
1.0
,
60 66 65 65
iFV
++G-BR IG-BR IBR
0.1
2+ +Y
tY
1.0 0.1 0.02
1.0 0.1 0.02 0,005
4
..
, .
0.09
1.0 0.1 0.02 1.0 0.1 0.02 0.005
IBL
..
T+R +R i R
0.1 0.02
D-Mannose
+RL
iY ..
...
0.10 0.02 L-Arabinose
..
.. ..
...
0.20
.. , . ..
+++IT +Y 2;
..
.. .. ..
0.5
Xanthosine
Detection b b y C D 2 3
Values of s p o t Center and Limits
+
.,. ...
3++Y 6 i Y , ,
.
++ ++Yw
3 +Y 4 i Y
+Y
z;
w
i
+iw+IT-
+
,,
.
3++Y
4iY
, .
...
2++Y
IW
3 i Y 3++Y 61Y
t + W +\T
+TF
+w
=ktW
E; {755;!; 67 70 70 70 73 72 74
(75-58) (75-65) (75-65) (79-62) (78-69)
. .. (76-68) ... (77-71) 2 + +W-Y 70 (79-60)
++Y ++FT
08 (75-60) 69 (74-63) 65 (68-61)
7 3 (78-67) 73 (77-69) 73 (76-69)
71 (78-64) 7 3 (78-69) 74 (78-69) 7 5 (87-0445) 70 (89-50) 74 (82-65) 7 5 (79-70) 7 5 (78-71)
... l + + W ' - Y 7 2 (81-61) 7 5 (82-07) 75 (79-70)
Substance X-dcetylgluposamine D-Ribose
Zlicromole" Spotted 1.0 0 2 0.04 1.0 0.1 0.02
Detectionb b y C D 2 3
1 +BR IG-BR
+Y
++ R+ R
++ Y+Y
...
... ...
0.1 0.02
meso-Erythri- 1 . 0 t 01 0.1 0.02 0.005 Gulonic 1.0 lactone 0.1 ( + acid)
i W
..
.. ..
IBR
...
Glycerol
+
+BR +BR i
.~. . ~ ...
i R
..
..
~, .
.. .. .. .. ..
, . .
...
...
...
fBR
I R
+R
1.0 0.1 0.02
+BR +R
Glucuronic lactone ( + acid)
1.0
++ P+ P
0.02
.. ..
... ...
Dihydroxyacetone
0.1
+ +
++ ++TW f W
...
1.0 0.1
1.0 0.1 0.02 rhamnose 1 . 0 0.1 0.02 ~ ~ ~ 0 .~5 0.1 0.02 L-ridine 0.5 0.1 0.02 L-Ascorbic 1.0 acid 0.1 0.02
++Y-R +R I R +Y-W ++W +W
IW
...
, . .
2-Deoxy-Dglucose
... ...
..
i R
Shikimic acid 0 . 5
4
5fY
iw
...
..
IU' 4-+ Y +Y +W +W
+
+ U'
++W +W fW i d. .
.. , .
:$;
1.0
IBR
:$;
77 (80-73)
O++W-Y 1 W-Y 1 i Y 34- +Y 31Y ... ... 3 4- W-Y 3++W-Y 31Y 3 i Y
77 78 78 73 77 77 77 75 40 80 43
1+ + Y
7 8 (90-69) 81 (86-77)
3++T 6kY .,. 3++F-T 9 i Y ..,
79 82 82 81 83 82
2++Y 2+Y 4+Y l++W-Y 1 +W-Y 3itY
84 (90-79)
++
+
1OiY
+
+
++Y
+W +Y fW
0 1 0.5 0.1 0.02
IBR
... ... ...
iR
.. .. .. ..
...
2 + +U'-Y 75 76 (84-67) (82-70)
1+
iz:
(85-67) (84-70) (82-73) (80-66) (82-70) (81-72) (80-75) (85-64) (49-30) (84-76)
(47-38)
(86-69) (86-78) (85-79) (90-72) (88-76) 186-77) . . (84-75) (86-78) (91-74) (89-78) (88-82) (91-71) (90-78) (91-82)
+ W-Y 82 (87-77) 86 (91-82)
2++W-Y 3++Y 3fY 7IY 81Y
86 (89-82) 84 (91-75)
33(38-28) 86 (91-81) 34 (38-30) 86 (90-82)
++Y
O++W
cf. Gjuconic Acid. R j(93-760.38 85
i W
l I Y
57) 90 (95-85)
Gluconolactone Dehydroascorbic acid Thymidine
71 (80-62) 7 5 (81-68)
80 82 ++W 82 +lT 84 iW 84 ++Y-W O + tTV-Y 82 +W l+Y 85 *W 4 i Y 86
++W U' *I?' +tW
+R I R i R
RI X 100 Values of S p o t Center and Limits
.. .. ..
1++Y
l+Y 2+Y
90 (96-83) 91 (96-86) 89 (94-84)
a Where calcium salts are used calculations are based on one half the molecular iveight. T h e number oi micromoles is therefore t h a t of the acid, not t h e salt. b Reagents used for detection a r e : CD-1 0.1M 2-aminobiphenyl hydrogen oxalate; CD-2, O.ld'd a\r-(l-naplithyl)-e~hylenediamine dihydrochloride; CD-3, 0.005M periodic acid followed by 0,OlM benzidine, and CD-4 0.01M potassium perrnangana'te. Colors of spots are abbrevirt'ted as follovs': B L , blue BL-W, blue-white B R , brown G - B R , greenish brown P , purple C-BR! brownish purple fading to brown K , red R - B R , reddish broivn R-Y. reddish yellow IT, white
W-Y, white center, yellow periphery Y, yellow Y-R, yellowish red Y-W, yellow center, white periphery No color symbol, color too faint t o identify Spot intensity is coded a s follows: niaximum intensity distinct spot, of less t h a n niaximum intensity zt f a i n t spot, near or a t limit of detection For spots detected b y CD-4, the intensity symbol is preceded b y a number from 0 t o 10. This is t h e number of minutes elapsed between t h e time of application of t h e reagent and the time of appearance of t h e spot. c Solution or spot treated with pyridinium (ethylenedinitri1o)tetraacetate to eliminate interference bv cations. d Solution or spot treated with pyridinium sulfate t o eliminate interference by cations.
substance. *4t about 10 minutes, the spots are outlined in pencil, and the color and intensity are noted. The background soon fades to a light tan on which many spots become undetectable. Some substances reduce permanganate only to manganese dioxide. Many substances quickly reduce i t to manganese dioxide, and then very slowly reduce this to the manganous form; the spots are yellow a t the end of 10 minutes, but fade t o white after 1 or 2 days. Minute quantities of these substances may not give a perceptible yellow spot in 10 minutes, but may show up
as white spots after 1 or 2 days. The sensitivity of detection for many substances is of the order of 0.01 pmole per square centimeter, but some substances (such as the nonreducing disaccharide trehalose) are almost undetectable even a t 0.5 pmole per square centimeter. The low specificity of permanganate limits its usefulness, for it detects alcohols, amines, amino acids, and many unsaturated and aromatic compounds. CD-4 is a useful auxiliary test on chromatograms of unknown mixtures of organic compounds.
+ ++
854
ANALYTICAL CHEMISTRY
I n alkaline medium permanganate is a weaker and more specific osidizing agent, and has been used to detect certain reducing amino acids such as methionine and tyrosine ( 3 ) . RESULTS AND DISCUSSION
Table I summarizes the data on the movement of carbohydrates and related compounds in the isopropyl alcohol-pyridiiie-wateracetic acid solvent. Mobilities are given in units of 100 R,; measured from the starting line to the estimated mass center of the spot, and the numbers in parentheses give a more complete description of the spot. For example, the numbering 26 (32-2112) indicates that the estimated mass center is a t R j 0.26, the main mass lies between K.j 0.32 and 0.21, while a “tail” extends from lii 0.21 to 0.12. \Inst of the spots show no tailing, however. Searly all of the r p t s were detected by more than one carboliydrate-detcc,tor soliition, and many were detected b ~ all four detector solutions; t,he numerical description is usually identical for at least two of the detector solutions, but the value listed is always t h a t of the more sensitive detection, The description, therefore, gives the actual location of the compound on a developed chromatogram, so that the possibility of separating and isolating in pure form varying quantities of two or more compounds can be checked. R/ values can be reproduced very well. As a test, eight strips were spotted with 1 pmole of Larabinose, and two strips each detected with detector solutions CD-1, CD-2, CD-3, and CII-4, Sumerical (100 R / ) descriptions of the eight spots were: 69 (76-GO), 68 (75-60), 68 (74-62), 69 (75-62), 69 (78-61), 69 (79-57), 69 (76-GO), 68 (75-60). In careful work, R j values can nearly always be duplicated to ,C 0.01 unit. This makes possible a clear analysis of the nature of the systematic variations in Xi due to variations in the quantity of the substance in the spot (load effects) or in the quantity arid quality of other substances t h a t may affect its movement on the chromatogram (interference effects). One important load effect shows u p with compounds thitt are completely ionized. This has been previously described for cations like sodium or magnesium ( 5 ) )and is also clearly shown in Table I by the data for tartaric acid ( R i 0.36) and o t h w strong acids. T h e R , values both for the mass center and for the upper limit of the spot decline, but the value for the l o w r limit is relatively constant, as the load is decreased. This foraard elongation of the spot as the load increases may be due t o a change in the water-concentration gradient’ along the strip caused by the competition for solvent unter molecules between the moving ions and the stationary cellulose fibers (5, 6 ) . A second load effect is shown by most of the nonionized compounds listed in Table I-e. g., by adonitol ( R j 0.70). > i t the heaviest (1 pmolej load, the R , values for the mass center and the lower limit of the spot are several units lower than the values a t loads of 0.5 pmole or less; at the l o m r loads, all Rj values become relatively constant-Le., the size and location of the spot do not vary with changes in load. It is clear that the upward-moving and outward-spreading of the original spot by the ascc,nding solvent flow are unaffected by the load u p t o a critical value near 0.5 pmole; a t loads abore 0.5 pmole there is a backrvard-elongation of the spot because the solvent cannot carry the full load, and the esress load lags behind. T h e carrying capacity of the solvent for most substances seems t o be about 0.5 pmole, hut for some substances it is nearer 1 pmole (cf. L-fucose, Rj 0.74), and for others i t is nearer 0.1 pmole (cf. dulcitol, R f 0.65). Ionic Interference Effects. T h e most serious interference effect is the interaction between cations and anions. T h e developing solvent normally moves cations as the acetate salt$ and anions as the pyridinium salts, b u t when the load of ions is heavy or the ions are polyvalent the solvent may be unahle to effect separation (6). This failure is shown b y glucosamine hydrochloride ( R , 0.38) a t the 1-pmole level, \There the glucosaminium and chloride ions move together t o a higher level ( R j 0.53)
than chlol,ide ions alone (8,. 0.50). At the 0.1-pmole level, the ions are separated by the solvent and glucosamine moves as the acetate t o its “true” Rj value of 0.38. A similar effect is shown by sodium glycerophosphate a t 0.25 pniole, where the unseparated salt moves to R/ 0.20, a value lower than sodium ions alone ( R j 0.30). At 0.1 pmole, the solvent effects a separation, and pyridinium glycerophosphate moves to Rf 0.40. .%pparently, ionic int’erference between nionovalent ions can be corrected simply by lowering the load to 0.1 pmole or less. With polyvalent ions the interionic attraction is greater, and a variety of chelate complexes and salts may be formed, so that a streak rather than a conipart spot is likely to show u p on the chromatogram-e.g,, calcium 2-ketogluconate in Table I, R/ 0.40. This is best corrected (as described in the section on Procedure) by overspotting on pyridinium sulfate before development of the chromatogram. The sulfate spot stays near the starting line and holds polyvalent cations strongly, but doev not affect the movement of anions. Pyridinium (ethyleiiedinitri1o)tetraacetate is also effective, but has several disadvantages, and should be used only ivhen needed to dissolve insoluble salts. It has a higher R, than sulfate and covers a larger useful area on the developed chromatogram. I t reacts strongly with permanganate. I t s positively caharged nitrogen atoms attract and hold back some anions. I t id a weak acid, and is less effertive than sulfate when competing for calcium ions with st!’(Jiig acid chelating agent3 such as tartaric arid: at the p H (xhout 5 ) of the chromatographic solvent. . There is no mutual interference betLveen neutral molecules (such as ribose, galactose, or lnctose) and ions. The presence of 0.1.11 potassium, sodium, calcium, and magnesium in the carbohydrat,e solution has no effect on the R , values. Overspotting a carbohydrate solution on pyridinium sulfate, pyridiniuni (ethylenedinitrilo)tetraacetate, or barium acetate does not alter the R / values. There are striking interference effects, however, in chromatograms of ionized molecules. I n the presence of calcium and magnesium a lactic acid spot shifted from K j 0.83 t o 0.80, a citric acid spot from R f 0.6; to 0.14, and a tart’aricacid spot from R f 0.35 t o 0. This interference was rectified by overspotting the solution on pyridinium sulfate; t’heacids then moved as pyridinium salts to their normal R f values, while the calcium and magnesium were retained by the sulfate spot. Overspotting the solution on barium acetate displaced the lactic spot t o Rj O.ti!l, while the citric and tartaric acids were mostly held a t R,. 0 with slight upward streaking t o R j 0.24. Glucono-6-lactone also shows serious distortion in high-salt chromatograms, but the normal pattern is restored b y overspotting on pyridinium sulfate; overspotting on barium acetate causes complete retention of the gluvonic acid in the barium spot belmv R j 0.20. The most complete study of ionic interference was carried out with calcium 2-ketogluconate (cf. Table I! Rj 0.40). A spot containing 0.5 pmole of 2-lcetoglrironic arid plus 0.25 pmole of calcium gives a long streak from Rj 0.46 to RJ 0.1;. \\-hen detected with CD-1, the upper portion of the streak is purplta i n color, and the lower portion is purplish red; chromatograms twated with 8-quinolinol to reveal cnlcium (5j &ow t h a t the calcium is located in the lor\-er portion of the streak. There ia therefore a n imperfect separation of the free arid and a calcium complex (probably the calcium 2-ketogluconate acetate). If the calcium 2-ketogluconate is dissolved either in 131 pyridinium sulfate or in 0.65M pyridiniuni (ethylenedinitri1o)tetraacet:ite iristead of in water, the calcimn is almost completely retainrtf near the starting line, and all of the 2-ketogluconic acid move?; :is the pyridinium salt to a compact spot a t R.i 0.39, which gives the characteristic purple color of uronic acids n h e n deterted hy CD-1. Overspotting of a water solution of calcium 2-ketogluconate on either pyridiniuni sulfate or pyridinium (ethylenedinitri1o)tetraacetate gives chromatograms identical with those obtained by single spotting of ii mixed solution of the salt and the
855
V O L U M E 28, NO. 5, M A Y 1 9 5 6 precipitant. I n preliminary esperinieiits nsing the overspotting technique, a l d l polution of (ethylenedinitri1o)tetraacetate in ammonium hydroxide was used as the prespotted precipitant ; in this inettinee, the 2-ketogluconic acid moved as a compact spot to I?, 0.28. Presumably, the acid moved as the ammonium salt, ivhich has a much loner mot)ility than the pyridinium salt. Overspotting the Lvater solution of calcium 2-ketogluconate on I pmole of barium acetate leads to complete retention of the acid in the rrgion from Rj0.14 to Ri 0.03, while the calcium moves freely to its normal popition. Correlation of Molecular Structure and Rj Value. 11:xnj- wnpirical generalizations can be derived from the data in Table I. Desosyaldohesoses have R J valurs f r C m0.75 to 0.83, aldopentoses from 0.70 to 0.76, ketohesoscs from 0.6‘3 to 0.70, aldohexoses from C.63 to 0.70. These ranges are probably too narrow to e the rarer sugars-e.g., the aldohexose idose n.ould include s ~ m of probably have an Rj of 0 . i 5 , hg conipariwn with the data gii-en by Ishenvood and Jermyn ( 7 ) for niovenicnt in an ethyl acet:itepyridine-water solvent. These authors have sho~vn th:it the R J values for cnrbohydratcs follow the wine sequence in :ill rhromatographic solvents (except phenol 1. Galxc*tose and its homomorphs (arabinose, fucose, dulcitol, g:tiarturonic arid) hxvc the lowest R i values Lvithin the group to ivhic*hthey hcloiig. Gliirose and its homomorphs (xylose, sorliitd, glucuronic a ( % ) have and ~ its homomorphs (Iysose, slightly higher R J values. 1 1 a r i n o ~ rh:ininow, nimnitol, mnnnuronir ;wid) h;ive still higher R,
values, near the median value for each structural group. The configuration of the hydroxyls within each homomorphic group apparently determines the relative water affinity and thereby the relative mobility of the molecule. Xonophosphoiic esters have R , values from 0.37 to 0.43 unit lower than those of parent carbohydrates such as glycerol, glucose, or adenosine. Carboxylic acids have R , values 0.20 t o 0.30 unit lower t h a n the parent polyol, but usually also show a strong lactone spot with an Rj value 0.20 unit higher than the parent polyol. .4mino sugars (the only one tested, glucosamine) have an R J about 0.25 unit Ion-er than the corresponding hydrovyl foim. LITERATURE CITED
Buuhanan. J. G . , Dekker, C . .I..Long, A . G . , J . Chem. Soc. 1950, 3182. Cifonelli, J. .%., Smith, F.,A x a ~CHEM. . 26, 1 1 3 2 (1954). Dalgliesh, C . E., .l-atiue 166, 1076 (1950). Feigl, F., “Chemistry of Specific, Selective, and Sensitive Ileactions,” Academic Press, New York, 1949. Gordon, H. T..Hewel. C . .i..d s a ~CHEY. . 2 7 , 1 4 7 1 (1955). Isherwood, F. A., Brit. M e d . Bull. 10, 2 0 2 (1954). Isherwood, F. A . , Jermyn. 11..I., Biochem. J . 48, 515 (1951). Lederer, E., Lederer. 11.,“(‘hromatography, a Review of Prillciples and .Ipplications.” Elsevier, S e w York, 1953. Partridge, S. 11., Binchein. SOC.Symposia (Cambridge, E n o l . ) , S o . 3, p. 5 2 (1950). R E C L ILD \ for reyiew J u n e 1 4 , 1955
Accepted Deceinber 28, 1955.
Determination of Pipecolic Acid in Biological Materials 0 . 0.SILBERSTEIN’, R. M. ADJARIAN, and J. F. THOMPSON
u. S. Plant,
soil, end Nutrition ~!eborstory,Agricultural Research Service, khaca,
Because pipecolic acid has videspread occurrence in plants, a quantitative method for its determination in biological materials is desirable. A sensitive quantitative method utilizes the color formed between pipecolic acid and ninhydrin under acid conditions. The reaction is subject to interference by salts and alpha amino acids. These interfering materials are separated from pipecolic acid by chromatography on Dower 5O-Xl2 in the sodium form. The method has been applied successfidly to urine and to plant extracts.
P
IPEXOLIC acid has been isolated from and identified in a number of plants (3-5, 7 , 1 7 , 1 8 ) . I n both plants and animals ( 2 , 8 , 9 )pipecolic acid is formed from lysine. I n order t o study the possible utilization of pipecolic acid bv animals, a method for its determination in animal tissues, feres, and urine was developed and has also been applied t o plant tissues. Since the inception of this work, Schxeet ( I f ) has published a method t h a t has been applied to the analysis of proteins, but in nonprotein fractions salts .and amino acids interfere. The method proposed here separates pipecolic acid from these interfering substances and is more sensitive than Schiveet’e. The first attempts t o measure pipecolic acid Tvere made bg the method for the quantitative determination of amino acids on paper ( I S , Id), b u t this reaction lacked sensitivity and reproducibility. The colorimetric method described here is based on the reaction of pipecolic acid in vitro with ninhydrin under acidic conditions. The pipecolic acid is first freed of interfering materials (amino acids and salts) by chromatography on ion exchange resins. This report is conveniently considered in two parts: the proPresent address, T h e Welch Grape Juice Co., W’estfield, N. Y.
N. Y.
duction of color between pipecolic acid and ninhydrin and the separation of pipecolic acid from interfering materials. PRODUCTIOY OF COLOR BETWEEh PIPECOLIC ACID ANI) NIYHYDRIV
Reagents. Phosphoric acid, 1 N . Distilled 1-butanol. Ethyl acetate; practical grade is satistactory. Sinhydrin solution, 4% (weight/volunie) solution of ninhydrin in a mixture of 9 parts of 1-butanol and 1 part of 1M phosphoric acid. This is prepared fresh just before use (Table I). Procedure. A sample containing 1 to 20 y of pipecolic acid is evaporated to dryness in a test tube (12 X 175 mm.) in flowing air a t room temperature. One milliliter of ninhydrin solution is added and the tube is capped and shaken. After being heated in boiling water for exactly 15 minutes, the tube is cooled rapidly and the solution is diluted to a convenient volume (5 to 10 ml.) with ethyl acetate. During and after heating, the tube must be kept out of intense light because light causes fading of the color. T h e color is measured a t 575 mp (Figure 1) within 1 hour. T h e quantity of pipecolic acid is obtained from a standard ciirve made from the pure acid treated in the same way. If proline is also present, the color at 510 mp (Figure 1) must also be measured, T h e quantities of proline and pipecolic acid can be calculated from the absorption coefficients of these tlvo acids a t the two wave lengths. D578 = APi BPr. Djl0 = CPi DPr. DST8 Dslo = density measurements a t 575 and 510 n u . respectively. Pi and P r are the micrograms of pipecolic and proline. -4 = density from 1 y of pipecolic acid at 575 mp. B = density from 1 y of proline at 575 mp. C = density from 1 y of pipecolic acid at 510 mp. D = density from 1 y of proline a t 510 mp. T h e blank color is negligible. -4ccurate determinations of pipecolic acid up to 100 y can be made by further dilution with ethyl acetate, b u t larger quantities produce a n insoluble precipitate.
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