Anal. Chem. 1982, 5 4 , 2605-2607
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Automatic Dialyznng-Injection System for Liquld Chromatography of Ions and Small Molecules Francis I?.Nordmeyer* and Lee D. Hansen Department of Chemistty isnd ThermochemicalInstitute, Brigham Young University, Provo, Utah 84602
The presence of soluble polymeric materials in a sample often makes it difficullt, to analyze the sample by liquid chromatography because the polymeric material must first be removed or it will foul the chromatographic column. Also it is often of interest to know the equilibrium or free concentration of the species being analyzed as opposed to the total concentration. Both of these problems can be solved by dialysis of the sample aind injection of the dialysate into the chromatograph. However, normal dialysis procedures are slow, require relatively large smples, and usually result in severe dilution of the sample. We have developed a dialyzing-injection system which u,ses as little as 40 pL of sample and delivers the precisely measured, total volume of dialysate to the eluent stream. The system described provides an attractive way to handle any chromatographic samples containing undesired ultrafllterable material. This injection system should be useful for the analysis of ions and drugs in blood, small molecules and ions in soil extracts, and ions and small molecules in polymer-containing pharmaceuticals. There is also an interest in the equilibrium concentrations of unbound ions and small molecules in complex fluids (1-4).We have chosen to demonstrate the use of this new injection system by measurement of free Ca2+in human blood serum.
EXPERIMENTAL SECTION A Dionex Model 10 ion chromatograph was used. Standard solutions containing analytical reagent grade NaC1, Mg(NOa)g, and Ca(N0J2 were prepared. The sulfate-suppressed barium eluent system (5) was used. A typical dialysis chamber and associated fittings are shown in Figure 1. The dialysis chamber consists of a length of 0.022 in. i.d. TFE tubing with a single strand of hollow fiber dialysis membrane inside. The recipient solution, which after equilibration is transferred to the sample loop of an auxiliary injection valve, is inside the hollow dialysis fiber. The sample solution is drawn into the dialysis chamber through the lower 0.015 in. i.d. TFE tubing until it fills all or part of the annular space inside the 0.022 in. i.d. TFE tubing. The machined Lexan fittings and TFE tubing were assembled with Altex fittings as shown. The 0.015 in. i.d. tubing extends about 5 cm from the Lexan fitting. A volume of 13 pL is required to fill the entire length of *this0.015 in. i.d. TFE needle. Lengths of 0.022 in. i.d. tubing from 10 to 18 cm were used to construct various dialysis chambers. A length of 200 pm i.d., 222 pm o.d., type ClIM Cuprophane dialysis fiber (Enka Glanzstoff, West Germany) is strung through the axis of the assembly, taking care that it pulls freely and is not flattened as it passes by the 0.015 in. i.d. TFE tubing. It is allowed to protrude several centimeters from each end. Silicone rubber glue is introduced beside the hollow fiber from each end of the apparatus until the cavity inside the Lexan is filled. The flow of glue can most easily be observed if a black silicone glue is used. Care must be taken not to seal over the inside ends of the 0.015 in. i.d. TFE tubing and to leave a mound of glue on each end of the apparatus surrounding and centering the hollow fiber. After at least 24 h of curing, the mound of glue and the excess embedded hollow fiber are trimmed off with a razor blade. The clhamber was connected to Altex slider valves with standard in. Teflon tubing and fittings. Care must be taken not to damage the end of the hollow fiber as the fittings are tightened. The type ClIM hollow fiber is designed for dialysis of the middle molecular weight range (5OC-2000). With the fiber strung through 0.022 in. i.id. TFE tubing, the ratio of annular 0003-270O/82/0354-2605$0 1.25/0
volume to volume inside the hollow fiber is such that 13% of the total volume is inside the hollow fiber. The volume inside the hollow fiber is transferred to the eluent stream of the chromatograph. The dialyzing injection system is diagrammed in Figure 2. Altex slider valves A, B, and C have pneumatic actuators which are driven by compressed air controlled by solenoids which in turn are operated by a digital controller. A syringe clamped in a device with mechanical stops measures reproducible sample volumes. Depressing the plunger of the water-filled 100-pL Hamilton gastight syringe F to position 1expels the contents of the annular space. A 10-pL bubble of air is introduced by removing the drop of liquid from the tip of the sample intake tubing and allowing the spring-driven plunger to retract to the trigger stop (position 2). The syringe is shown in this position. A predetermined volume of sample is introduced into the dialysis chamber by positioning the sample at the intake tubing and triggering the syringe. The spring-driven syringe will withdraw to position 3. Transfer pump E is a Model 396 Lab Data Control Instrument Minipump set at 46 mL/h. When the dialysis time has elapsed, this pump transfers the recipient solution through the 7 cm of tubing to the 50-pL sample loop of valve B. Injection of the recipient solution is accomplished by valve B which is in the eluent stream just ahead of the normal sample injection valve of the ion chromatograph. Rinse pump D is a peristaltic pump set to deliver 2.5 mL/min. This pump expels the old sample solution and rinses the annular space with water or other rinse solution. Water from pump E is again pumped through the inside of the hollow fiber at the same time the annular space is rinsed. At the completion of the rinse cycle, dialyzable solutes present in the rinse solution will equilibrate with the water inside the fiber to provide the recipient solution for the next sample. These dialyzable solutes may be omitted from the rinse solution if it is desired to have pure water as the recipient medium. A 12 in. length of 3.5 in. 0.d. (3/la in. wall thickness) aluminum pipe serves as the thermostating vessel for the dialysis chamber. The pipe is wrapped with 24 ft of 0.047 in. i.d. TFE tubing from pump D to provide for thermal equilibration of the rinse solution. Heat sink compound was used to improve thermal contact with the TFE tubing. The pipe was further wrapped with aluminum foil, a 9 ft, 63-W heating tape, a second layer of aluminum foil, and closed-cellfoam insulation. The ends of the pipe are plugged with closed-cell foam insulation. Temperature was regulated at 37 "C by a Tronac Model PTC 40 temperature controller with a thermistor probe mounted on the outside surface of the aluminum pipe. The dialysis-injection cycle is shown in Figure 3 as it was programmed into a digital controller built especially for this purpose. The timing of the dialysis, transfer, injection, and rinse portions of the cycle is based on the electrical signal generated by an optical revolution counter attached to transfer pump E. This pump runs continuously although water is transferred beyond valve A only during the transfer and rinse portions of the cycle. All switching occurs while this pump is on the back stroke and transfer liquid is motionless. This pump operates at 89 strokes/min. Typical numbers of pump strokes are 100,5,3, and 30 for dialysis, transfer, injection, and rinse, respectively. These settings must be optimized for a particular dialyzing injection system. The equilibration and rinse times depend on the length and diameter of the dialysis chamber, whereas the number of transfer strokes depends on the volume of the tubing connecting the dialysis chamber with valve B. The dialysis-injection cycle is triggered simultaneously with release of syringe F which draws sample into the annular space. 0 1982 American Chemical Society
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ANALWICAL CHEMISTRY, VOL. 54. NO. 14, DECEMBER 1982
0.015 In 1.d. TFE TUBING
LEXAN FITTING
Figure 1. Hollow fiber dialysis chamber. Details of assembly are shown in the inset.
F . .. .. .!. .? ..
r? . .. ~--. . .. . ..
Y
I-
S1"PLE ,WT.KE
Figure 2. Dialyzing injection system. Valves and pumps are shown diagrammatically.
RESULTS Forty-eight microliters of sample is required to just fa the 0.015 in. i.d. needle and a dialysis chamber having a 147 mm length of 0.022 in. i.d. TFE tubing. Repeatability of sampling was checked by using a 0.154 M NaCl solution dialyzing into water. Peak heights were measured without any intervening chromatographic column. With sample volumes of 40,50,and 60 pL the observed average peak heights were 666 f 5 (8),720 f 6 (ll), and 734 f 3 (4)(abritrary units) respectively, where the errors given are fl standard deviation and the number of peaks averaged are given in parentheses. During the course of this work there has been no significant correlation of precision with sample size. A distilled water sample immediately following a 0.154 M NaCl sample showed a carry-over peak of less than 0.25% of the former peak. After a dialysis time of 27 s (40pump strokes), dialysis of 0.154 M NaCl into water a t 37 "C was 90% of that for complete equilibration. If the dialysis time is increased to 54 s (80strokes) dialysis was 98% of equilibration. Ca(N0J2 and Mg(NO& dialyze into water a t the same rate as NaCI. However, for dialysis of Ca2+and Mg2+from 0.154 M NaCl into 0.21 M Tris buffer ([Tris] + [HTris+] = 0.21 M), 62 s (92 strokes) are required for 90% of equilibration, and for dialysis into 1.05 M Tris buffer, 135 s (200 strokes) are required. Calcium and magnesium ions in protein solutions (human serum)dialyzeinto 0.21 M Tris buffer at the same rate as they do from 0.154 M NaC1. Repeatability of measurements with protein containing samples was checked by thawing and analyzing h e n aliquots
A
VALVESPUMP B C D
RESET SYRINGE F POSITION SAMPLE SOLUTION
TRIGGER SYRINGE AND CONTROLLER
DIAL1SIS
TRINSFER
INJECT RlNSE
REPEAT CYCLE
Figure 3. Dialysis-inmion cycle. Dashed and solid lines correspond to valve positions in Figure 2.
of a pooled human serum sample. The peak heights for magnesium and calcium were calibrated against a standard containing 0.154M NaCI, 0.808 X M Mg(NO&, and 1.73 X lo-' M Ca(NO& Fifteen measurements of the pooled
Ar7aI. Chem. 1982, 54, 2607-2610
serum over a 2-week period gave (0.59 f 0.03) X W3M Mg2+ and (1.62 f 0.07) X M Ca2+. Errors given are d:l standard deviation. The relative standard deviations, 5 % and 4%, respectively, are characteristic of ion chromatographic measurements made by direct injection. Peak heights are linear with concentration in the range M Ca2+,with a relative standard measured, from 0 to 1.7 8: deviation of 3%. Eight measurements were made at three different Ca2+concentirations. An earlier dialysis chamber was constructed with a 55 mm length. The hollow fiber was surrounded by a 0.038 in. i.d. (in place of the 0.022 in. i.d.) TFE tubing for most of ita length and by Lexan at each end. The hollow fiber volume represented 6% of the total volume in this device and peak heights obtained were proportionately smaller than thogie with the dialysis chamber described above and shown in Figure 1. A dialysis time of 40 s (60 pump strokes) was required for dialysis of 0.154 M NaCl into water at 37 "C to be 90% of equilibration. In all other respects this earlier dialysis chamber gave results of the same quality as those reported above.
CONCLUSIONS The dialyzing injection system described is a substantial improvement over previous methods of sample preparation by dialysis or filtration. The dialyzing time is not lengthy (about 1 min) and can be overlapped with the elution of the previous sample. There is little loss of precision introduced by the dialysis manipulation. Buffers or background com-
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pounds can be added to the material to be injected into the chromatograph simultaneously with the dialysis, if desired, by adding them to the water of the dialysis chamber rinse solution. Analysis of low volume samples is facilitated because a minimum volume of sample is required by this method. The system will also lend itself to fully automated sample handling.
ACKNOWLEDGMENT The authors thank John Lamb for helpful discussion as this work proceeded and Hal Rushton for his experimental work. The authors also thank Ludwig Wolf, Jr., of Travenol Laboratories, Inc., for supplying the hollow fiber dialysis membrane.
LITERATURE CITED (1) Borondy, P.; Dill, W. A.; Chang, T.; Buchanan, R. A.; Glasko, A. J. Ann. N . Y . Aced. Scl. 1973, 226,82-87. (2) Klnnlburgh, David W.; Boyd, Nlgel D. Clln. Pharmacol. Ther. 1981, 29. 203-210. --- - (3) Ladenson, Jack H.; Lewls, John W.; McDonald, Jay M.; Slatopolsky. Eduardo; Boyd, James C. J. Clin. Endocrlnol. Metab. 1979, 48, 393-397. (4) Toffalettl, John; Bowers, George N. Clin. Chem. ( Winston-Salem, N . C . ) 1979, 25, 1939-1943. (5) Nordmeyer, F. R.; Hansen, L. D.; Eatough, D. J.; Rollins, D.K.; Lamb, J. D, Anal. Chem. 1980, 52,852-858.
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RECEIVED for review June 28,1982. Accepted August 30,1982. This research was supported in part by the Thrasher Research Fund. Portions of this work are described in patent application No. 387 357 which was filed on June 11, 1982.
Generation of Pe!rfluorolsobutylene Reference Sample and Determination by Gas Chromatography wlth Electron Capiture and Flame Ionization Detection John S. Marhevka," Glenn D. Johnson, and IDonald F. Hagen Central Research Laborataries/3M, 3M Center, Box 33:?27, St. Paul, Minnesota 55 144
Richard D. Danielson Commerclal Chemicals Division/3M, 3M Centor, St. Paul, Minnesota 55 744
Perfluoroisobutylene (PFIB) is reported to be a byprodud obtained during heating of tetrafluoroethyleneat temperatures in excess of 750 "C ( I ) and from the synthesisr of tetrafluoroethylene from chlorodifluoromethane, bromodifluoromethane, or tetrafluoromethane ( I ) . Small amounts of PFIB may also be formed during condensation reflow soldering which utilizes various fluorinated organic compounds as high-temperature nonfl,smmable primary fluids ( 2 , 3 ) . The most common synthetic routes to PFIB utilize reactions of perfluorocyclobutane (4)or perfluoropropylene (5) at high temperatures. These routes are only feasible for generating large quantities of this highly toxic material. Studies on the thermal decomposition of polytetrafluoroethylene (PTFE) have been reported as early as 1947 when C a d , C3F6,and C4F8(pedluorocyclobutane) were identified as pyrolysis products (6)l. Perfluoroisobutylene was first reported as a decomposition product in 1953 (7). Work by Atkinson et al. (8) indicated that PFIB is formed from perfluoropropene, tetrafluonoethylene, or perfluorocyclobutane at temperatures in excew of 600 "C. They also report that at 725 "C, PFIB pyrolyzes to form perfluoromethane. The catalytic effect of oxygein (and other gases) on the pyrolysis of PTFE was noted by Michaelson et a1.-(9). More recently, PTFE and polyfluoroethylenepropylene (PFEP) were p y r o 0003-2700/82/0354-2807$01,25/0
lyzed in both nitrogen and air under both dry and humid conditions in a flowing system by Arito et al. (10). They reported that oxygen suppressed the formation of PFIB and postulated its formation from radical recombination. They also suggest that PFIB may be more readily produced in a static system. The analyses of highly volatile fluorocarbons have long presented a challenge to chromatographers. Fluorocarbons are not retained or well-separated by most stationary phases because of their high vapor pressure and poor solubility. Gas chromatography of fluorocarbons has been reported on fluorocarbon (11,12)or chlorofluorocarbon (11)stationary phases, on n-hexadecane on Chromosorb P (23), on silica gel (14,15), on Porapak (16),and on SPlOOO on Carbopack C (17). The utilization of electron capture detection in chromatography has recently benefited from an excellent monograph (18) as well as a comprehensive review (19). One can conclude from these references that the change in response of various compounds with respect to detector temperature can vary from no change to over 1000-fold over the normal range of detector temperatures. Therefore, detector temperature control is critical; the operating temperature should be maintained at f0.2 "C (20). T o further complicate the situation, Hattori et al. (21) reported response vs. temperature 0 1982 Amerlcan Chemical Society
