experiment with 64 kilo-bytes of core memory, it took about 3 seconds for a search of 110 reference spectra. This speed requires about 45 minutes for a search of 100,000 reference spectra, but this time can be reduced sharply by using the following three procedures. The first is the speed-up owing to utilization of largescale computers. Computing speeds of existing large-scale computers are five to ten times greater than that of the one used here. The second is the speed-up by the improvement in the retrieving program. The computer program in this study was written in FORTRAN IV language, but programming in machine languages such as BASIC ASSEMBLER will lead to a t least two or three times speed-up. The third is the speed-up owing to utilization of some preliminary retrieving methods. Making use of data of the average and the standard deviation of spectral patterns will be available for such a preliminary retrieving method, which may give some reduction of machine time for the retrieval. Consequently, these procedures will produce a t least one order of magnitude speed-up in machine time for the computer retrieval, and a search of 100,000 reference data by this method will take only a few minutes, which is quite practicable for the retrieval of unknown spectra. From these considerations, it can be concluded that the correlation coefficient method studied here will be sufficiently practicable for the retrieval of unknown samples from numerous reference spectra. For existing methods which use only peak position data, a key to the success in the retrieval predominantly lies in the correct choice of absorption peaks used for the retrieval, and much attention has to be paid to the treatment of weak or shoulder bands associated with strong absorption bands in spectra. On the other hand, the correlation coefficient method calls to account spectral data a t every wavenumber point, and there may be no room for human deliberation on spectral data of
unknown samples in the retrievl. In this respect, the correlation coefficient method does not require much skill for the retrieval, and it will be successful even for inexperienced users. When the compilation of digitized IR spectra is carried out for numerous chemical substances, this will be a promising method for the identification of chemical substances using their IR spectra.
LITERATURE CITED ( 1 ) R. A. Sparks, "Storage and Retrieval of Wyandotte-ASTM Infrared Spectral Data Using an IBM 1401 Computer, ASTM, Philadelphia, Pa., 1964. (2) L. D. Smithson, L. B. Fall, F. D. Pins, and F. W. Bauer, "Storage and Retrieval of Wyandotte-ASTM Infrared Spectral Data Using a 7090 Computer," Technical Documental Report No. RTD-TDR-63-4265, Research and Technology Division, Wright-Patterson Air Force Base, Ohio, 1964. (3) T. A. Entzminger and E. A. Diephaus, "Storage and Retrieval of Wyandotte-ASTM Infrared Spectral Data Using a Honeywell 400 Computer."
(4) (5) (6) (7) (8)
U S . Public Health Service, Robert Taft Sanitary Engineering Center, Cincinnati, Ohio, 1964. D. H. Anderson and G. L. Covert, Anal. Chem., 39, 1288 (1967). D. S. Eriey, "Fast Searching System for the Wyandotte-ASTM Infrared Data File," Chemical Physics Research Laboratory, The Dow Chemical Company, Midland, Mich., 1967. D. S.Erley, Anal. Chem., 40, 894 (1968). L. H. Cross, J. Haw, and D. J. Shields, "Retrieval of Infrared Data," Mol. Spectrosc., Proc. Conf. 4th 189 (1968). Yu. P. Drobyshev, R. S. Nigmatullin, V. I. Lobanov. I. K. Korobeinicheva, V. S. Bochkarev, and V . A. Koptyug, Vestn. Akad. Nauk SSSR, 40, 75
(1970). D. S. Eriey, Appl. Spectrosc., 25,201 (1971). G. A. Massios, Amer. Lab., 3, 55 (1971). R. W. Sebesta and G. G. Johnson, Jr., Anal. Chem., 44, 260 (1972) C. S. Rann. 44. 1669 11972). , Anal. . Chem.. ~. S. Shimizu, Annual Meeting of the Chemical Society of Japan, 2F14, Hiroshima, 1973. (14) S. Kihara, K. Takahashi, K. Fukaya, and J. Ishida, Tokyo Conference on Applied Spectrometry, 1B11, Tokyo, 1973. (15) K. Tanabe, S.Saeki, and T. Tamura, Jap. Anal., 23,626 (1974). (16) J. C. Reid and E. C. Wong, Appl. Spectrosc., 20, 320 (1966). (17) The Coblentz Society Board of Managers, Anal. Chem., 38 (9), 27A (1966). (9) (10) (1 1 ) (121 (13) \
-
I
-
-
RECEIVEDfor review February 19, 1974. Accepted October 1, 1974.
Ligand-Exchange Chromatography of Amino Sugars James
D. Navratil, Eduardo Murgia, and Harold F. Walton
Department of Chemistry, University of Colorado, Boulder, Colo. 80302
The amino sugars, glucosamine, galactosamine, and mannosamine, are retained strongly on copper- and nickel-loaded ion-exchange resins and eluted selectively with aqueous ammonia in the order quoted. They are easily separated from carbohydrates and all but one or two amino acids. Detection and measurement are done by ultraviolet absorbance of the copper complexes eluted from the resin. Best results were obtained with an acrylic-type resin, but resins based on polystyrene were also used. To test the analytical method, glucosamine was determined in chitin from lobster shells. The method is simpler and faster than published methods and can detect one microgram of amino sugar.
Studying the ligand-exchange chromatography of ethanolamines, we noted that glucosamine and galactosamine were retained strongly by a nickel-loaded cation-exchange resin ( I ). We decided to investigate the ligand-exchange chromatography of amino sugars more thoroughly for two 122
reasons. First, they are important biochemically. They occur as units of natural polymers in the shells of crabs and lobsters ( 2 ) ,marine biological adhesives ( 3 ) , and bacterial cell walls ( 4 ). Mucopolysaccharides, containing hexosamine units, are excreted in the urine of patients with liver disorders and diseases of connective tissue ( 5 - 7 ) ;hepatitis is induced experimentally by feeding d- galactosamine (8). Second, the amino sugars are ideal subjects for ligand-exchange chromatography. Their strong binding by metalloaded resins, due apparently to a synergistic effect of amino and hydroxy groups, offers the possibility of selectively absorbing them from complex mixtures. The analytical method for hexosamines most often mentioned in the literature is that of Elson and Morgan (9) modified by Boas ( 1 0 ) . I t uses a color-producing reaction that is hard to control and does not distinguish between different hexosamines. The hexosamines are separated beforehand from sugars and amino acids by cation exchange. Glucosamine and galactosamine can be separated from one
ANALYTICAL CHEMISTRY, VOL. 47, NO. 1, J A N U A R Y 1975
another by chromatography on Dowex-50 cation-exchange resin, using 0.3 N HCl as eluent (11 ). Using mixed boratecitrate buffers in a commercial amino acid analyzer, seven hexosamines were separated in 11 hours (12). In normal amino-acid analysis by cation-exchange chromatography, glucosamine is eluted along with the amino acids and appears as one peak among many others ( 4 , 1 3 ) . Thin-layer chromatographic methods for the amino sugars have been described; one of these has very high sensitivity ( I d ) , and another uses ligand exchange on silica gel sprayed with copper sulfate (3, 15).
A
EXPERIMENTAL Materials. The following ion-exchange resins were used: BioRex 70, with functional carboxyl groups on an acrylic matrix, and irregularly-shaped particles of rated diameter 37-44 pm; Aminex Q-150S, with sulfonic groups on a polystyrene matrix and spherical particles of diameter 20-30 pm; Poragel PT, a spherical resin of the polystyrene type, 37-75 pm diameter. The Bio-Rex and Aminex resins were supplied by Bio-Rad Laboratories, Richmond, Calif., the Poragel PT by Waters Associates, Milford, Mass. The Poragel resin, as supplied, has no ionic groups. We hydrolyzed it in boiling 10% alcoholic KOH for one hour, according to the procedure of N. Madamba and D. H. Freeman (to be published), liberating carboxyl groups. The resins were converted to the copper-ammonia form by stirring with excess ammoniacal copper sulfate solution before placing in the columns. In preliminary tests, resins were used in the nickelammonia and zinc-ammonia forms. Hydrochlorides of glucosamine, N - acetylglucosamine, galactosamine, and mannosamine were purchased from ICN Pharmaeeuticals, Inc., Cleveland, Ohio. Amino acids and sugars were obtained from several sources. A standard calibration mixture of 17 amino acids was obtained from Bio-Rad Laboratories. Equipment. Glass columns, 6.3-mm and 2.8-mm internal diameter, were used, along with sample introduction valves, connectors, ultraviolet absorption detectors, a pulseless pump, and a Model 3100 liquid chromatograph, all obtained from the Chromatronix Division of Spectra-Physics, Inc., Berkeley, Calif. A differential flowing refractometer, Model R401 and a high-pressure pump, Model 6000, from Waters Associates, Milford, Mass., were also used. Ultraviolet Detection. Amino sugars do not absorb in the ultraviolet, but, as they travel along the column of copper-loaded resin, they pick up copper from the resin as soluble complexes that absorb much more strongly than the copper-ammonia complex at the wavelengths used for detection (254 and 280 nm). The absorbances of the copper-glucosamine and copper-galactosamine complexes are about the same, while that of the copper-mannosamine complex is greater. This finding was an unexpected stroke of good fortune, yet, by analogy with the uncharged copper complexes of diethanolamine, it should not have been surprising. It was thus possible to use ultraviolet absorption detectors routinely. Ultraviolet detection is more sensitive than refractometric detection and less subject to artifacts such as changes in solvent composition. Tests showed that the areas of the ultraviolet absorbance peaks were directly proportional to the amounts of glucosamine injected. Figure 1 shows a simultaneous recording of refractive index and ultraviolet absorbance during elution of four amino sugars. The sharp rise in absorbance and refractive index a t one void volume is due to the conversion of hexosamine chlorides to ammonium chloride, which displaces cupric ions from the resin. General Chromatographic Procedure. The influent was aqueous ammonia, usually lM, containing copper sulfate of concentration 1 X to 5 X 10-4M. The copper ions in the influent replaced those removed from the resin by the injected amino sugars and acids, and also prevented the gradual stripping of copper ions by the ammonium ions of the influent. A portion of influent solution was used as a static reference in the ultraviolet detector. The amino sugars were injected as solutions of their hydrochlorides. Solutions that were injected could be weakly acid. At first we added excess ammonia to the solutions before injection, but we found that ammoniacal solutions of amino sugars are oxidized appreciably in one hour or less on standing in air. The pressures and flow rates that can be used are limited by the mechanical stability of the resin. Hydrolyzed Poragel and Aminex Q-150s are sufficiently rigid to stand high pressure gradients, but
0
100
50
m l 0.5
I50
NH3
Figure 1. Column, hydrolyzed Poragel-Cu, 1.5 meter X 2.8 mm, 60 O C , 36 ml/hr; absorbance at 254 nm. Injected, 0.3 mg of each com-
pound as hydrochloride
1 1
O 0
1 -
30
ml I . O M
60
NH3
Column, Bio-Rex 70, 21 cm X 6.3 mm, room temp., 30 ml/hr; eluent contained 6 mg Cull. injected (micrograms):tryptophane 6, glucosamine HCI 40, galactosamine HCI 60, histidine 80, mannosamine HCI 60. (Note that glycine eluted at the same volume Figure 2.
as tryptophane.) Bio-Rex 70, which gave the best chromatographic separations, is a soft resin whose granules are fractured by high pressures, giving fine particles that plug the column. As long as the pressure drop did not exceed 100 psi (7 bars) along a 25-cm column, this resin could be used continuously without deterioration. A t this pressure, the linear flow rate is about 100 cm/hour. Theoretical-plate heights, based on band widths for glucosamine and galactosamine and on corrected elution volumes, are about 0.5 mm under these conditions.
RESULTS Preliminary Tests with Ni-Bio-Rex 70. These showed that sugars were not absorbed, and that there were great differences in retention between different amino sugars. Relative retention volumes with 0.68M ammonia were: glucose (at void volume) 1.00, glucosamine 3.05, galactosamine 4.30, mannosamine 5.10. Copper-Loaded Resins. With Cu(11) as the coordinating ion, there is less difference between glucosamine and galactosamine than is observed with Ni(II), and more difference between galactosamine and mannosamine. Figures 1 and 2, which show the behavior of two different resins, are typical. Glucosamine and galactosamine have neighboring OH and NH2 groups trans, whereas mannosamine has these groups cis to one another. We made potentiomet-
ANALYTICAL C H E M I S T R Y , VOL. 47, NO. 1 , J A N U A R Y 1975
123
I
TRYPTOPHANE 0 .I
E
0
In N W
u
z 4
m
a
0
Lo m 4
0
L
-
2
10
0
I 20
1 30
I 40
I l
50
l
60
70
80
90
m l I M AMMONIA
Figure 3. Column, hydrolyzed Poragel-Cu, 1.5 meter X 2.8 mm, 60 OC, 36 mVhr to 54 ml, then 64 ml/hr. Injected, 0.5 mg lysine, 0.3 mg glucosamine HCI, 0.5 mg galactosamine HCI, 0.5 mg tryptophane, 0.3 mg mannosamine HCI
I
1
I
30 m l I.O_M N H 3
10
1
20
40
-5 0
Figure 4. Column, Bio-Rex 70, 21 cm X 6.3 mm, room temp., 18 ml/hr; eluent contained 6 mg Cull. Injected 20 pg glucosamine HCI, 30 pg galactosamine HCI, and 0.05 micromole of each of the following amino acids: lysine, histidine, arginine, aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, leucine, isoleucine, tyrosine, phenylalanine. Peaks ( a ) and ( b ) recorded at quarter-sensitivity, ( c ) at haif-sensitivity. Peak ( d )is probably glycine, ( e )histidine; see text
Table I. Elution Volumes on Hydrolyzed Poragel PTO
N- Acetylglucosani ine Glucosan1ine Galactosamine M anno samine Glucose Galactose Mannose
1.1 4.0 5.0 15.5 1.2 1.35 1.2
Beta-alanine
1.0 0 I-
Aspartic acid 1.0 Tyrosine 1.3 Cystine 1.45 Glycine 2.5 Lysineb 2.85 Phenylalanine 3.1 Tryptophane 7.1 Histidine 8.7 Arginine very l a r g e
E
e
ul N
0 005w
z a
m
I I :
0 v,
Eluent, 1.OM NH3 a t 60 "C. Units are multiples of void volume.
m
* The elution volume of lysine a t 24 "C was 4.8 void volumes. Other
a
elution volumes were little affected by temperature.
i ric and absorptiometric measurements that showed mannosamine to form a somewhat more stable complex than glucosamine with copper in aqueous solution. As noted above, the copper-mannosamine complex absorbs much more strongly in the ultraviolet than do the copper complexes of glucosamine and galactosamine. The corrected elution volumes of the amino sugars are inversely proportional to the first power of the ammonia concentration, indicating that one ammonia molecule replaces one molecule of amino sugar. Remembering that the amino sugar is displaced from the resin as a copper complex, we might speculate that the process is, by analogy with ethanolamines ( 1 6 ) , Res-CuAOH'
+ NH, + H,O
+== Res-NH4'
+
CuA(OH),
where A means the amino sugar molecule. The copper concentration of the influent affects elution volumes, but the effect is considerably less than an inverse first power relation. Resin Matrix Effect: Separations from Amino Acids. Figures 2 and 3 show the behavior of two different resins. The theoretical-plate height is much smaller for the acrylic resin (Figure 2) than for hydrolyzed Poragel; in part, this is due to the larger particle size and greater variation in size of the Poragel, though it should also be noted that the linear flow rate was six or seven times faster in the Poragel column. A more significant difference appears in the reten124
.
/ I
0
I
20
I 40
I 6C
ml
LO!
.
I 20
I 40
I
60
NH3
Figure 5. Column, Bio-Rex 70, 32 cm X 6.3 mm, room temp., 24 ml/hr; eluent contained 29 mg Cull. First curve, 0.1 mg glucosamine HCI, 0.4 mg lysine HCI; second curve, same as first pius 0.08 mg galactosamine HCI
tion of the amino acids. Tryptophane, which has an aromatic structure, is much more strongly retained on Poragel, whose polymer network is partly aromatic. Phenylalanine is more strongly retained on Poragel than on Bio-Rex 70. The retention of various amino acids on Poragel is shown in Table I. One expects strong retention of aromatic amino acids on an aromatic resin, and one expects strong retention of basic amino acids on both types of resin. No similar table was prepared for Bio-Rex 70, but an experiment was made in which a standard solution containing 17 amino acids was injected along with glucosamine and galactosamine. The result is shown in Figure 4. Peak ( e ) ,due to histidine and perhaps also lysine, falls comfortably between galactosamine and mannosamine (not shown). Glycine elutes a t ( d ) , and tryptophane would elute a t the same volume if it were present. The other acids elute soon after the void volume. The only amino acid that causes trouble is lysine. Figure 5 shows the lysine peak in 1M ammonia and its proximity to the galactosamine peak. Now it happens that the elution
ANALYTICAL CHEMISTRY, VOL. 47, NO. 1, J A N U A R Y 1975
volume of lysine is very sensitive to the concentration of ammonia. The corrected elution volume (observed volume minus void volume) is inversely proportional to the square of the ammonia concentration. With 1.5-2.OM ammonia, the lysine peak overlapped the hexosamine peaks. The volumes depend somewhat on the degree of saturation of the resin with copper, and presumably on the batch of resin too. Each experimenter must find his own conditions for avoiding interference. The unsymmetrical shape of the lysine peak shows that the distribution ratio increases as the lysine concentration increases. This relation and the fact that the corrected retention volume depends on the square of the ammonia concentration are consistent with this absorption-desorption mechanism:
CUL,
-
cu
21
+
2L-
where HL is the uncharged form of lysine, H2N(CH2)4CHNH2C02H. Coordination of Cu2+ with ammonia is implied, but not shown. The resin Aminex Q-150s was not studied as carefully as Poragel and Bio-Rex 70. It holds copper ions satisfactorily and will tolerate much higher flow rates than Bio-Rex 70, but the theoretical-plate heights are not better than with Bio-Rex 70, and the differences in absorption strengths are much greater. Thus, with 1M ammonia, the elution volumes of the amino sugars (uncorrected) were: glucosamine 3.7, galactosamine 5.4, mannosamine 21.0 void volumes. The basic and aromatic amino acids were retained very strongly indeed. Lysine, for example, eluted at 24 void volumes, and arginine stayed in the column indefinitely. This resin should be used with an ammonia concentration gradient, whereas Bio-Rex 70 works well with isocratic elution. A Simple Quantitative Analysis: Glucosamine in Chitin. T o test the use of a Bio-Rex 70 column for analysis of a natural product, a quantity of chitin was prepared from lobster shells according to the method of Purchase and Braun ( 1 7 ) and air-dried. A 100-mg portion of chitin was hydrolyzed to glucosamine by placing it in a dry test tube, adding 1 ml 12M hydrochloric acid, and heating the test tube to 95 "C in boiling water for 30 minutes in one experiment, for 60 minutes in another. The chitin disintegrated and dissolved within 5 minutes to yield a brown solution. After heating for 30 to 60 minutes in the test tube, the contents were poured into an evaporating dish and evaporated over steam in a current of air. In 5-8 minutes the excess acid was removed and a moist solid remained, which was dissolved in water and transferred to a 10-ml volumetric flask. Two ml of this solution was further diluted to 10 ml. Portions of the diluted solution (corresponding to 2.0 mg chitin per ml) were injected through a 0.1-ml sample loop into a column of Bio-Rex ~ O - C U32 , cm X 0.63 cm, passing 1.5M ammonia which contained 30 mg Cu per liter as CuSO4. The flow rate was 0.5 ml/min; monitoring was done
by UV absorption a t 254 nm, with full-scale deflection 0.32 absorbance unit. Several injections were made of each sample, and of standard solutions containing 1.0 and 2.0 mg pure glucosamine hydrochloride per ml (in 0.01M HCl). Peak heights were considered to be proportional to peak areas, because the standard deviations of the glucosamine peaks remained constant during a day's run. Two different hydrolyzates, A and B, hydrolysis times 30 and 60 minutes respectively, gave the following percentages of glucosamine base in the chitin: A: 55.2, 56.0, 54.8, 54.2, 53.5, 54.2; av. 54.7. B: 55.4, 58.6, 54.8, 54.8, 52.3; av. 55.2. These percentages agree with the glucosamine yields in Ref. ( I 7 ) .Evidently our hydrolysis conditions were correctly chosen. Conditions that are too drastic cause glucosamine to decompose (10 1. This is an easy analysis because the chromatogram has only two major peaks, an intense, narrow peak at one void volume and the glucosamine peak a t four void volumes. We could therefore introduce three samples in succession before the first glucosamine peak emerged, and obtain three glucosamine peaks in 11/2hours from the first injection. Sensitivity. With the detector at maximum sensitivity we obtained distinct peaks with 0.5 microgram of glucosamine, injected as a solution of the pure hydrochloride. ACKNOWLEDGMENT We thank Stephen Levine, Veterans' Hospital, Denver, for suggesting this problem. The help of Jerry Harder in the laboratory is gratefully acknowledged. LITERATURE CITED (1) K. Shimomura, Tong-Jung Hsu, and H.F. Walton, Anal. Chem., 45, 501 (1973). (2) R . A. A. Muzzarelli, "Natural Chelating Polymers," Pergamon Press, Elmsford, N.Y., 1974. (3) A . F. Krivis and M. D. Martz, Microchem. J.. 17, 456 (1972). (4) R. S. Steele, K. Brendel, E. Scheer, and R. W. Wheat, Anal. Biochem., 34, 206 (1970). (5) S. Roseman, Amer. J. Med., 1959, 749. (6) A. Linker and K. D. Terry, Roc. SOC.Exp. Bioi. Med., 113, 743 (1963). (7) D. Kaplan, V. McKusick, S. Trebach, and R. Lazarus, J. Lab. Clin. Med., 1966, 48. (8) D. Keppler, R . Lesch, W. Reutter, and K. Decker, Exp. Mol. Patho/., 9, 279 (1968). (9) L. A . Elson and W. T. Morgan, Biochem. J., 27, 1827 (1933). (10) N. F. Boas, J. Bid. Chem., 204, 553 (1953). (11) S. Gardell, Acta Chem. Scand., 7, 207 (1953). (12) M. Yaguchi and M. B. Perry, Can. J. Biochem., 48, 386 (1970). (13) R . M. Zacharius and E. A . Talky, Anal. Chem., 34, 1551 (1962). (14) Y. Vladovska-Yukhnovska, C. P. Ivanov, and M. Malgrand, J. Chromafogr., 90, 181 (1974). (15) M. D. Martz and A. F. Krivis, Anal. Chem., 43, 790 (1971). (16) J. F. Fisher and J. L. Hall, Anal. Chem., 39, 1550 (1967). (17) E. R. Purchase and C. E. Braun, Org. Syntheses, 26, 36 (1946).
RECEIVEDfor review July 29, 1974. Accepted August 29, 1974. Preliminary accounts of this work were presented at the ACS Regional Meeting in Albuquerque, N.M., July 1974, and the 3rd International Symposium on Ion Exchange, Balatonfiired, Hungary, May 1974. The research was supported by the National Science Foundation under Grant GP-37779X. The Dow Chemical Co., Rocky Flats Division, provided financial assistance to one of us (J.D.N.).
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