Anal. Chem. 2000, 72, 4973-4978
Isolation of n-Decyl-r(1f6) Isomaltoside from a Technical APG Mixture and Its Identification by the Parallel Use of LC-MS and NMR Spectroscopy Patrick Billian,† Walter Hock,‡ Reinhard Doetzer,‡ Hans-Ju 1 rgen Stan,*,† and Wolfgang Dreher‡
Institute of Food Chemistry, Technical University of Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany, and Agricultural Center Limburgerhof, BASF AG, 67114 Limburgerhof, Germany
The identification of n-decyl r(1f6)isomaltoside as a main component of technical alkyl polyglucoside (APG) mixtures by the parallel use of liquid chromatographymass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy is described. Following enrichment on a styrene-divinylbenzene-based solid-phase extraction material, unknown components were separated by reversed-phase liquid chromatography (LC). Chemical characterization was achieved by both mass spectrometry and NMR spectroscopy. It is demonstrated that the combination of LC-MS with various NMR techniques is very suitable for stereochemical assignment of unknown components in technical APG mixtures. Alkyl polyglucosides (APGs) represent a new class of nonionic surfactants. APGs are manufactured using two different synthetic routes, namely, direct synthesis and transacetalization. Because of higher equipment costs, direct synthesis is often preferred in industrial APG production.1 Monomeric glucose, obtained from starch, reacts in an acid-catalyzed one-stage reaction with longchain fatty alcohols obtained from renewable resources such as coconut oil or from petrochemical sources. In contrast to stereospecific pathways using protective groups, which results in welldefined components, the industrial process leads to a complex mixture of alkyl monoglucosides as well as higher oligomers. Figure 1 represents the general structure of alkyl polyglucoside surfactants. Technical APG mixtures consist of species with differences both in alkyl chain length (C8-C16) and in the number of glucose units (1-10). Alkyl monoglucosides are the main group forming ∼50% of a mixture, followed by alkyl diglucosides at ∼10%. Because of the formation of R- and β-anomers, furanosides, and pyranosides, as well as binding isomers, technical products contain a large number of different components and stereoisomers. NMR spectroscopy is the most powerful technique in structural elucidation, but it shows a lower sensitivity than mass spectrometry (MS) or tandem mass spectrometry (MS/MS). It requires more sophisticated chromatographic enrichment and separation for the measurement of compounds at low concentration levels. * Corresponding authors: (tel, fax) ++49 (0)30 314 72702; (e-mail)
[email protected]. † Technical University of Berlin. ‡ BASF AG. (1) Hill, K.; von Rybinski, W.; Stoll, G. Alkyl Polyglucosides; VCH Weinheim, 1997. 10.1021/ac0004005 CCC: $19.00 Published on Web 09/13/2000
© 2000 American Chemical Society
Figure 1. General chemical structure of APGs (binding and ring isomers not indicated).
In recent years, many applications have been published in the fields of pharmaceutical,2 natural product,3 and surfactant research. Carminati et al.4 gave an overview of surfactant mixture analysis by 13C NMR. It was applied as a preliminary screening for the analysis of commercial products. The coupling of liquid chromatography to NMR spectroscopy is becoming increasingly popular. Schlotterbeck et al.5 investigated nonionic surfactants from the alcohol ethoxylate type by on-line LC 1H NMR. Running one single on-line experiment, information on the number of components, their chemical compositions, and substitution patterns could be obtained. Strohschein et al.6 separated and identified tocotrienol isomers by LC-MS and LC NMR coupling. Preiss et al.7 used LC-MS and LC NMR to characterize dyes and other pollutants in the effluent of a textile company. In these papers, it was demonstrated that the combined use of both techniques provides complementary structural information. A recent review on LC NMR was presented by Albert.8 LC-MS is well suited to analyze the composition of APG mixtures because the components can be identified by their quasimolecular ions [M - H]-. The best results were obtained with atmospheric pressure chemical ionization mass spectrometry in the negative ion mode. All components exhibit abundant [M H]- ions. The identity can be confirmed with LC-MS/MS by means of indicative product ion spectra.9 It turned out that the (2) de Koning, J. A.; Hogenboom, A. C.; Lacker, T.; Strohschein, S.; Albert, K.; Brinkmann, U. A. Th. J. Chromatogr., A 1998, 813, 55-61. (3) Bringmann, G.; Ru ¨ ckert, M.; Saeb, W.; Mudogo, V. Magn. Reson. Chem. 1999, 37, 98-102. (4) Carminati, G.; Cavalli, L.; Buosi, F. J. Am. Oil Chem. Soc. 1988, 65, 669677. (5) Schlotterbeck, G.; Pasch, H.; Albert, K. Polym. Bull. 1997, 38, 673-679. (6) Strohschein, S.; Rentel, C.; Lacker, T.; Bayer, E.; Albert, K. Anal. Chem. 1999, 71, 1780-1785. (7) Preiss, A.; Sa¨nger, U.; Karfich, N.; Levsen, K.; Mu ¨ gge, C. Anal. Chem. 2000, 72, 992-998. (8) Albert, K. J. Chromatogr., A 1999, 856, 199.
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product ion spectra of APGs consist mainly of fragments resulting from the carbohydrate portion of APG molecules. Small intensity differences between product ion spectra of different isomers were not suitable for differentiating isomeric conformation. This demonstrates that LC-MS cannot be used for analyzing stereoisomeric compounds because the fragmentation pattern of stereoisomers is very similar. However, by the parallel use of LC-MS providing molecular mass information and NMR spectroscopy resulting in stereochemical information, the greatest possible structure assignment of unknown components was achieved.10 In this paper, the isolation and identification of n-decyl-R(1f6) isomaltoside will be described. After solid-phase extraction and separation by reversed-phase liquid chromatography, both negative atmospheric pressure chemical ionization (APCI) mass spectrometry and NMR spectroscopy were used to characterize the unknown component. Structural elucidation of the isolated component by the combined use of 1H NMR and 13C NMR techniques resulted in finding n-decyl-R(1f6) isomaltoside to be one of the main diglucosidic APG components. EXPERIMENTAL SECTION Reagents. LC solvents were of Lichrosolve LC grade (Merck, Darmstadt, Germany). Solvents for LC NMR separation were acetonitrile NMR Chromasolv (Riedel-de Haen, Seelze, FRG) and deuterium oxide (99.9% D, Cambridge Isotope Laboratories, Andover, MA). The other chemicals used (cyclohexane, methanol, ethyl acetate) were of analytical grade and were from Merck. APGs such as n-alkyl β-D-monoglucosides and n-alkyl β-D-maltosides from Anatrace were used as reference substances and were chromatographically pure. All substances were used as received. Glucopon 225 (produced by Henkel, Du¨sseldorf, Germany) was obtained as liquid formulation. SPE material ENV+ were from Separtis (Grenzach-Wyhlen, Germany). Prior LC all samples were filtered using disposable 0.2-µm polyamide membrane filters (Muder & Wochele, Berlin, Germany). Separation of Alkyl Diglucosides from Glucopon 225 by SPE. About 50 mg of Glucopon 225 was diluted in 20 mL of water. Alkyl diglucosides and higher oligomers were separated from the technical product by solid-phase extraction using ENV+. Before use, SPE material (200 mg in a 6-mL polyethylene cartridge) was washed and conditioned with 6 mL each of methanol and water. Alkyl monoglucosides were eluted with ∼15 mL of a mixture of cyclohexane/ethyl acetate (50 + 50, v/v), whereas alkyl diglucosides and higher oligomers were eluted in a second step by 2.5 mL of methanol. The methanol eluate was dried with a gentle stream of nitrogen, and the residue was dissolved in 5 mL water. The eluate was treated a second time by the same SPE procedure to improve the efficiency of enrichment. The second eluate was dried, dissolved in 1 mL of LC eluent (acetonitrile/water 35 + 65, v/v), and filtered using 0.2-µm polyamide membrane filters. Characterization of APGs by LC-MS and LC-MS/MS. A Hewlett-Packard (HP) 1100 series LC system was combined with a triple-quadrupole mass spectrometer by means of an orthogonal Z-flow interface (Micromass, Manchester, U.K.). Liquid chromatographic separation was carried out on a Nucleosil 100 RP(9) Billian, P.; Stan, H.-J. J. Chromatogr., A, submitted for publication. (10) Glaser, T.; Dachtler, M.; Albert, K. GIT Labor-Fachzeitschrift 1999, 9/99, 904-909.
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C8 column (250 × 4.6 mm, partikel size 5 µm) from Muder & Wochele (Berlin, FRG) with acetonitrile/water starting from 35 + 65 (v/v) and changing to 50 + 50 (v/v) within 15 min. These final conditions were held isocratically for 5 min. Eluent flow was 1.0 mL/min. Column temperature was held at 25 °C. LC-MS was operated using an APCI probe in the negative ion mode. Probe and source temperature were set to 400 and 120 °C, respectively. Cone voltage was set to 40 V and corona discharge voltage to 3.3 kV. Nitrogen as drying gas and nebulizing gas was generated from pressurized air in a Whatman model 75-72 nitrogen generator. The nebulizer gas flow was set to ∼600 L/h and the drying gas flow to 250 L/h. APGs were detected by selected ion recording (SIR) as [M - H]-. Data acquisition and mass spectrometric evaluation were performed by use of MassLynx-Software 3.2 (Micromass. MS/MS mode was used with LC using collision gas argon (99,999%) with a pressure of 1.0 × 10-6 bar in the collision cell. Collision energy (CE) was set to 25 eV to obtain maximum sensitivity. Analysis of Alkyl Oligoglucosides by On-Line LC 1H NMR. For the analysis of alkyl oligoglucosides by on-line LC 1H NMR, a Varian 9012 solvent delivery module was used. The separating column, composition of mobile phase, and LC run conditions were identical to those used for LC-MS. The Varian Unity Inova 600 spectrometer was operated at 599.82 MHz. For on-line LC NMR experiments, it was equipped with a 1H {13C} 60-µL inverse detection pulsed field gradient (PFG) microflow probe. Shimming was performed on the lock signal (D2O) before starting the experiment. Eight transients with 18 208 complex points and a spectral width of 10 kHz were recorded per retention time increment. An acquisition time of 1.82 s/transient with a repetition rate of 1.93 s was used. The pulse angle was set to 70°. A total of 33 free induction decays (FIDs) with an acquisition time of 15.4 s/FID were recorded during the separation. Data were treated as 2D matrix (t1 direction ) retention time) and processed with Varian VNMR software (without zero filling and exponential function). CH3CN and residual HOD signals were suppressed by means of a combination of multiple frequency selective excitation (WET) with PFG.11,12 Static NMR Experiments. For static measurements, a 1H 13 { C} 3-mm indirect detection PFG probe was used. The 1D 1H NMR spectra were acquired using 20 886 complex points with a spectral width of 5960 Hz; the acquisition time was 3.5 s. The pulse angle was set to 75°. Total acquisition time was 7.5 min, recording 128 transients. Data were processed with Varian VNMR software, using 64k zero filling and multiplied by an exponential function (lb ) 0.2 Hz). The 2D gradient-HSQC (Heteronuclear Single Quantum Coherence) spectrum was acquired using 512 complex points with a spectral width of 5290 Hz (1H) and 20000 Hz (13C), respectively. The acquisition time was 0.193 s/transient (delay 1.2 s), with 13C decoupling during acquisition. Adiabatic WURST decoupling was carried out as described by Kupce and Freeman.13 A total of 128 transients were recorded in F1 leading to a total acquisition time of 6.8 h. The spectrum was processed without zero-filling but linear prediction to 256 increments in F1. (11) Smallcombe, S. H.; Patt, S. L.; Keifer, P. A. J. Magn. Reson. A 1995, 117, 295-303. (12) Ogg, R. J.; Kingsley, P. B.; Taylor, J. S. J. Magn. Reson. B 1995, 104, 1. (13) Kupce, E.; Freeman, R. J. Magn. Reson. A 1995, 117, 246.
Apodization was achieved by a 90° shifted squared sine bell both in F2 and F1. RESULTS AND DISCUSSION Enrichment of Alkyl Diglucosides from Technical APG Mixtures. Preconcentration of the unknown compounds was required to address the lower sensitivity of LC NMR. Typically, solid-phase extraction (SPE) is used for the concentration of nonionic surfactants from water samples. The successful enrichment of APGs using C8-, C18-, and styrene-divinylbenzene-based sorbent materials was reported.9,14 Since APG mixtures consist of about 50% alkyl monoglucosides and 50% alkyl oligoglucosides, in a first step, the alkyl monoglucosides have to be separated. The separation is necessary to avoid overloading of the LC column and to exploit the separation capacity for the resolution of the alkyl diglucosides and higher oligoglucosides. From the large number of diglucosidic stereoisomers, it can be estimated that concentration levels of about 0.11% per individual component may be expected. Up to an estimated amount of 500 µg of diglucosidic stereoisomers was injected within an injection volumn of 100 µL. Conventionally reversed-phase LC on RP-C8 adsorbent has been employed to separate and characterize chemically and structurally different components. In preliminary experiments with various sorbents including C18, graphitized carbon, and styrene-divinylbenzene-based materials, solid-phase extraction on ENV+, a styrene-divinylbenzene copolymer-based sorbent was found to be best suited for the extraction of APGs from water samples.9 Different eluents were tested for their capability to separate alkyl monoglucosides from the other APG components. A mixture of cyclohexane/ethyl acetate (50 + 50, v/v) was found to give the best results for the separation of two alkyl monoglucosides and two alkyl maltosides available as pure reference substances. The use of defined test substances allowed a check of recovery completeness. In the first step, alkyl monoglucosides were eluted with 15 mL of cyclohexane/ethyl acetate, whereas higher oligomers were almost completely retarded. After a short dry time, these components were eluted with 2.5 mL of methanol. Depending on the alkyl chain length, ∼80% of monoglucosides were found in the first eluate but only 10% of maltosides were eluted by cyclohexane/ethyl acetate. Variations of the separation parameters did not improve the final resolution. With the higher elution power of methanol, the remaining APGs were eluted into the second (“methanol”) fraction. The SPE procedure was repeated to increase the efficiency of enrichment. This leads to a further relative increase of alkyl diglucosides. The analysis of the “methanol fraction” resulted in less than 3% of alkyl monoglucosides while only ∼15% of alkyl diglucosides were lost using this two-stage SPE procedure. Out of 50 mg of Glucopon 225, 20 mg of alkyl diglucosides and higher oligomers was isolated. Characterization of APGs by LC-MS and LC-MS/MS. LC is currently the most suitable method for the analysis of surfactants.15 It may be successfully applied to the separation of oligomers and homologues of nonionic surfactants, i.e., alcohol ethoxylates and APGs. Using reversed phases (C8, C18), nonionics were separated according to their alkyl chain length. APGs were (14) Eichhorn, P.; Knepper, T. P. J. Chromatogr., A 1999, 854, 221-232. (15) Vogt, C.; Heinig, K. Fresenius J. Anal. Chem. 1999, 363, 612-618.
Figure 2. RP-C8 LC separation of APGs in Glucopon 225 (original) and after two-stage SPE with ENV+. Alkyl monoglucosides and alkyl diglucosides were detected using APCI in negative mode by their deprotonated molecular ions [M - H]-. The peaks A and B correspond to the two major decyl diglucosides, which were further investigated by tandem mass spectrometry.
also separated on C8 phases according to their degree of polymerization by applying acetonitrile/water mixtures as mobile phase. Due to the absence of chromophoric groups in the APG molecules, the common UV and diode array detectors (DAD) are not useable. More selective detection methods such as mass spectrometry are required for analyzing APGs. Using liquid chromatography-electrospray mass spectrometry14 or liquid chromatography-atmospheric pressure chemical ionization mass spectrometry in the negative ion mode,9 separation and identification of individual components in technical APG blends were achieved. The total ion chromatogram (TIC) for Glucopon 225, obtained under APCI conditions for detecting APGs, is shown in Figure 2. It is dominated by the two alkyl monoglucosides C8G1 and C10G1 with their [M - H]- ions at m/z ) 291 and 319, respectively. Only a single peak was observed in the ion traces of monoglucosides, which means R- and β-anomers were not separated under the conditions used. In the ion traces of alkyl diglucosides C8G2 and C10G2 at m/z ) 453 and 481, however, a large number of incompletely resolved peaks were observed. It has to be noted that n-decyl R(1f4)maltoside (formula presented in Figure 3), the only available reference substance, shows a retention time of ∼ 9.5 min which is different from all the major peaks detected in the ion trace of decyl diglucosides. This demonstrates that the n-decyl R(1f4)maltoside obviously is not one of the major diglucosidic isomers. The effect of the enrichment by the twostage SPE is also shown in Figure 2. At the concentration level applied to the LC-MS analysis, no remaining alkyl monoglucosides can be spotted. Only diglucosidic components were found. In a series of experiments, various eluents were tested to improve the chromatographic resolution but without success. Due to their stereoisomeric structure, all peaks show very similar retention characteristics. The best chromatographic separation was achieved using a linear gradient of acetonitrile/water from 35 + 65 (v/v) to 50 + 50 in 15 min. Using isocratic conditions with an eluent system of acetonitrile/water 31 + 69 (v/v), the best resolution was achieved but retention times dramatically increased. For further chemical characterization, the peak patterns of decyl diglucosides were analyzed by tandem mass spectrometry. Maximum structural information was obtained using APCI Analytical Chemistry, Vol. 72, No. 20, October 15, 2000
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Table 1. MS/MS Data (m/z) of Decyl Diglucosides Separated on RP-C8
tR (min)
ion 1 base peak
8.10 8.34 8.58 (A)d 9.01 9.27 (B)d 9.48
101 101 101 89 101 101
rel abundance ion 2 ion 3 71 (45) 89 (80) 89 (80) 119 (80) 89 (90) 89 (60)
89 (40) 119 (70) 119 (60) 101 (60) 119 (70) 119 (40)
other fragmentsa
179
185
113, 319 71, 319 71, 319 113 113, 319 113, 319
ndb +c + + + +
nd + + nd nd nd
a Fragments with a relative abundance more than 25%. b nd, fragment was not detectable. c Fragment was observed in the MS/MS spectrum with a relative abundance of more than 5%. dThe peaks A and B correspond to the MS/MS spectra presented in Figure 4.
Figure 3. Structures of (a) n-dodecyl R(1f4)maltoside (reference diglucoside) and (b) n-decyl R(1f6)isomaltoside (title compound).
Figure 4. LC-MS/MS experiments with APCI in negative mode. Product ion spectra (m/z 50-490) of the two major decyl diglucosides A + B (see also Figure 2).
in the negative ion mode and MS/MS. The analytes were characterized by their molecular mass and specific fragment ions together with their retention time. The product ion scans of two major decyl diglucosidic peaks are compared in Figure 4. As expected, both spectra consist only of fragments resulting from the carbohydrate moiety of the APGs from m/z 179 down to m/z 60. This fragmentation pattern appears very similar to that of alkyl monoglucosides. Comparing the two spectra presented, only small intensity differences with all fragment ions were observed in both spectra. Only the fragment ion m/z 185 seems to be characteristic for the analyte A, because this ion could not be found in the spectral pattern of analyte B. Due to the lack of reference material, no further structure elucidation was possible. Characteristic fragment ions of analytes investigated by tandem mass spectrometry are summarized in Table 1. It can be stated that LC-MS(/ MS) provides information about molecular mass and the presence of glucose moieties. From these data, the alkyl chain length and the degree of polymerization can be calculated. However, there is no information about structural assignment available to elucidate stereochemistry of diglucosidic APGs. The daugther ion spectra 4976 Analytical Chemistry, Vol. 72, No. 20, October 15, 2000
Figure 5. Contour plot of the RP-C8 LC separation of alkyl diglucosides with the on-flow 1H NMR spectrum (600 MHz) of analyte A with a retention time of ∼8.50 min. Ordinate, retention time of eluting analytes; abscissa, chemical shift of 1H.
obtained from the two most intense chromatographic peaks are not only almost identical but also identical with those of the less intense isomers. Analysis of Alkyl Diglucosides by On-Line LC 1H NMR Coupling. For determining the regiochemistry and stereochemistry of isomeric compounds, NMR spectroscopy is the method of choice. Because of the complexity of the isolated diglucoside mixture, on-line coupled LC 1H NMR was applied to obtain spectra of single isomers. LC NMR in flow mode gave some insight into the stereochemistry of the isomers (Figure 5). The acetalic (“anomeric”) protons H1 and H1′ yield peaks well separated from the other signals, and the characteristic difference in their coupling constants to the adjacent protons (H2, H2′) allows assignment to R (with H1/H1′ in equatorial position, J1,2 ) 3-4 Hz) and β anomers (H1/H1′ axial, J1,2 ) 7-8 Hz). The most abundant compound in the mixture was thus found to be of R configuration on both glucose units. This compound, being chromatographically quite well separated from its isomers, was further focused upon, and full elucidation of its structure pursued.
Table 2. Assignments of the Chemical Shifts (δ, ppm) and Representative Coupling Constants (J, Hz) in the 1H and 13C NMR Spectra of n-Dodecyl r(1f4)Maltoside and n-Decyl r(1f6)Isomaltosidea reference: n-dodecyl R(1f4)maltoside (n ) 6) 1H
analyte A: n- decyl R(1f6)isomaltoside (n ) 4)
13C
position
δ
multip
J
δ
1 2 3 4 5 6 OH-2 OH-3 OH-4 OH-6 1′ 2′ 3′ 4′ 5′ 6′ OH-2′ OH-3′ OH-4′ OH-6′ a b c d (n) e f g
4.62 3.24 3.64 3.31 3.42 3.59/3.64 4.70 5.32
d m m m m m/m d d
3.6
97.9 71.0 72.8 79.7 70.4 59.9
4.40 4.98 3.22 3.38 3.07 3.47 3.46/3.61 5.36 4.82 4.84 4.46 3.31/3.58 1.51 1.30 1.24 1.23 1.26 0.86
t d m m m m m/m d d d t m/m m m br m m t
5.6 3.6
1H
multip d d d d d t
6.4 3.2 100.3 72.2 72.9 69.8 72.9 60.4
d d d d d t
6.2 4.8 5.6 5.9
6.9
66.6 28.6 24.9 28.5 31.0 21.8 13.6
t t t t, br t t q
δ
13C
multip
J
δ (ppm) 98.3 71.7 73.0 70.2 70.4 66.2
d d d d d t
98.2 71.5 73.0 69.9 71.9 60.5
d d d d d t
66.7 29.0 25.5 28.4 31.0 21.7 13.4
t t t t, br t t q
4.59 3.17 3.38 3.09 3.52 3.54/3.66 4.55 4.72 4.86
d m m m m m d d d
3.6
4.63 3.18 3.39 3.08 3.42 3.45/3.56 4.46 4.65 4.75 4.33 3.28/3.59 1.49 1.31 1.24 1.23 1.25 0.84
d m m m m m d d d t m m m m m m q
3.6
multip
6.2 4.6 5.5
5.8 4.5 5.1 5.8
6.8
a
Multiplicities: m, multiplet; t, triplet; d, duplet. For position names, see also Figure 3. Most of the signals named as multiplets are overlaid by one another or by a solvent signal and cannot be further interpreted. Signal d represents atoms of the alkyl chain whose resonances are all very similar and undistinguishable. n gives the number of chain CH2 groups summarized under signal d (6 respectively 4 in the two compounds).
Figure 6. Comparison of 1H NMR spectra of the reference substance dodecyl R(1f4)maltoside and the isolated compound A (4.2-5.6 ppm). For further details, refer to Experimental Section. Chemical structures with numbering of dodecyl R(1f4)maltoside and decyl R(1f6)isomaltoside are given in Figure 3.
However, information about regiochemistry, respectively, the position of the glycosidic linkage between the rings, was obtained from neither flow nor stopped-flow LC NMR, for three reasons: (1) Proton chemical shifts of sugar methyne or methylene groups in ring-linkage isomeric disaccharides do not differ enough to be of analytical value. (2) Couplings of ring protons to OH protons are invisible in the D2O-containing solvent because of H/D exchange. Thus, the ring proton(s) next to an ether instead of an alcohol group cannot be identified from coupling patterns in COSY.
Figure 7. Two-dimensional 1H-13C HSQC NMR spectrum of dodecyl R(1f4)maltoside with corresponding 1H spectrum on the horizontal axis. Ordinate (F1), chemical shift of 13C, abscissa (F2), chemical shift of 1H.
(3) The amount of substance collectible in the active volume of the NMR flow probe head was rather limited by the chromatographically demanding nature of the sample. It was, hence, impossible to record a heteronuclear multiple bond connectivity (HMBC) spectrum, which would have revealed interannular 3JCH couplings. Analytical Chemistry, Vol. 72, No. 20, October 15, 2000
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Therefore, it was decided to collect more material of the major isomer A by repeatedly cutting the respective chromatographic fraction and pooling the material. Since the analytes were lacking a UV-absorbing chromophore, flow LC NMR by itself had to be used to monitor the separation: When flow NMR spectra indicated that the desired peak had approximately reached maximum concentration in the probe, the HPLC pump was stopped immediately, the capillaries were disconnected, and the substance was flushed from the probe into a flask with acetonitrile. The eluent was removed on a rotary evaporator, the residue taken up in excess methanol to achieve D/H back-exchange, and the methanol evaporated to dryness of the sample. Finally, the sample was dissolved in 200 µL of DMSO-d6 and transferred to a 3-mm NMR tube for conventional, “static” NMR measurements. Structural Assignment of n-Decyl R(1f6)Isomaltoside by the Use of 1H NMR and 13C NMR. In tube NMR, with DMSOd6 as solvent, sugar OH protons gave not only peaks but also interpretable coupling patterns. Six of the seven OH proton signals were doublets, meaning attachment to a CH group, and only one showed a triplet indicating a directly connected CH2 group (Figure 6). Furthermore, no couplings of OH groups to the acetal protons H1 and H1′ were observed. These findings together determined the ring linkage to be 1,6-glycosidic with the alkoxy group attached to C1. The R configuration of the anomeric carbons had already been shown in the LC NMR experiment. COSY data further support the assignments. Finally, an HSQC spectrum yielded a full set of the molecule’s carbon resonances, showing the signal of the linkage carbon atom (C6) shifted 5 ppm downfield from the terminal methylene carbon C6′ (66 vs 61 ppm), while in the
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1,4-glycosidic reference substance n-decyl R-maltoside, both sugar methylene carbons were at 61 ppm (Figure 7). The combined data (Table 2) identified the prominent isomer A in the diglycoside mixture as n-decyl R(1f6)isomaltoside. CONCLUSION LC-MS in combination with NMR spectroscopic techniques permits the identification of stereoisomeric compounds in complex APG mixtures. Detection by mass spectromery results in information about molecular mass, whereas NMR spectroscopy allows the stereochemical characterization. To assign the chemical structure of minor components such as n-decyl R(1f6)isomaltoside, SPE on styrene-divinylbenzene-based sorbent material was needed for the enrichment of di- and oligoglucosides by elimination of the monoglucoside excess. Using this preconcentrated mixture, it was possible to isolate and identify the title compound from the technical APG product Glucopon 225. ACKNOWLEDGMENT The financial support of these investigations by the German research Council (DFG, Bonn) as part of the special joint research program Sfb 193 “Biological treatment of industrial and commercial waste water” is gratefully acknowledged. Special thanks to the BASF AG for the valuable help with the NMR spectroscopy instrumentation. The authors thank Mr. R. Hatton for his help in preparing the manuscript. Received for review April 6, 2000. Accepted July 21, 2000. AC0004005
