346
Anal. Chem. 1985, 57,346-348
Danielson, N. D.; Taylor, R. T.; Huth, J. A.; Slergiej, R. W.; Galloway, J. G.; Paperman, J. B. Ind. Eng. Chem. Prod. Res. Develop. 1983, 22, 203. Huth, J. A.; Danielson, N. D. Anal. Chem. 1982, 54, 930. Siergiej, R. W.; Danielson, N. D. Anal. Chem. 1983, 5 5 , 17. Siergiej, R . W.; Danielson, N. D. J. Chromatogr. Sci. 1983, 21, 362. Smlth, L. I . "Organic Synthesis"; Wiley: New York, 1943; Vol. 23, p 83. Snyder, L. R.; Kirkland, J. J. "Introduction to Modern Liquid Chromatography"; Wlley-Interscience: New York, 1979; p 418. Mattern, D.; Danielson, N. D.; Hercules, D. M., unpubllshed results. Horvath, C. "HPLC, Advances and Perspectives"; Academic Press: New York. 1980: Vol. 2. OD 215-217. (23) Siergiej, R. W. Ph.D. Dissertation, Miami University, 1982, p 76. (24) Rlley, C. M.; Tomlinson, F.; Jefferies, T. M. J. Chromatogr. 1979, 185, 197. (25) Horvath, C.; Melander, W.; Molnar, 1. J . Chromatogr. 1976, 125, 129.
(26) Kawabata, N.; Ohlra, K. fnvlron. Sci. Techno/. 1979, 13, 1401. (27) Tanaka, N.; Goodelf, H.; Karger, B. L. J . Chromatogr. 1978, 158, 233. (28) Willlams, R. C.:Baker, D. R.; Schmlt, J. A. J. Chromatogr. Scl. 1973, 1 1 , 618.
RECEIVED for review July 9,1984. Accepted October 29, 1984. This work was supported in part by grants from the Research Corporation and the donors of the Petroleum Research Fund, administered by the American Chemical Society. Donation of the Model 9533 liauid chromatoaraDh bv IBM to the Chemistry Departmentis greatly appreciated. This work was presented at the 35th Pittsburgh Conference on Analytical Chemistry and Spectroscopy, Atlantic city, NJ, March 8, 1984.
Simultaneous Gas-Liquid Chromatographic Determination of Aldonic Acids and Aldoses Jacob Lehrfeld Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604
A method has been developed for the slmultaneous quantltatlon of several aldonic aclds and aldoses. The aldoses are converted to aldltol acetates, and the aldonic aclds are converted to N-propylaldonamlde acetates. A standard mixture contalnlng the derlvatlzed rlbonlc, mannonlc, gluconlc, and galactonlc aclds and arabinose, xylose, mannose, glucose, and galactose was separated by GLC In 18 mln. Phenyl P-o-glucopyranoslde was used as an Internal standard.
Analysis of complex mixtures, such as those found in fermentation broths and pulping residues, often requires multiple analytical metbodologies. For example, a kinetic evaluation of the production of gluconic acid by Pseudomonas ovalis (1) required separate analyses of glucose and gluconic acid. A simple method which would simultaneously quantitate both components would be useful. A differential method (2) for quantitating mixtures such as this is available. However, it is tedious and time-consuming because it requires three borohydride reductions and a duplicate analysis. A new procedure, described here in Figure 1,is capable of simultaneously analyzing a wide variety of aldose and aldonic acid mixtures. Aldoses are reduced to their corresponding alditols, and the aldonic acids are converted into the corresponding Npropylaldonamides. The new derivatives are acetylated by pyridine-acetic anhydride; the peracetates are readily separated by GLC in 18 min on a SP 2340 column. EXPERIMENTAL SECTION Materials. Propylamine, L-arabinose, D-Xylose, D-mannose, D-glucose, and D-galactose were obtained from Aldrich Chemical Dco. Phenyl 0-D-glucopyranoside, ~-g~ucono-1,5-lactone, galactono-1,4-lactone,L-mannono-l,4-lactone,sodium D-glUCOnate, and myo-inositol were obtained from Pfanstiehl Laboratory, Inc. Potassium ribonate and ammonium xylonate were a gift from M. E. Slodki. GLC-coated support (3% Sp 2340 on 100/120 mesh Supelcoport) was obtained from Supelco, Inc. Cation-exchange resin (AG 5OW-XB 200-400 mesh H') was obtained from Bio-Rad Laboratory. GLC Analysis. Analysis by GLC was performed on a Packard Instrument Model 428 gas chromatograph equipped with dualflame ionization detectors and dual electrometers. Peracetylated derivatives were separated on a glass column (1m x 2 mm i.d.)
packed with 3% SP 2340. The temperature was programmed from 190 to 260 "C at a rate of 5 "C/min and held there until the last peak was eluted. Helium flow rate was 20 mL/min. Conversion of Aldoses and Aldonic Acids to Peracetylated Alditols and N-Propylaldonamides. A 0.1 M sodium carbonate solution (0.6 mL) was added to a mixture containing approximately 2 mg each of L-arabinose, D-Xylose, D-mannOSe, D-glucose, D-galactose, myo-inositol, D-ribonolactone, ~-mannono-1,4-lactone, D-galactono-1,4-lactone, and phenyl p-DD-glucono-~,~-~actone, glucopyranoside in a 16 X 125 mm culture tube. The solution was maintained at 30 "C for 1 h and then treated with sodium borohydride (0.5 mL of a 4% solution) for 1 l/z h at 22 "C. Excess sodium borohydride was decomposed by the dropwise addition of acetic acid (25%) until bubbling stopped (6-8 drops). To remove sodium ions, the solution was poured onto a column of cation-exchange resin (2 mL) and eluted with 7 mL of water. The eluate was evaporated to dryness in a Buchler vortex evaporator (45 "C in vacuo). Borate was removed by twice evaporating methanol (3 mL) from the residue. Heating at 85 "C in vacuo for 2 h converted the aldonic acids into aldonolactones. This residue was dissolved in pyridine (1mL), 1-propylamine (1 mL) was added, and the tube was capped and heated at 55 "C for 30 min. The solution was cooled (under 45 "C), the cap was removed, and nitrogen was bubbled through the reheated solution (55 "C) until dry. The residue was dissolved in pyridine (0.5 mL) and acetic anhydride (0.5 mL) and heated at 95 "C for 1h. Sodium gluconate, when treated as above, gave similar results. The sample is suitable for GLC as is; usual injection size is 0.8 pL. If sample size is small (less than 1-2 mg), tailing from pyridine and acetic anhydride can interfere with detection and/or quantitation. If so, remove them by bubbling nitrogen through the solution until dry and reconstitute with 100 pL of acetone, chloroform, or methylene chloride. Optimization of Lactone Hydrolysis. A mixture of Lmannono-l,4-lactone (39 mg), ~-glucono-1,5-lactone(41 mg), ~-galactono-l,4-lactone(39 mg), and phenyl P-D-ghcopyranoside (36 mg) was dissolved in 0.1 M sodium carbonate (8 mL) and kept at 30 "C. Aliquots (0.5 mL) were removed at 15, 30,45,60, 90, and 120 min and treated with a 4% sodium borohydride solution (0.5 mL). The aliquots were then treated as above. Similar experiments were performed at temperatures down to 22 "C and with more dilute base. RESULTS AND DISCUSSION The standard methods for the analysis of aldoses as alditol acetates (3) or aldononitrile acetates ( 4 , 5 )and aldonic acids
This article not subject to U S . Copyrlght. Published 1984 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985 Step A Aldose
I
__________ Aldon,c Ac!d
---_-----Aidoflolacrone
Step Aldose
Na2C03 H20
B
__-_-----NOBHl Ma Aidmale
_-___ ____
-
Table I. Hydrolysis of Aldonolactones in 0.1 M Na2C03'
Aiditol Acetate
_____________ N Piopylaldunamide . p I w v l ~ m ~ n ~_____________ - ________ 3.4 1Pyridine
--C Ma Aidmale
_____---Nu Aidunare
Step C Alditol
Ne Aidonare
347
1 H'IMeOH
Ace[(( Anhydride
N.ProprieIdunumd8
Figure 1. Method for the simultaneous analysis of aldonic acids and
aldoses.
time, min 0 9
% lactone in solution mannono-1,4- galactono-1,4- glucono-1,5lactone lactone lactone 22 OCb 30 "Cb 22 "C 30°C 22 "C 30
"c
100
100
33
100 10
100
100
100
c
15
14
5.2 2.0
3 0.5 0.2
0.4
30 45
5.0 0.2 c
0.6 0.1
0.1
c
C
C
C
60 90
0.8
c
c
C C
C
0.2
C C
C C
C
"The ratio of milliliters of 0.1 M Na2C03to milligrams of lactone was about 0.07. bTemperature at which hydrolysis occurred. cIndicates less than 0.1%.
Tim8
Imml
Figure 2. Separation of peracetylated N-propylaldonamides and aklitols by gas-liquid chromatography on a 3% SP 2340 packed glass column (1 m X 2 mm Ld.) and temperature programmed from 190 to 260 "C at a rate of 5 'Clmin: (1) arabitol, (2)xylltol, (3)mannitol, (4)galactitol, (5)glucitol, (6) inositol, (7) ribonamide, (8)phenyl P-o-glucopyranoslde, (9) mannonamide, (10) gluconamide, and (1 1) galactonamlde.
as N-propylaldonamides are simple and accurate. However, the application of these methods to a mixture containing aldoses and aldonic acids will yield inaccurate or incomplete data. The method for the analysis of aldonic acids by conversion to N-propylaldonamides (6) is not compromised by the presence of aldoses. However, accurate information regarding the types and amounts of aldoses present is lacking because their conversions to the N-propylglycosylamine are not stoichiometric; an exception to this is glucose. Aldonic acids do not interfere with the determination of aldoses by the aldononitrile procedure. The aldonic acids do not form a volatile derivative, and no information regarding their type or amount is obtained. However, quantitation of aldoses in the presence of aldonic acids by the alditol acetate procedure is inaccurate. Aldonic acids easily form aldonolactones, which are readily reduced to alditols. The alditols measured will no longer reflect the quantity of aldose previously present but rather will reflect the sum of aldose and aldonolactone. In addition, under most circumstances, the ratio of lactone to acid will be variable (7,8)except under special conditions (9). To overcome these difficulties, a modified procedure was developed (Figure 1) that now permits the simultaneous quantitation of aldonic acids and aldoses. Initial treatment of the mixture with a base converts all the lactone to the sodium salt of the acid, which is not reduced by sodium borohydride. Consequently, the reduction step converts the aldoses to alditols, and the aldonic acids are not reduced. The aldonic acids are then quantitatively converted into the lactones and then into the N-propylaldonamides. Alditol acetates and N-propylaldonamides are easily separable by GLC in 18 min (Figure 2). Alkaline Hydrolysis of Lactone (Step A). The problem of interference by uronic acids in the alditol acetate procedure (3)has been dealt with previously. Reduction of the lactone to the alditol can be eliminated by converting the lactone to the sodium salt of the aldonic acid. Sodium borohydride does not reduce acids or their salts (10). The recommended (3) 20-min saponification period and adjustment to 0.01 M Na2C03was not satisfactory for aldonic acids. Table I summarizes the results of a hydrolysis ex-
periment performed at 22 "C. An excess of base was ensured by the addition of about 1.3 mmol of Na2C03/mmolof lactone. After 15 min, 0.1% D-glUCOnO-1,5-~aCtOne,14% mannonol,.l-lactone, and 3% galact~no-1,4-lactonewere left. Even after 45 min, detectable amounts of mannono-l,4-lactone (2%) and galactono-1,4-lactone (0.2 %) remained. The rates of hydrolysis could be speeded up by increasing the temperature, by increasing the concentration of alkali, or both. A large increase in either would cause epimerization and degradation of the carbohydrates; consequently, small incremental changes were made in the reaction conditions until the best compromise was found. A temperature of 30 "C and the same alkali concentration resulted in essentially complete hydrolysis in 45 min (Table I). To determine the degree of epimerization and degradation, the aldoses and the aldonic acids were dissolved in 0.1 M sodium carbonate and kept at 30 "C for 2 h. Degradative losses for the aldoses and aldonic acids were less than 0.5%. After 2 h the sample of gluconic acid contained 0.3% mannonic acid and the sample of mannonic acid contained around 0.4% gluconic acid. Galactonic acid was remarkably stable. Essentially, the solution now contained only aldoses and sodium aldonates. Reduction with Sodium Borohydride (Step B). Sodium borohydride quantitatively reduces aldoses ( 1 0 , I I ) and aldonolactone (12) but not free acids or their salts (13). A key assumption in the new procedure is that sodium aldonates will not be reduced. To determine to what extent this is true, a sample of sodium gluconate, sodium galactonate, and sodium mannonate was treated with 4% sodium borohydride solution for 18 h at 22 "C. The sample was then treated in the manner described under the Experimental Section. No alditols were detected. Alkali stabilizes aqueous sodium borohydride (14);however, increased pH also decreases the hydride's reactivity toward sugars (15). To confirm the complete reduction of the aldoses, within 1.5 h, a mixture of L-arabinose, D-xylose, D-mannose, D-glucose, and D-galactose was dissolved in 0.1 M sodium carbonate and treated as described under the Experimental Section. None of the aldoses could be detected after 11/4 h; i.e., complete reduction had occurred. Similar results were obtained from the same mixture dissolved in water. Essentially after the borohydride reduction (step B, Figure l),a mixture remains in which the original aldoses are now alditols and the original aldonic acids and aldonolactones are now sodium aldonates. Conversion to Peracetylated Alditols and N-Propylaldonamides (Step C). The reduced mixture was treated with acetic acid to decompose residual borohydride and to neutralize the sodium carbonate. The solution was poured through a cation-exchange column to remove sodium ions, the water was removed by evaporation, borate was removed as
340
Anal. Chem. 1 9 ~ 557, , 348-352
the volatile trimethyl borate by codistillation with methanol, and the residue was heated 2 h at 85 "C, in vacuo to completely convert the aldonic acids into aldonolactones. Lower temperatures and/or shorter time periods resulted in partial conversion and the isolation of some free aldonic acids. Complete conversion is necessary because aldonic acids will not form N-propylaldonamide when treated with l-propylamine. Even though the amine is very basic, it did not cause epimerization under the conditions studied. Acetylation with pyridine and acetic anhydride requires 1h and is quantitative. Dehydration reactions occur with the lactone (16) but not with the amide. Essentially the original aldoses have been converted into alditol acetates, and the original aldonic acids and aldonolactones have been converted into corresponding N-propylaldonamides. Calibration curves constructed by plotting detector response vs. milligrams of sample (1.2-6.7 mg) have a correlation coefficient greater than 0.995. A wider range of sample sizes can be accommodated by suitable concentration or dilution of the injected sample. There are a number of advantages to the new procedure. The method is able to handle complex mixtures of aldonic acids and aldoses. Alduronic acids can be determined by suitable modifications. Because the carboxyl group is kept, the stereochemical configuration of the sugar is maintained, unlike the differential method (2)wherein the lactone is reduced to the alditol. The new method adds a degree of versatility not found in most methods. The N-propyl group can be substituted easily by an N-butyl, N-amyl, or even an N-hexyl by replacing 1-propylamine by some other amine. The lactone will react under basically the same conditions to give a homologous derivative. The advantages are readily apparent in that the larger the N-alkyl group, the greater the elution time relative to the alditols or other possible compo-
nents, e.g., phenyl P-D-ghcopyranoside. In fact, N-propylxylonamide coelutes with phenyl 0-D-glucopyranosides; by changing to N-amylamine, one is able to separate them into two peaks while maintaining the resolution seen in Figure 2. Registry No. Ribonic acid, 17812-24-7; mannonic acid, 6906-37-2; gluconic acid, 526-95-4; galactonic acid, 13382-27-9; L-arabinose, 5328-37-0; D-xylose, 58-86-6; D-mannose, 3458-28-4; D-glucose, 50-99-7; D-galactose, 59-23-4; myo-inositol, 87-89-8; ~-mannono-1,4-lactone,22430-23-5; D-galactono-1,4-lactone, 2782-07-2; D-g~ucono-1,5-~actone, 90-80-2.
LITERATURE CITED Humphrey, A. E.; Reiiiy, P.J. Biotechnol. Bioeng. 1965, 7,229-243. Lehrfeld, J. Anal. Biochem. 1981, 115, 410-418. Sloneker, J. H. Methods Carbohydr. Chem. 1972, 6 , 20-24. Easterwood, J. M.; Huff, B. J. L. Sven. fapperstidn. 1969, 72, 768-772. (5) Dmitriev, B. A.; Backinowsky, L. V.; Chizhov, 0. S.; Zoiotarev, 8. M.; Kochetkov, N. K. Carbohydr. Res. 1971, 19, 432-435. (6) Lehrfeld, J. Anal. Chem. 1984, 5 6 , 1803-1806. (7) Blake, J. D.; Richards, G. N. Carbohydr. Res. 1968, 8 , 275-281. (8) Blake, J. D.; Richards, G. N. Carbohydr. Res. 1970, 14, 375-387. (9) Morrison, I. M.; Perry, M. B. Can. J. Biochem. 1968, 44, 1115-1126. (10) Sawardeker, J. S.; Sloneker, J. H.; Jeanes, A. Anal. Chem. 1965, 37, 1602-1604. (11) AbdeCAkher, M.; Hamilton, J. K.; Smith, F. J. Am. Chem. SOC.1951, 73,4691-4692. (12) Sjostrom, E.; Haglund, p.; Janson, J. Acta Chem. Scand. 1968, 2 0 , 17 18-17 19. (13) Brown, H. C. "Hydroboration"; W. A. Benjamin: New York, 1962; p 243. (14) "Sodium Borohydride"; ThiokoilVentron: Danvers, MA, 1979; p 4. (15) Lee, J. B. Chem. Ind. 1959, 1455-1456. (16) Nelson, C. R.; Gratzl, J. S. Carbohydr. Res. 1978, 6 0 , 267-273. (1) (2) (3) (4)
RECEIVED for review June 25,1984. Accepted October 9,1984. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S.Department of Agriculture over other firms or similar products not mentioned.
Analysis of Complex Mixtures by Gas Chromatography/Mass Spectrometry Using a Pattern Recognition Method Mingjien Chien
Givaudan Corporation, Clifton, New Jersey 07014
Analysis of a complex mlxture Involves a gas chromatographlc separation followed by a mass spectral search for Identification of each component. Thls Is an extremely tedlous and time-consumlng process for fragrances and flavors whlch sometimes contaln hundreds of components. Thls presentatlon describes a computer pattern recognttlon method for the rapid ldentlficatlon of components in a complex mixture, the comparlson between the complete GC profiles of different samples, and the recognltlon of a group of compounds as a single entity. The central part of thls method is a comparlson algorlthm which compares the components In two mixtures uslng the K nearest neighbor classification rule. I t is Illustrated by comparing essential oils of dlfferent sources and by detectlng essentlal 011s In perfumes. A varlety of procedures deslgned to expedlte the search process are also dlscussed.
In the past decade, with the development of automated computer processes, gas chromatography/mass spectrometry
has become one of the most powerful tools in the analysis of complex mixtures. Today, computer-processed data acquisition, quantitation, and consequently the search of a data base for each peak have become a routine practice. However, for a sample containing no less than 100 components, the conventional approach of an exhaustive mass spectral search for each GC peak is still very tedious and time-consuming. Besides, mere peak identification of such complex mixtures often provides us with information of little value. On the other hand, there is indeed much valuable information concealed under the complicated GC pattern and can be brought out only through more sophisticated data processing. In the analysis of flavor and fragrance meterials, complex mixtures are routinely encountered. However, most components in the sample are very likely to be known compounds. When the same type of flavor or fragrance is analyzed, virtually the same set of compounds is found each time. Complete mass spectral search for identification is often unnecessary. Furthermore, many components in the mixture may have originated from a single source, such as an essential oil.
0 1984 American Chemical Society 0003-2700/85/0357-0348$01.50/0
