.-
0
I
L
12
8
r t m e / m r n / 16
Figure 1. C4 hydrocarbons at 50 "C on a 2.2-m X 2-mm column
0
+
0.19% picric acid: containing Carbopack C (100-120 mesh) pressure drop: 4.8 Kg/cm2: linear carrier gas velocity, 10.9 cm/sec
1
2 3 time lmin)
-
Figure 2. C4 hydrocarbons at 47 "C on a 2.2-m X 2-mm column containing Carbopack C (100-120 mesh) 0.7% picric acid: pressure drop 2 Kg/cm*; linear carrier gas velocity, 5.75 cm/sec
+
RESULTS AND DISCUSSION In previous paper ( I ) we showed that adding picric acid to Vulcan-G resulted in considerable modifications of the elution order of hydrocarbons as compared to that on a bare GCB surface. Moreover, just by varying the relative amount of picric acid added to the carbon surface, a large range of selectivity could be made available. When separating a typical C4 hydrocarbon mixture of interest in the petroleum industry, methane, ethylene, acetylene, and ethane are generally not present. There may be some propane and propylene, but also some iso- and n-pentane. In making this separation, 0.19% wlw was found to be the optimum percentage of picric acid to be added to Carbopack C. As shown in Figure l, base-line separation of all C4 hydrocarbons is accomplished within 16 minutes. From a practical point of view, it is noteworthy that this column packing can be operated at 50 "C. This is a realistic column temperature which can be easily controlled by common gas chromatographic apparatus. To obtain high efficiency columns, the GCB particle size range used was 100-120 mesh. As a matter of fact, a t the
optimum carrier gas velocity, the calculated column efficiency was seen to be more than 3300 plates per meter. Figure 2 shows a chromatogram of the same hydrocarbon mixture eluted on Carbopack C coated with a monolayer of picric acid. As can be seen, in this instance, the elution time is greatly reduced. However, butene-2-cis and butene-2trans are eluted as one peak. Picric acid-coated Carbopack C columns are thermally stable up to 110-120 "C. We have used the columns continuously at 50 " C for some weeks without observing any alteration. A flame ionization detector was used. At the maximum sensitivity of the amplifier system, we had no problem of base-line stability.
LITERATURE CITED (1) A. Di Corcia and R. Samperi, J. Chromatogr., 107, 99 (1975).
RECEIVEDfor review December 9, 1974. Accepted May 19, 1975.
Separation of Oligosaccharides by Partition Chromatography on Ion Exchange Resins Jaroslav Havlicek and Olof Samuelson Department of Engineering Chemistry, Chalmers University of Technology, 5-40220 M t e b o r g 5, Sweden
Oligosaccharides are important constituents of beverages, food, and waste water from industries which produce products containing polysaccharides, e.g., the cellulose industry. Adsorption chromatography on charcoal-Celite ( I ) and paper chromatography (2) have been used to separate these compounds for preparative and analytical purposes. Efficient separations which depeGd on differences in the degree of polymerization (DP) of the oligosaccharides are achieved by gel permeation chromatography (3-6) and partition chromatography in aqueous ethanol on ion exchange resins (6, 7). In a recent investigation of oligomeric sugar alcohols by this method it was shown that the distribution 1854
coefficients were dependent not only on the D P and the sugar moieties but also on the type of glycosidic linkages (8). The present paper deals with the separation of different types of oligosaccharides containing glucose units linked by various types of glycosidic linkages.
EXPERIMENTAL The B-(1+3)-linked D-glucose oligomers were prepared by acid hydrolysis of laminarin with 0.1M hydrochloric acid a t 100 "C for 1 hr (50 cm3 of acid per gram of laminarin). Oligosaccharides of p(1-6)-linked D-glUCOSe series were kindly supplied by J.-C. Janson, Uppsala, Sweden, The other saccharides were the same as used in previous work (6, 7 ) .
ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975
!-CHART READING.mm
100
2.0
2
1.5
1.o
100
200
300
LOO
500
600 700 ELUATE VOLUME,cm3
Figure 1. Partition chromatography of a-(1-+6)-linked o-glucose oligosaccharides in 65 % (w/w) aqueous ethanol at 75 OC
0.5
Resin bed: 4 X 925 rnm, Dowex 1-X8, SO4*-, 12-18 yrn. Nominal linear flow: 3.8 crn min-' (1) D-glUCOSe (3 rng); (2) di- (4 rng); (3) tri- (4 rng); (4) tetra- (7 rng); (5) penta- (8 rng): (6) hexa- (12 mg): (7)hepta- (12 mg); (8) octa-saccharide (20 rng)
The chromatographic separations were performed in jacketed glass columns containing anion exchange resins in the sulfate form or a cation exchange resin in the lithium form under conditions similar to those described previously (9). The eluate was analyzed automatically by the orcinol method ( I O ) , using 1 g of orcinol per liter of 60% (volume by volume) sulfuric acid as reagent. The volume distribution coefficients (D,)were calculated from the peak elution volumes as usual (11).
I
I
1
I
I
1
1 2 3 L 5 6 7 NUMBER OF MONOMERIC RESIDUES Figure 2. Influence of the type of glycosidic linkages on the distribution coefficients of some oligomeric series Lithium resin (Aminex A-6, 15-19 yrn), 8 5 % ethanol, 75 OC. (A)@-(1+4)linked o-xylose, (X) @-(1+3)-linked D-glucose, (0) a-(1+4)-linked D-glucose. (A)@-(1-+4)-linkedD-glUCOSe, and ( 0 )a-(1+6)-linked o-glucose series: (0) 6-O-~-~-giucopyranosyl-D-glucose(gentiobiose)
RESULTS AND DISCUSSION A chromatogram obtained on elution of a sample containing glucose and oligosaccharides of the a-(1+6)-linked D-glucose series in a run on the sulfate resin is given in Figure 1. The ethanol concentration was chosen so that all species were well resolved. This resulted in fairly long retention times for the higher oligomers. In this run, the octasaccharide appeared after 23 hours. The time was reduced by 60% after a decrease in column length of 40%. Under these conditions, the first two peaks overlapped slightly while the other peaks were completely separated. In agreement with earlier studies (6),with a high concentration of ethanol, the oligomeric sugars were eluted in the order of increasing molecular size; whereas, at a low concentration, the elution order was reversed. A straight-line relationship exists between the logarithm of the distribution coefficient and the number of monomeric units in oligomers both with the anion exchanger in the sulfate form and with the cation exchanger in the lithium form (Figure 2). The slope of the lines which corresponds to the incremental decrease in free energy when one monosaccharide moiety (hypothetical) is transferred from the external solution to the resin phase, depends on the type of glycosidic linkage. With most sugars, even the monomer fits well into the straight line. The only exception observed among the oligomeric series separated at 75 "C was the P-(l-+3)-linked D-glucose series (Figure 2). With oligomeric sugar alcohols, there exists a similar relationship but here the straight lines start a t d p = 2 in most oligomeric series (8). As expected, the slope of the straight line valid for each series of oligomeric sugars was the same as that observed for the corresponding series of sugar alcohols. The results obtained with different series of glucose oligomers indicate that, in aqueous ethanol, the slope of the straight line increases with increasing length of the oligomer. At low ethanol concentration, the differences are leveled out and, in pure water, the differences in D, between sugars of the same d p are hardly significant. Since the partial
i
I
I
I
I
I
I
I
I
I
1
2
3
L
5
6
7
8
9
NUMBER OF D-GLUCOSE RESIDUES
Figure 3. Relationship between log 0,and number of o-glucose residues at various concentrations of ethanol Sulfate resin (Dowex 1-X8. 12-18 prn), 75 'C. ( 0 )oligosaccharides of the a-(1-+6)-linked D-glucose series, (0)oligosaccharides of the a-(1+4)-linked D-glucose series
niolar volume in water is not affected significantly by the type of glycosidic linkages ( 1 2 ) ,the results support the conclusion (13)that, in aqueous solution, the distribution coefficients of strongly polar compounds are mainly determined by the partial molar volume of the solutes. Since a straight-line relationship is valid both within the range of ethanol concentration where the sugars are held strongly in the resin phase and at low ethanol concentration where the net effect is a marked exclusion, there must exist a critical eluent composition a t which all oligomers of the same series exhibit the same distribution coefficient. This was shown in previous experiments with xylose oli-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975
1855
Table I. Chromatographic Data for Some Series of Oligosaccharides Dowex 1-X8, S042-, 12-18 urn,
Oligomeric series
/3-(1-4)-linked /3-(1-3)-linked cy -(l-4)-linked /3-(1-4)-linked a -(1- 6)-linked
D
-xylose
glucose D
-glucose
g glucose D
-glucose
Critical concn of ethanol,
Aminex A - 6 , L i L , 15-19 urn, 85% Et3H
70% EtOH
%
(W/W)
DV disaccharide
d log
DV
d log
Dvlddp
disaccharide
4I d dp
5042- resin
Lit resin
2.98 3.89 4.66 4.81 6.71
0.084 0.035 0.14 0.12 0.30
2.58 3.56 4.60 4.82 8.54
0.20 0.15 0.21 0.24 0.49
60 65 60 59 49
70 77
1.5
69 70 62
J "5
1.5 -
/
5"CJ
1.0 1.0 0.5 0.5
0 I
l l I , I I I I I 1 2 3 4 5 6 7 8 9 NUMBER OF D-GLUCOSE RESIDUES
Flgure 4. Effect of the degree of crosslinking on the distribution coefficients of a-( 1+6)-linked D-glucosides Anion exchange resins in the sulfate form, 60% ethanol. 75 OC.(A)Durrum DA-X4. 15-25 pm; (A)Bio-Rad Ag 1-X4, 15-35 pm; (0)Dowex 1-X8, 12-18 pm; and (0)Dowex 1-X10, 14-17 prn
gomers (6) and oligomeric sugar alcohols (8) and confirmed by the results given in Figure 3. The greater the slope of the straight line (at high ethanol concentration), the more effectively is an oligomer of a given d p held in the resin phase. I t was therefore expected that the critical ethanol concentration should decrease with increasing length of the oligomer a t a given dp. The results given in Table I confirm that this is true for the three series of glucose oligomers having the glycosidic linkages in 3, 4, and 6 positions. The differences in distribution coefficients between the a(1-4) and p-(1+4) oligomers were very small and the plots of log D , vs. d p were found to intersect. The observed differences in critical ethanol concentration for these series were small and hardly significant. Separation of the individual sugars from complex mixtures of oligomers from these two series is therefore very difficult by this method. A comparison of the critical ethanol concentration for the oligomeric sugar series (Table I) and those previously reported for the corresponding sugar alcohols (8) shows that the values coincide. As shown in Figure 4, the distribution coefficients depend on the degree of crosslinking of the anion exchange resins. All resins were of the benzyltrimethylammonium type and the degree of crosslinking is given by the manufacturers. The greater the uptake of glucose, the greater is the incremental increase in log D , for the next oligomer, Le., the higher the separation factor of the oligomers. The resins X 4 (4% nominal divinylbenzene) exhibited a higher 1856
*
I
I
I
I
I
I
I
1 2 3 L 5 6 7 8 9 lUMBER OF D-GLUCOSE RESIDUES
Flgure 5. Influence of temperature on the distribution coefficients of a-(1+6)-linked D-glucosides in 4 9 % (full line) and 60% aqueous ethanol (broken line) on anion exchanger in its sulfate form (Dowex 1-X8, 12-18 pm)
uptake of glucose and increased separation factors in comparison to the resins with a higher degree of crosslinking. This is in agreement with studies of the equilibrium distribution by Ruckert and Samuelson ( 4 ) who ascribed the larger distribution coefficient of glucose t o the fact that, a t a given ethanol concentration in the external solution, the mole fraction of water was much higher in a resin of low crosslinking than in a resin of the conventional type (X8). Similar improvements in separation factors can be obtained with resins containing 8% nominal divinylbenzene by increasing the ethanol concentration (Figure 3). In separations of lower oligomers, no advantages were gained by using the resins of lower crosslinking. With oligomers with a high d p (about 10 and higher), precipitation occurs a t the ethanol concentrations required for efficient separations on Dowex 1-X8. For such separations, the resins of lower crosslinking offered great advantages. The results given in Figure 5 show that the temperature has a great influence on the distribution coefficients and that effective temperature control is a prerequisite for reproducibility. At a given ethanol concentration, the separation factors of the oligomers increase with a decreasing temperature. On the other hand, the elution bands become much broader a t low temperature. This can be ascribed to a slow diffusion inside the resin particles (15),and too low a rate of interconversion of anomeric species (16).Since similar improvements in the separation factors can be obtained by increasing the ethanol concentration, it is strongly recommended to carry out the separations a t high tempera-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975
150
HART READING, mm GLC. MAL, MAL-3
100
50
C I
I
30
40
ELUATE VOLUME,
503
cm
Figure 6. Separation of oligosaccharides of maltose and isomaltose series in 60% (w/w) ethanol at 75 OC Resin bed: 4 X 1300 mm Dowex 1-X8,Sodz-,8-13 pm. Linear flow rate: 1.7cm min-'. Glucose, maltose, maltotriose, isomaltose, and isomaltotriose, 10 mg of each
ture (75 "C or higher) although the equilibrium conditions are less favorable. The lower part of Figure 5 refers to the critical concentration of ethanol at 75 "C for the isomaltose series. No separation was obtained a t this temperature whereas, as expected, both the distribution coefficients and the separation factors increased by lowering the temperature. The results show that the critical concentration depends on the temperature. As expected, the critical concentration was lowered when the temperature was decreased. This can at least partly be ascribed to an increased mole fraction of water inside the resin a t low temperature. The dependence of the critical ethanol concentration on the type of glycosidic linkages (and also on the type of
sugar moieties) can be taken advantage of in separations of complex mixtures containing oligomers belonging to different oligomeric series. Figure 6 illustrates the separation of a mixture containing the first four members of the maltose series from isomaltose and isomaltotriose on a resin in the sulfate form. The substances were eluted a t the critical ethanol concentration for the maltose series (60% ethanol) and, as expected, the oligomers belonging to this series were obtained together with glucose in a single peak well separated from the individual oligomers belonging to tlie isomaltose series. In a subsequent run at higher ethanol concentration (70%), glucose and the oligosaccharides of the maltose series can be easily separated. From a practical point of view, it is important that when working a t 75 O C , the peak elution volumes were reproducible within f 1 % for a period of a t least 30 days and that the results were the same in runs with single species and with mixtures. The areas of the peaks were proportional to the amounts applied and, when the areas were compared to those in calibration runs made on the subsequent day with approximately the same amounts as those present in the sample, the precision was f 1 . 5 % or better.
LITERATURE CITED (1) R. L. Whistler and C. C. Tu, J. Am. Chem. SOC.,74,3609 (1952). (2)C. T. Bishop, Can. J. Chem., 33,1073 (1955). (3)M. John, G.Trenel, and H. Dellweg, J. Chromatogr.,42,476 (1969). (4)K. Chitumbo and W. Brown, J. Polym. Sci., 36,279 (1971). (5)W. Brown and 0. Andersson, J. Chromatogr.. 67, 163 (1972). (6)J. Havlicek and 0. Samuelson, Carbohyd. Res., 22,307 (1972). (7) E. Martinson and 0. Samuelson, J. Chromatogr.,50, 429 (1970). (8)J. Havlicek and 0. Samuelson, Chromatographia, 7, 361 (1974). (9)P. Jonsson and 0. Samuelson, Sci. Tools, 13, 17 (1966). (IO) B. Arwidi and 0. Samuelson, Sven. Papperstidn.. 68, 330 (1965).
(11) 0.Samuelson, "Ion Exchange Separations in Analytical Chemistry," Almqvist and Wiksell, Stockholm: Wiley, New York, 1963. (12)W. Brown and K. Chitumbo, Chem. Scr., 2, 88 (1972). (13) M. Mattisson and 0. Samuelson, Acta Chem. Scand., 12, 1386 (1958). (14)H. Ruckert and 0. Samuelson, Acta Chem. Scand., 11,315(1957). (15)0.Samuelson, in "ion Exchange 11," J. Marinsky, Ed., Dekker, New York. 1969. (16)0.Ramnas and 0. Samuelson, Acta Chem. Scand., 28,955 (1974).
RECEIVEDfor review March 25, 1975. Accepted May 12, 1975. Work supported by the Swedish Board for Technical Development.
Determination of Dimethylnitrosamine and Nitrosoproline by Differential Pulse Polarography Shaw Kong Chang and George W. Harrington Department of Chemistry, Temple University, Philadelphia, Pa. 79 722
Nitrosamines have been shown to be among the most potent chemical carcinogens ( I , 2) and, since nitrites are widely used as food preservatives, nitrosamines may be readily derivable from a variety of edible proteins during processing, cooking, or the digestive process. In view of their well established toxic effects, it is important that sensitive and accurate analytical procedures be developed for the determination of nitrosamines in a variety of media. The analytical chemistry of nitrosamines has received considerable attention. The subject has been discussed, in general, in a recent report by Wasserman ( 3 ) .Many analytical methods have been applied such as gas chromatography (31, mass spectrometry (31, spectrophotometry ( 3 ) , thin layer chromatography ( 3 ) and polarography (3-12).
While each of these methods can be highly sensitive, and reliable, they each'have certain unique disadvantages. Gas chromatography, for example, is restricted to volatile nitrosamines. Mass spectrometry and spectrophotometry, while highly sensitive, can be very difficult to interpret in the case of mixtures. Ordinary dc polarography which has been studied extensively in the case of nitrosamines has a limited sensitivity and rather poor resolution. The disadvantages associated with the polarographic technique can be minimized or eliminated by the use of differential pulse polarography. This technique has the further advantage of being capable of examining both volatile and nonvolatile nitrosamines. Differential pulse polarography has been thoroughly studied and is well established as a highly sensi-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 1 1 , SEPTEMBER 1975
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