vestigated showed the presence of the same 3 hexa-CDD isomers; traces of a fourth isomer were found in some P C P samples. The chromatograms further show the presence of 1 major and several minor hexa-CDF isomers. Incomplete separation of the ‘first hexa-CDD isomer and the major hexa-CDF component was obtained on OV-101 and Silar 1oc. In Figure 6, partial chromatograms of a combined pyrolyzate sample show the separation achieved on these glass capillary columns with 8 hexa-CDD isomers. Isomer assignments of these hexa-CDD peaks, previously based on a simple condensation scheme of polychlorophenates (13),are omitted because this condensation was later shown to possibly result in rearranged products ( 1 5 ) . The elution patterns of the hexa-CDD isomers on these capillary columns were slightly different. OV-17 again showed the best separation of these isomers. The hexa-CDD isomers found in all PCP samples of high PCDD content a t almost identical isomeric ratios did correspond to peaks 1, 3, and 6 of the combined pyrolyzate sample (Figure 6). Some PCP-Na samples of low PCDD content showed the unexpected presence of a tetra-CDD a t levels of 0.1 to 0.2 ppm (8). The elution of this compound is indicated in Figures 3a-5a. Addition of highly toxic 2,3,7,8-tetra-CDD and reanalysis of these samples on the glass capillary columns revealed that the tetra-CDD originally present was not the 2,3,7,8substituted isomer. As indicated in Figure 7, the unknown isomer elutes before and after the 2,3,7&substituted compound on OV-101 and OV-17, respectively. The two isomers are not separated on Silar 1Oc under the operating conditions used.
CONCLUSIONS It has been demonstrated that high-resolution gas chromatography is applicable to the analysis of PCDDs and
PCDFs. The high separation efficiencies of glass capillary columns could be maintained for the trace analysis of these very high-boiling chlorinated compounds using an isothermal splitless injection technique and an electron capture detector of low internal volume. Although the quantitative aspects have not yet been fully investigated, this technique may offer an attractive alternative to conventional gas chromatography with highly specific detection techniques (mass fragmentography) for the routine determination of PCDDs and PCDFs. The use of high-resolution gas chromatography with glass capillary columns should also be seriously considered for the detection and determination of the various PCDD and PCDF isomers in industrial and environmental samples.
LITERATURE CITED (1) D. Firestone, J. Ress, N. L. Brown, R. P. Barron, and J. N. Damico, J. Assoc. Off. Anal. Chem., 55, 85 (1972). (2) C. Rappe and C. A. Nilsson, J. Chromatogr.,87, 247 (1972). (3) H A . Buser, J. Chromatogr., 107, 295 (1975). (4) B. A. Schwetz, J. M. Norris, G. L. Sparschu, V. K. Rowe. P. J. Gehring, J. L. Emerson, and C. G. Gerbig, Environ. Health Perspect., 5, 87 (1973). (5) G. I. Sparschu, F. L. Dunn, and V. K. Rowe, FoodCosmet. Toxicob, 9,405 (1971). (6) J. P. Seiler, Experientia, 29, 622 (1973). (7) E. A. Woolson, R. F. Thomas, and P. D. J. Ensor, J. Agric. FoodChem., 20, 351 (1972). ( 8 ) H.-R. Buser and H.-P. Bosshardt, J. Assoc. Off. Anal. Chem., in press. (9) W. 8. Crummett and H. R. Stehl, Environ. Health Perspect., 5, 15 (1973). (10) R. Baughman and M. Meselson, Adv. Chem.. 120,92 (1973). (11) K. Grob and K. Grob, Jr., J. Chromatogr., 94, 53 (1974). (12) D. L. Stalling and J. N. Huckins, J. Assoc. Off.Anal. Chem., 54, 801 (1971). (13) H.-R. Buser, J. Chromatogr., 114, 95 (1975). (14) H.-R. Buser, J. Chromatogr., in press. (15) A. P. Gray, S. P. Cepa, and J. S. Cantrell, Tetrahedron Lett., 33, 2873 (1975).
RECEIVEDfor review March 26, 1976. Accepted June 1, 1976.
Gas Chromatographic Determination of Hydroxyethyl Derivatives of Glucose Johan W. Mourits, Henk G. Merkus, and Leo de Galan* Laboratorium voor Analytische Scheikunde, Technische Hogeschool, Delft, The Netherlands
The distribution of hydroxyethyi groups in glucose units resulting from the reaction between ethene oxide and starch is determined gas chromatographicallyusing a caplllary SE-30 column following siiylatlon of the hydrolyzed sample. The six rnonosubstltuted and twelve disubstituted anomeric forms are completely separated and identlfied from thelr mass spectra. Higher derivatives up to pentasubstituted products are separated in groups enabling quantitative determination of the degree of molar substitution with a precision of better than 5%.
The properties of starch can be improved through reaction with ethene oxide. For low degrees of substitution, the hydrolyzed reaction mixture consists mainly of monosubstituted glucose units in addition to nonreacted glucose. With increasing degree of substitution, an increasingly complex mixture of higher derivatives up to pentasubstituted glucose units is obtained. For the characterization of hydroxyethylated starches, the degree of molar substitution (MS, i.e., the
number of moles of ethene oxide per mole of anhydrous glucose) is very popular, because this quantity can be readily obtained through the classical titration of ethyl iodide and ethylene resulting from the reaction with hydrogen iodide (1-4). On the other hand, the performance of the products is also highly dependent upon the degree of substitution (DS, Le., the fraction of anhydrous glucose units substituted a t any of the three reactive hydroxyl groups). However the distribution of the hydroxyethyl groups over the individual glucose units is much more difficult to determine. From the reduced reactivity towards periodate and the increased tendency to form a tritylether, Husemann and Kafka ( 5 ) ,concluded that ethene oxide reacts preferentially with the hydroxyl group attached to the second carbon atom in the glucose ring. This was confirmed by Srivastava and Ramalingam (6) using a paper chromatographic separation of the products of hydroxyethyl starch subjected to Smith degradation. Paper chromatography has also been used by Bollenback e t al. (7) for the separations of the monosubstituted hydroxyethyl glucoses, but this method fails to separate
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
1557
"able I. Instrumentation and Operating Conditions Gas chromatographs Becker 409 with flame ionization detector and electronic integrator HP 3370 A F&M 700 coupled to a Riber quadrupole mass spectrometer Columns 2-m, 2.5-mm i.d. packed with 60-80 mesh silanized gaschrom Q, loaded with 6% SE 30; injection volume, 1 p1 25-m, 0.31-mm i.d., coated with 0.5 pm SE 30 (L.K.B), splitless injection of 0.1 111 (Hamilton 7001), flow rate 20 cm/s Injection block and detector 260 "C Temperature program Column from 180 to 290 at 1.2 "C/min Mass spectrometer Riber Q156 quadrupole analyzer, direct inlet, total ion current registration used to trigger 2-s mass spectra from m / e = 100 to 600, analyzer pressure Torr, mobile phase (He) flow rate 100 cm/s ~
'i
./
\
\
-.-*'
k
*/
/*
.-
furfuraldehyde
-Normality
H,SO,
Figure 1. Hydrolysis yield vs. acidity during hydrolysis of starch; hydrolysis time 4 h
(m) Remaining disaccharides, ( 0 )furfuraldehyde production (A)glucose yield
the monoderivatives from higher derivatives (8). Such separations are possible by liquid chromatography using a carbon column (6,9),but sample preparation is time consuming and the lengthy chromatographic separation is practically limited to mono- and disubstituted products. Gas chromatographic analysis of the hydroxyethyl derivatives of glucose is possible after volatility enhancement through reaction with trifluoracetic acid and hexamethyldisilazane. After optimizing the latter procedure, Lott and Brobst ( 1 0 , I I ) were able to identify all three monosubstituted hydroxyethyl glucoses by comparison with synthesized reference compounds. Quantitative determinations were inaccurate due to incomplete separation on their packed SE-52 column. Improved separation and determination of all six anomeric monohydroxyethyl glucoses has been realized by Roberts and Rowland (12) using a packed SE-30 column. The identification and determination of higher derivatives has not been reported. This paper describes the separation, identification, and quantitative determination of all mono- and disubstituted hydroxyethyl glucose units in starch using a capillary SE-30 column coupled to a quadrupole mass spectrometer for component identification or a flame ionization detector for quantitative analysis. The group separation and determination of higher substitution products are also reported. EXPERIMENTAL Instrumentation. The main components and instrumental conditions are collected in Table I. The packed SE-30 column was used only for comparative purposes but was found to be inferior to the
capillary column. Temperature programming was necessary for the simultaneous determination of mono- up to pentasubstituted hydroxyethyl glucoses. Splitless injection and direct coupling to the mass spectrometer were used to avoid systematic errors and improve the sensitivity. Even then, reliable mass spectra could only be obtained from components present over 3% in the original sample (30 ng). The Total Ion Current recording was practically identical to the Flame Ionization Detector signal proving that the GC-MS coupling entailed no loss in chromatographic resolution. Chemicals. Trisyl Z from Pierce Chemical Corp. was used for the silylation reaction. Representative samples of hydroxyethyl starch with degrees of molar substitution from 0.03 to 2.3 were obtained from a Dutch starch company (Scholten-Honig). Analysis Procedure. About 150 mg of hydroxyethyl starch is hydrolyzed for 4 h at 100 O C in 15 ml of 1.3 N sulfuric acid. After cooling, the hydrolysate is deionized with an Amberlite IR 45 ion exchange column; 150 ml are collected in half an hour and freeze dried overnight. About 15 mg of the freeze dried sample is dissolved in 1ml of Trisyl Z, if necessary by gentle heating. Then 0.1 p1 of the solution is injected onto the capillary column of the gas chromatograph using the conditions stated in Table I. Temperature programming is started as the solvent peak (pyridine) appears and the final temperature is reached after 90 min, by which time all glucose derivatives including pentasubstituted products have been eluted from the column (Figure 2). The upper temperature limit of 290 "C is dictated by the Teflon connections used in the present equipment. With stainless steel connections, an upper temperature of 320 O C is feasible. DISCUSSION Hydrolysis a n d Silylation of Hydroxyethyl Starch. After Lott and Brobst (10, 11) the hydrolysis of starch is carried out in acid solution at 100 "C. Good results were obtained by heating small sealed reaction vessels in an oil bath thermostated to 100 f 1 O C . The reaction time and the acidity of the solution must be optimized to avoid either incomplete hydrolysis (evident from the appearance of disaccharide peaks in the chromatogram of pure starch) or partial degradation of glucose units into 5-hydroxymethyl-2-furfuraldehyde(as evidenced by its yellow colored dimer). The data in Figure 1 demonstrate that glucose yields of better than 98% are obtained for hydrolysis times between 3 and 5 h in 1.1 to 1.5 N sulfuric acid. This is considered sufficient for the accurate determination of the degree of molar substitution and the distribution of hydroxyethyl groups in glucose units. After preparation of this paper, an extensive discussion of new silylating agents has been published (13).In the present study, silylation is achieved through a mere dissolution of the dried hydrolysis product in a solution of trimethylimidazole in pyridine (commercially available as Trisyl Z). This rapid procedure is insensitive to traces of water and yields stable reaction products. Test experiments with pure glucose showed that a t least 1ml of Trisyl Z per 15 mg of sample is required for a complete reaction. Because of rapid mutarotation, the reaction mixture always shows an equilibrium composition of nearly equal amounts of CY and p glucose, irrespective of the composition of the original product. Gas Chromatographic Separation. For the simultaneous separation of glucose and its hydroxyethyl derivatives, a nonpolar stationary phase is to be preferred ( 1 4 ) . In agree-
1558 * ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
glucose I
a
II
23 29
1
trisubstitution
I
I
I
1
I
I
I
70
60
50
40
30
20
10
Time. minutes
disubstitution
Qlucor
I
b
I
rnOnOSubStitution
pentasubstitution
tetrasubstitut1on
290
270
I 1
I
90
I
80
Temperature, centigrade 250 230 70
I
I
I I
50
60
190
210
~~
1
I I
40
I
30
I
I
20
10
T i m e , minutes
Figure 2. Chromatograms of TMS-derivatives of hydrolyzed hydroxyethyl starch on a capillary SE 30 column with temperature programming: (a) M S c l,(b)MS=2 Elution order: 1. Glucofuranose; 2. 1,2-0-.ethylene-a-~-glucofuranose;3. a-E-glucose; 4. 1,2-0-ethylene-o-~-glucopyranose; 5. 1,2-Oethylene-~-o-glucopyranose; 6 . @-glucose; 7. 3-0-hydroxyethyl-a-~-glucose; 8 and 9? 10. 2-0-hydroxyethyi-a-D-glucose;11, 12, 13, 17. cyclization products of 2,3- and 2,6-Odihydroxyethyl 18. 6-0-hydroxyethyl-~-o-gIucose; 19. 2,3-0glucose; 14, 6-0-hydroxyethyl-a-~-gIucose; 15. 3-0-hydroxyethyI-~-D-glucose; 16.2-0-hydroxyethyl-~-~-glucose; dihydroxyethyl-a-D-glucose; 20. 3-0-hydroxyethoxyethyl-a-o-glucose; 21. 3,6-0-dihydroxyethyl-a-~-glucose; 22. 2,3-Odihydroxyethyl-~-~-glucose;23. 2-0hydroxyethoxyethyl-a-o-glucose; 24. 2,6-0-dihydroxyethyl-a-~-glucose: 25. 3,6-0-dihydroxyethyl-~-~-glucose?;26. 6-0-hydroxyethoxyethyl-a-~-glucose; 27. 2,6-O-dihydroxyethyl-~-~-glucose; 28.3-0-hydroxyethoxyethyl-~-~-glucose;29. 2-0-hydroxyethoxyethyl-@-~ glucose; 30. 6-0-hydroxyethoxyethyl-~-~-glucose, 31? 32-41. Trisubstituted ethylglucoses
ment with the results of Lott and Brobst ( I O ) , a 3% SE-52 packed column provided insufficient separation of the monosubstituted anomeric forms, so that experiments with this stationary phase were discontinued. Better results were obtained with a 2-m, 6% SE-30 packed column (12).Using isothermal elution at 200 "C, a nearly complete separation of all six anomeric monosubstituted hydroxyethyl glucoses was achieved in 70 min. However, simultaneous elution of higher substituents is only possible with temperature programming, but this severely deteriorates the separation of the monosubstituted glucoses. A tenfold better efficiency is provided by a commercially available 25-m capillary column coated with a 0.5-wm layer of SE-30. Again using temperature programming, Figure 2a demonstrates that this column provides a complete separation of all six monohydroxyethyl anomeric glucoses and all 1 2 anomeric dihydroxyethyl glucoses. An additional advantage of the capillary column is that the
smaller sample volume appreciably reduces the detrimental influence of deposited silicon oxide upon the detector sensitivity. The nearly unit degree of molar substitution of the sample run in Figure 2a excludes the presence of large amounts of trisubstituted products, although it is clear that the 20 possible anomeric forms are not completely separated. It is possible, however, to determine the total contribution of trisubstituted products, so that the degree of molar substitution of the original hydroxyethyl starch is easily derived from the chromatogram. This is further illustrated by the example in Figure 2b from a sample with a degree of molar substitution as high as 2.2. The shift from monosubstitution to di- and trisubstitution is clear from the well separated groups of peaks representing consecutive amounts of substitution ranging from nonreacted glucose to pentasubstituted hydroxyethyl derivatives.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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~~
Table 11. Relative Intensity of Characteristic Mass Peaks Used for the Identification of Eluted Components m / e values
Elution order (Figure Za) 1 1 7
204
191
147Q
217
235
248
-
305
349
361
-
-
5 10
