Automated Chromatography of Sugars on Cation Exchange Resins P e r Jonsson and Olof Samuelson Department of Engineering Chemistry, Chalmers Tekniska Hogskola, Goteborg, Sweden As DEMONSTRATED in earlier papers, partition chromatography on anion exchange resins in water-ethanol is a useful method for the separation of various monosaccharides ( I ) . From the same medium, sugars can be taken up effectively by means of cation exchange resins (2), but no attempts have previously been made to apply this sorption mechanism for analytical purposes. The aim of this paper is to study the application of a sulfonated styrene-divinylbenzene resin in its lithium, sodium, and potassium forms for the automated separation of monosaccharides. EXPERIMENTAL
The ion exchange resin was of commercial origin (Amberlite IR 120; 8 % crosslinkage) and had been subjected t o grinding in a gear pump a t high pressure. The particles had been fractionated by repeated sedimentations in water a t constant temperature. The particle diameter was 3 t o 17 microns, determined for the Kf-resin in 92.4% ethanol, The resin was prepared by B. Lindqvist and supplied by LKB-Produkter, Stockholm-Bromma. The resin was converted into the desired form by treatment with aqueous chloride solutions and conditioned with the eluent in a separate column before filling the chromatographic column. When working a t high pressure, breakage occurred with several glass columns flanged a t the top and bottom. Unflanged cylindrical glass columns were therefore used. The coupling used a t the top of the column is illustrated in Figure 1. A similar coupling was employed at the bottom with a porous Teflon plug inserted in a hole drilled in the coupling. The column dimensions were 460 X 6 mm. Because the swelling of the resin in aqueous ethanol increases in the order K < Na < Li (3),the weight of dry resin in the column decreases in that order. The pressure a t the head of the column was read o n a Bourdon type manometer with a circuit breaker that stopped the pumps if the desired pressure was exceeded and also if (because of leakage) the pressure dropped below a certain value; in most runs the pressure was 30 t o 40 atm. In other respects the chromatographic system was the same as described previously ( 4 ) . The eluate was analyzed continuously using the anthrone method. Anthrone in sulfuric acid was pumped into the analyzer using a piston pump made of Hastelloy C. The other equipment and the working conditions were the same as described in a previous paper ( 4 ) . In the experiments carried out a t very high temperature (above 100” C ) certain precautions were taken t o prevent boiling in the chromatographic column. The whole system was kept under pressure by inserting a steel capillary in the outlet tube from the flow cell in the photometer. To prevent reagent solution from being pressed backwards into the column, a small bullet valve of stainless steel was inserted in the eluate tube between the column and the analyzing system.
(1) L.-I. Larsson and 0. Samuelson, Acta Chem. Scand., 19,1357 (1965). (2) H. Riickert and 0. Samuelson, Svensk Kern. Tidskr., 66, 337 (1954). (3) H. Ruckert and 0. Samuelson, Acta Chem. Scand., 11, 303 (1957). (4) P. Jonsson and 0. Samuelson, Science Tools, 13, 17 (1966).
1 156
ANALYTICAL CHEMISTRY
Figure 1. Fitting at top of chromatographic column A . Teflon coupling between glass column and Teflon tubing with conical connection, B , for attachment of Teflon tubing C . Aluminum nut for securing Teflon coupling D. Glasstube E. PVC-sleeve sealed to glass tube with epoxy resin F. Nitrile rubber O-ring
RESULTS AND DISCUSSION
The peak elution volumes of 16 monosaccharides were determined in 92.4 ethanol at 75 O and 100” C and the volume distribution coefficients ( D o )were calculated (5). The results obtained with the lithium, sodium, and potassium forms are given in Table I. All the species, with the possible exception of digitoxose, were held more strongly by the sodium form than by the lithium form. The ethanol concentration inside the resin is much lower with the lithium form than with the sodium form (3). [With the resin used in previous work (Dowex 50 X-8) the sorption was much larger with the potassium form than with the sodium form although the waterethanol ratio was about the same.] Interaction forces between the ions inside the resin and the sugar molecules have a marked influence upon the sorbability (6). The results given in Table I show that glucose exhibited a similar behavior with the resin used in this work. Most other sugars behaved like glucose-Le., they were retained more effectively by the potassium form than by the sodium form. An exception was (5) 0. Samuelson, “Ion Exchange Separations in Analytical Chemistry,” Wiley, New York, 1963. ( 6 ) H. Riickert and 0. Samuelson, Acta Chem. Scand., 11, 315 (1957).
[Chart reading, m m
I
Chart reading, mm
I
/i
,
Ta so 100.
Ga
Fr
b
0
Figure 2. Separation of various monosaccharides in 92.4 ethanol at 75 "C on Li +-resin
x
Flow rate 1.2 ml cm-2 min-' Rh = rhamnose, 0.2 mg; 2-deGl = 2-deoxyglucose, 0.4 mg; X = xylose, 0.6 mg; A = arabinose, 0.4 mg; Ta = tagatose, 0.4 mg; GI = glucose, 0.8 mg;Ga = galactose, 1.5 mg
Figure 3. Separation of ketoses in 92.4 at 7 5 ° C on K+-resin
min-1. Ta = tagatose, 0.3 mg; Flow rate 4.4 ml So = sorbose, 0.3 mg; Fr = fructose, 0.3 mg Chart reading
ribose, which was held more strongly by the resin in the sodium form than by the potassium form. Tagatose exhibited a similar behavior, although the difference was much less. Calculated on a weight o r equivalent basis of resin, the effects are larger. For the two deoxy sugars with the lowest distribution coefficients, the experimental errors are larger and n o significant differences could be detected. F o r all sugars and all ionic forms of the resin, the distribution coefficient decreased markedly when the temperature was raised from 75" t o 100" C. This is in agreement with the results obtained with anion exchange resins ( 7 ) . With anion exchange resins in the sulfate form the distribution coefficients increased with a n increased number of hydroxyl groups in the sugar and were lower with deoxy sugars than with sugars containing the same number of hydroxyl groups ( I ) . With a few exceptions, these rules hold true also for cation exchange resins in the ionic forms studied. Hence, digitoxose, which is a dideoxyhexose, appeared first in the eluate followed by deoxyribose and the deoxyhexoses. The next group consisted of the four pentoses; here the rule was found to hold true for the lithium resin, whereas for the sodium and potassium resins the elution behavior of fucose a t 75" C was more similar to that of the pentoses than to the deoxy sugars. With the lithium form, the ketohexoses appeared after the pentoses, but with the sodium form tagatose appeared before ribose in the run at 75 " C. Among the individual sugars within each group, there was in several instances a reversed order of elution when the counter ion in the resin was changed. With some species the order of elution was the same as observed with anion exchange resins in the sulfate form, whereas with others the order was reversed. Similarly, a few sugars changed position when the temperature was raised from 75" t o 100" C. The results confirm that the sorption mechanism is complex, and with the data available a t present no rigorous theoretical treatment can be given. In separations of various monosaccharides by partition chromatography o n anion exchange resins, it was found that a n increased temperature resulted in a considerable sharpening of the elution curves (7). This was confirmed in experiments with cation exchange resins where, with some sugars, the most rapid separations were achieved at 110" C. An interfering (7) B. Arwidi and 0. Samuelson, Suensk Kern. Tidskr., 77, 84 (1965).
ethanol
,rnm
50
Figure 4. Influence of ionic form on the separation of 2-deoxy sugars in 92.4x ethanol at 75 "C Flow rate 1.3 ml min-'. Di = digitoxose, 0.16 mg; 2-deRi = 2-deoxyribose, 0.32 mg; 2-deG1 = 2-deoxyglucose, 0.16 mg; 2-deGa = 2-deoxygalactose, 0.16 mg. Upper chromatogram K +-resin; lower chromatogram Lit-resin
Table I. Volume Distribution Coefficients (D,)in 92.4% Ethanol at 75" and 100" C Determined on Columns with Resin in Lithium, Sodium, and Potassium Forms
Li
Na
75" 100" 75"
Digitoxose 0.8 2-Deoxy-~-ribose 1 . 5 2-Deoxy-~-glucose 2 . 1 2-Deoxy-~-galactose 2.9 Rhamnose 1.4 Fucose 2.4 Lyxose 2.9 Xylose 3.0 Arabinose 3.8 Ribose 4.0 Tagatose 4.5 Sorbose 4.6 Fructose 5.6 Glucose 5.4 Mannose 5.3 Galactose 6.8
0.6 1.3 1.7 2.3 1.2 1.8 2.4 2.4 3.0 3.1 3.7 3.7 4.3 4.4 4.4 5.3
0.8 2.0 2.9 4.3 3.3 7.1 6.5 7.3 11.2 12.3 11.3 13.0 18.7 16.8 17.4 22.0
K 100"
75"
100"
0.6 1.4 2.2 3.1 2.3 4.2 4.4 4.5 6.2 6.8 7.6 8.1 10.0 10.3 10.7 12.6
0.8 2.0 3.2 4.3 4.1 8.9 8.1 8.6 14.2 9.2 10.9 13.4 20.1 22.5 25.0 27.5
0.6 1.4 2.4 2.9 2.7 5.1 5.5 5.7 7.9 5.6 7.4 8.5 11.3 13.6 14.6 15.2
VOL. 3 9 , NO. 10, AUGUST 1967
1157
decomposition occurred with several sugars above this temperature. Glycoside formation is known to occur very easily in solutions containing 2-deoxy sugars (8). Stepped chromatograms were recorded for 2-deoxy sugars at 100" C. To avoid any detectable formation of ethyl glycosides of 2-deoxy sugars the temperature had to be decreased to 75" C. A typical application of partition chromatography on a cation exchange resin in its lithium form is given in Figure 2. The seven monosaccharides applied in this run were separated in 5 hours, including the retention time in the analyzing system. Among these sugars rhamnose and 2-deoxyglucose cannot be separated o n anion exchange resins in the sulfate form. The separation of xylose and tagatose is also extremely difficult in the latter system, whereas the other sugars can be separated as well on this type of resin as on the cation exchange resin. Another example of failure to achieve satisfactory separation on an anion exchanger in its sulfate form is that of tagatose and fructose. As can be seen in Figure 3, these two ketoses can be easily separated on a cation exchanger in the potassium form. Sorbose was also included in this run. With the short column used in this work sorbose overlapped with tagatose but fairly accurate evaluation of the chromatogram can nevertheless be made. If greater accuracy in the quantitative analysis of the three ketoses is desired, the separation can be made on a longer column. The application of the resin in its potassium and lithium forms is compared in Figure 4. In the separation of 2-deoxy (8) W. Pigman, "The Carbohydrates," Academic Press, New York, 1957.
sugars the lithium form is superior to the potassium form. The separation is more effective and the time of elution is shorter. With other sugars the other ionic forms are sometimes superior to the lithium form. The distribution coefficients reported in Table I can serve as a guide in the choice of resin form. One disadvantage of the lithium form in comparison with the sodium and potassium forms is that, compared at constant flow rate, the pressure drop in the column was much higher. For this reason all runs with the lithium resin were carried out a t moderate flow rate t o avoid breakage of the column. Some simple separations-for instance, that of xylose and glucose-could be made a t very high flow rates by using the potassium form of the resin. When very rapid separations are desired, a high temperature should be chosen whenever possible. This permits lower peak elution volumes and a decreased pressure drop in the column. A practical example is the separation of xylose and glucose, which could be made in 45 minutes at 100" C. Compared with anion exchange resins in the sulfate form, cation exchange resins in the forms studied in this work have certain disadvantages. Most striking is the fact that mannose cannot be separated from glucose on the cation exchanger. Cation exchange resins in lithium, sodium, and potassium forms can, therefore, not replace the anion exchange resin in all determinations of monosaccharides. The main advantages of the cation exchangers are in analyses of solutions containing deoxy sugars and ketoses. RECEIVED for review January 16, 1967. Accepted April 26, 1967. Work supported by the Swedish Technical Research Council.
Calculation of Essential Parameters in Programmed Temperature Gas Chromatography from Programmed Temperature Data Alone Robert Rowan, Jr. New Mexico State Uniuersity, Uniuersity Park, N. M .
METHODSHAVE BEEN PRESENTED (I) for the calculation, o r prediction, of retention temperatures in programmed temperature gas chromatography (PTGC). The methods most pertinent to the present work utilize the experimental variables along with two characteristic constants, here designated B and B (or In B). These are the slope and intercept, respectively, of the well known linear equation: In
);(
-
-; --f1nB
where V , = net retention volume and T = absolute temperand is dependent ature. Theta is essentially AH(vanorilation)/R only o n the solute-solvent system. B is also a function of the solute-solvent system but in addition depends linearly o n the amount of solvent present. The reverse calculation-i.e., finding B and B from retention temperatures-is not a straightforward task because of the complexity of the equation relating the quantities involved. ( 1 ) P. Chovin, Ann. Chim. (Paris),7,727 (1962) (review containing
pertinent references). 1 158
0
ANALYTICAL CHEMISTRY
This paper describes a method for computing B and B from PTGC data alone and presents results confirming the validity of the method. Because these quantities are not entirely independent of temperature, the results obtained will be to some small degree a function of the temperature interval of the run and will be average values for this interval. THEORY
Significant variables and characteristic constants are related by the following equation ( 2 , 3 ) : rB e-#? 42 - = - _ -
FB
$2
$1
where r
=
temperature rise rate,
F
=
flow rate, ml/minute SjTi eIT2 air peak temperature, " K retention temperature
$1
=
$2
=
TI = Tz =
C/minute
(2) R. Rowan, Jr., ANAL.CHEM., 33, 510 (1961). (3) Zbid., 34, 1042 (1962).
