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ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
LITERATURE CITED
(5) J. Falbe, R. Paatz. and F. Korte, Chem. Ber., 97, 2544 (1964).
(1) J. S. Fritz, R . K. Gillette, and ti. E . Mishmash, Anal. Chem., 38, 1869 (1966). (2) E. M. Moyers and J. S. Fritz, Anal. Chem., 48, 1117 (1976). (3) J. S. Fritz and G. M. Orf, Anal. Chem., 47, 2043 (1975). (4) M. D. Arguello, Ph.D. Thesis, Iowa State University, Ames, Iowa, 1977.
RECEIVED for review December 19, 1977. Accepted May 12, 1978. This work supported by the U. S. Department of Energy, Division of Basic Energy Sciences.
Characterization of the Ion-Exchange Membrane Detector for Liquid Chromatography and Its Application to the Separation of Quaternary Ammonium Compounds John G. Dorsey, Mark S. Denton,’ and T. W. Gilbert” Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 4522 I
An LC detector which operates by measuring dimensional changes of a strip of ionexchange membrane has been further characterized and is shown to be useful for the detection of quaternary ammonium ions. The detector has a precision of better than 1 % standard error and a h e a r range for choline and acetylcholine of at least 2.5 orders of magnitude.
Quaternary ammonium ions are commercially used as antiseptics, antistatic agents, in detergent formulations of fabric softeners, as a water treatment biocide, and in secondary-recovery oil wells. They are also prevalent in biological systems, and are of great biomedical interest. The commercial products are usually mixtures, and the determination of individual compounds in them has long been a troublesome analytical problem. Most often only the total tetraalkyl ammonium content is determined. Trace amounts have been determined in both commercial (1) and biological (2) samples by ion-pair extraction and colorimetry. Larger amounts may be determined and differentiated from amines by nonaqueous titration (3). Some degree of selectivity may be achieved by careful control of ion-pair extraction conditions followed by two-phase titration with lauryl sulfate ( 4 ) . However, closely similar quaternary ammonium ions cannot be differentiated by the latter procedure and, for those that can be differentiated, qualitative knowledge of the sample composition is required for proper adjustment of the conditions of extraction (5, 6). For the separation of closely similar quaternary ammonium ions, a chromatographic method is required. Thin-layer chromatography on alumina (7, 8) and high voltage electrophoresis (9) have been used, and quantitation has been accomplished by spraying with color-forming reagents. Gas chromatography has been applied to the analysis of choline and its esters but, because of the nonvolatility of the compounds, either prior derivatization or pyrolysis techniques must be used (10-13). GC-MS, both electron impact ionization and chemical ionization, is also useful (14, 15). Liquid chromatography would be the separation method of choice because of the poor volatility of the compounds. Unfortunately, the lack of useful UV-visible absorption bands has made the detection and quantitation of quaternary ammonium ions difficult. Ion-pair partition chromatography ‘Present address, Oak Ridge National Laboratories, P.O. Box X, Room B8, Building 4500N,O a k Ridge, Tenn. 37830. 0003-2700/78/0350-1330$01 .OO/O
using picrate as the anionic species has permitted the use of IJV detection (16). However, this method has a very limited concentration range because of changes in the distribution ratios of the individual cations with concentration. Various other methods, all involving collected fractions and subsequent analysis, are still widely used for the detection of quaternary ammonium ions. These include precipitation with sodium triphenylcyanoborate (I 7), I4C labeling of the compounds (18), NMR analysis (19),and color formation with periodide (18, 20). It is clear that a continuous means of detecting quaternary ammonium compounds is badly needed for such studies. Recently a new general purpose detector was developed in this laboratory for continuously monitoring the effluent from a liquid chromatography column (21). This detector utilizes a phenomenon which is associated with all ion-exchange processes-namely, the volume change of the resin matrix which accompanies all ion-exchange reactions. An osmotically induced volume change also occurs for nonelectrolytes which partition into the resin phase. Thus, i t is seen that insofar as only species which are capable of exchanging with the counterions, or partitioning into the resin are considered, the detector is completely general, giving a response for all species. The magnitude of the response will depend on the identity of the pair of ions in the exchange reaction, the physical properties of the resin matrix, and the composition of the external solution. The effluent flows through a low volume glass tube in which a strip of ion-exchange membrane is suspended and attached to a linear voltage differential transformer (LVDT) in a Du Pont Model 941 thermomechanical analyzer (TMA). Extremely small changes in the length of the ion-exchange membrane during sorption and desorption of ions cause the LVDT core to be displaced from its electrical center, resulting in a signal which is recorded on the Y axis of a strip chart recorder. This paper describes the application of this detector to the separation and determination of some simple quaternary ammonium ions, and of choline and its esters. The synthesis of an ion-exchange membrane with a greater sensitivity than commercially available membranes is reported. The precision, linearity of response, flow rate sensitivity, and detection limits of this new detector are discussed.
EXPERIMENTAL Chromatographic System. Delivery of the mobile phase was made with a Chromatronix Model CMP-2 chemically inert piston pump. Samples were introduced into the flowing mobile phase C 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50,
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9
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Table I. Percent Polystyrene Grafting and Exchange Capacity of Membranes
& b --
radiation time, h
total dose, kR
1
188
4
752 1504 3130
ga
16.9
membrane capacity mequiv/g grafting, bequiv/ dry w/w cma membrane %
3.6 34.1 37.8 63.5
1.05 5.25 6.00 8.34
0.508 2.60 2.78 3.85
a A high degree of homopolymerization of this sample was indicated by a very viscous styrene liquid phase.
Figure 1. Inverted loop membrane detector. (a) Pb ball; (b) Pt ribbon; (c) ion-exchange membrane; (d) thick walled Pyrex tube; (e) Chromatronix microbore column fitting; (f) Teflon inlet tubing
stream with a Chromatronix R6OSV rotary injection valve having replaceable sample loops. An adjustable bed Chromatronix LC-9M column, 9.0-mm i.d., was used throughout. All components were interconnected with 0.79-mm i.d. Teflon tubing. The system was capable of operation up to a maximum pressure of 500 psi. Aminex A-4 resin (Bio-Rad Laboratories), 16-24 pm! was equilibrated three times with the mobile phase and then slurry packed. When the column was filled, the resin bed was compressed by pumping the mobile phase through the column at maximum flow rate for at least 1 h, and the column adjusted to this final bed height. The mobile phase was 0.035 M ethylene diammonium chloride (en.2HCl) made in distilled water and vacuum degassed. Reagents. The en.2HC1 was made using the procedure of Arguello and Fritz (23). The quaternary ammonium salts, tetramethylammonium bromide, ethyltrimethylammonium iodide, diethyldimethylammonium bromide, and triethylmethylammonium iodide (Eastman Chemical, Rochester, N.Y.); and choline and esters, choline chloride, acetylcholine chloride, propionylcholine iodide, and butyrylcholine iodide (all 9970,Sigma Chemical, St. Louis, Mo.) were used as received. Sample solutions were prepared by dissolving the solids in 0.035 M en.2HC1; solutions containing choline and its esters were stored at 4 "C. Membranes. Strips of polyethylene (12 X 0.15 cm, 26.7 pm thick) were cut from Glad Food Storage Bags (Union Carbide). They were washed overnight with a detergent solution, rinsed with distilled water and ethanol, and air dried. Pyrex tubes (ca. 35 cm long, 15-mm o.d., 12-mm i.d.) were half filled with styrene (Eastman, 9870, stabilized with tert-butylpyrocatechol), the polyethylene strips added, and the mixture was bubbled with N, for 10 to 15 min. The tubes were flame sealed and irradiated with a 195 kR/h y-ray source for varying lengths of time. After irradiation, the strips were washed three times with benzene, once with absolute ethanol, and dried. The percent grafting was established by the weight change of a pre-weighed square of the polyethylene film. The samples were sulfonated by immersing the polymer strips in chlorosulfonic acid at room temperature for 25 min. The strips were then placed successively in CC1, for 1 h to remove excess chlorosulfonic acid, concentrated H,SO, for 5 min, and finally in 2070 NaOH at 70 "C for 20 min. The capacities of the membranes were determined by titrating the hydrogen ions liberated by the membranes upon the addition of an excess of XaCl to a solution containing the membranes. Each membrane was first converted to the hydrogen form by soaking in 1 M HC1 followed by rinsing with water until the effluent was free of chloride ion. The titrations were performed with carbonate-free NaOH to a phenolphthalein end point. The titration volumes were corrected by a blank titration of NaC1. Monitor and Detector. The monitor and the adjustment of the TMA have been described previously (21). The detector design is that of Hansen (22) and is different from the one previously used. It is shown in Figure 1. A strip of sulfonated, 4-h irradiated copolymer, 12 X 0.15 cm, was looped in the shape of an inverted V inside a thick walled glass tube, 7.2 cm X 0.27
cm i.d., giving the detector a volume of 412 pL. A piece of Pt ribbon (0.009 x 0.15 mm) was placed through the membrane loop, and a P b ball was squeezed shut on the ends of the Pt ribbon. The Pb ball then rests in a cradle on the tension probe connected to the LVDT core. By using thick walled glass tubing and the fittings from a Chromatronix microbore column, the bottom of the membrane is easily held in place. A sealing disk with a centered hole holds the two ends of the membrane tightly against the end of the glass tube and the column effluent is pumped directly into the center of the detector body. Procedures. For the membrane response comparisons, the flow rate study, the calculations of precision of response, and the time constant measurements 0.276-mL samples of 0.1 M NaCl were injected directly into the detector w t h 1.0 M H2S04as eluent.
DISCUSSION Membranes. T h e primary considerations for an ideal membrane for this application are: high mechanical strength for resistance to tearing, flexibility for resistance to cracking, thinness for rapid equilibration with the external solution, resistance to inelastic deformation and low cross-linking for maximum swelling or shrinking response to ion-exchange reactions. These characteristics are antagonistic and compromises must be made. Of the many commercial membranes which have been tested, the cation membrane CC-60 and the anion membrane AA-60 (American Machine and Foundry Co.) which were used previously (21) continue to be the most responsive. These are of the graft copolymer type, consisting of a polyethylene backbone with grafted polystyrene which is then sulfonated or quaternized. LJnfortunately, these are no longer being manufactured, so similar membranes were prepared by a method similar to that of Chen et al. (24). Table I shows the percent grafting and capacity data for these membranes. The copolymer films from the 8- and 17-h irradiation times were grainy, showing localized styrene polymerization, and have not been investigated for detector response. T h e response of the 4-h irradiated sample was then compared t o that of the CC-60. The average probe displacement for the CC-60 was 4.74 pm (15 replicates) while that for the synthesized membrane was 14.0 wm (seven replicates). Thus, for a pure ion-exchange interaction, the newly prepared membrane gave a response about three times greater than that of the commercial membrane. It is believed that two factors contribute to this improved response. The new membranes are three to four times thinner than the commercial membranes, providing for more rapid equilibration during the exchange process. Second, the extent of cross-linking of the commercial membranes is not known, but the new membranes are not DVB cross-linked, and so are likely to show greater dimensional changes during an exchange reaction. Detector Design and Response. T h e new detector cell design is a n inversion of that previously reported (21). T h e previous design required very tedious ;alignment of the detector cell to prevent the TMA probe from binding on the cell walls.
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
F
W
z
220-
e
Lo
z
p i
--p1-i4i I i A~
'3
4c
30
I
02
04
10 FLOW RA'E
20 :rnilminl
Figure 2. Detector response to NaCl samples vs. flow rate Table 11. Precision Data
injection 1 2 3
4 5 6 7
peak height: mm
peak width,., , mm
peak area, mm2 b
71.5 69.5 70.5 69.5 70.5 71.5 71.0
10.0 10.0
715 695
9.8 10.0
691
10.0 10.0
9.5
695 705 71 5 67 5
A peak height of 70 mm corresponds to a probe displacement of 1 4 pm. Peak area standard and mean deviations. S = 14.3 mm2. S , = 5.40 mm2. a
T h e inverted loop membrane design is not as critical with respect to alignment, and setup time is on the order of a few minutes. The new design is also much less sensitive to flow pulsations. Figure 2 shows the response of the inverted loop membrane detector as a function of flow rate. Each data point represents the average of triplicate samples. The slower flow rates allow longer residence time of the solute in the detector cell and, hence a greater response, as more of the solute ions exchange onto the membrane. Present work is being directed toward a design giving more turbulent flow to minimize this diffusion limited response. Table I1 shows the peak height and peak area ( A = h x was) for seven sequential injections. The average area was 699 mm2 with a standard deviation of 14.3 mm2 and a standard error of 5.40 mm2. Thus, the detector is reproducible during any given period of operation. Day to day variations of 10 to 20% can occur, however, if the detector is dismounted at the end of the day's operation. Different membrane strips of the same capacity and similar in length show responses within the 10 to 20% variation range. Therefore, if the detector is dismounted, or a new membrane is used, two or more standards must be run to re-establish the calibration curve. These seven sequential injections were also used to measure the time constant of the membrane, defined as the time required for the membrane to reach 63% of its maximum value a t a flow rate of 2.00 mL/min. For the adsorption process, the value is 11 s, while for the desorption process, it is 25 s. Applications. Figure 3 shows a separation of simple quaternary ammonium ions with a 22.5-cm column of Aminex A-4 resin. The negative peak at 10 min is the "exchange peak" and has been discussed previously (21). Analogous to the refractive index detector, both positive and negative peaks can be obtained, corresponding to expansion and contraction of the membrane. Figure 4 shows the separation of choline and three of its esters with a 19-cm column of Aminex A-4. Two factors had
6C
70
( w nm
TIM:
10
&--A
5c
Figure 3. Separation of simple quaternary ammonium ions. (a) tetramethylammonium; (b) ethyltrimethylammonium; (c) diethyldimethylammonium; (d) triethylmethylammonium. Sample: 1.009 mL of 0.035 M en-2HCI which is also 0.015 M in each of (a),(b), (c),(d). Mobile phase: 0.035 M en.2HCI. Flow rate: 1.00 mL/min. Column: 22.5 X 0.90 cm Aminex A-4
'j\ ,J
;u..-~jr 8 -'
L
-
-
.
_
1
_
#
8
20
310 -lME
40
,
I
/
-
50
(mnl
Figure 4. Separation of choline and esters. (a) choline, 0.010 M; (b) acetylcholine, 0.01 0 M; (c) propionylcholine, 0.0060 M; (d) butyryicholine, 0.0060 M. Sample: 1.009 mL of (a),(b), (c),(d) at the above concentrations in 0.035 M en.2HCI. Mobile phase: 0.035 M en.2HCI. Flow rate: 2.00 mL/min. Column: 19 X 0.90 cm Aminex A-4
-E,
c 9-
I
-
;7 L
E 0
c
m -
L
.
,-
5-
31-
-lME
(m n)
Figure 5. Determination of choline in a soy meal extract. (a) LI'; (b) choline determined to be 2 6 mg/mL Sample: 0.456 mL of soy meal extract Mobile phase: 0.035 M en.2HCI Flow rate: 2.00 mL/min. Column: 17 X 0.90 cm Aminex A-4 to be considered in the selection of the mobile phase for this separation. First, it has been shown that the identity of the mobile phase and the sample affect the response of the detector (21,25). Second, the stability of the choline esters had to be considered. It has been shown that the maximum stability of acetylcholine with respect to hydrolysis at room temperature was obtained a t pH 4.7 (26). The rates of hydrolysis of the other choline esters should be similar. The pH of 0.035 M en.2HC1 is 4.4 at 22 "Cand the greatest membrane response to choline was obtained with this mobile phase. The linear range for choline under these conditions is from 10 M t o 5 X M. The average peak heights of triplicate samples of 1.009 mL were converted to probe displacement and a plot of log displacement vs. log concentration gave a slope of 1.00. This method for obtaining a quantitative estimation of detector linearity has been described by Fowliss and Scott (27). The lower end of the linear range is limited by the sensitivity of the detector and could probably be extended somewhat at a slower flowrate. With a mobile phase of 0.035 M en.2HC1 and a flow rate of 2.00 mL/min, the
'
ANALYTICAL CHEMISTRY, VOL. 50, NO. 9, AUGUST 1978
minimum detectable quantity, defined as the concentration of sample t h a t produces a signal equal to twice the baseline noise, is about 3 x lo-" g/mL for choline or acetylcholine. While this detector is significantly less sensitive for these compounds than GC (10-13), no lengthy sample pretreatment is necessary. Figure 5 shows an application of the membrane detector to the determination of choline in a soy meal extract. The large, off-scale peak is Li', from LiOH, used to raise the p H of the extract. The second peak is choline, determined to be 2.6 mg/mL of extract. Details of this analysis, including extraction conditions and comparisons with other methods will be presented elsewhere.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9)
(IO) (11) (12)
M. E. Auerbach, Ind. Eng. Chem., Anal. Ed., 15, 492-493 (1943). S. Eksborg and 6.A. Persson, Acta Pharm. Suec., 8, 605-608 (1971). St. J. H. Bhkeiey and V. J. Zatka, Anal. Chim. Acta, 74, 139-146 (1975). S. 0. Jansson, R. Modin, and G. Schill, Talanta, 21, 905-918 (1974). R. Modin and S. Back, Acta. Pharm. Suec.. 8, 585-590 (1971). B. A. Persson, Acta Pharm. Suec., 8, 217-226 (1971). C. Radecka, K. Genest, and D. W. Hughes, Arzneim.-Fwsch, 21, 548-550 (1971). W. F. H. McLean and K. Jewers, J . Chromatogr., 74, 297-302 (1972). S. E. Brooker and K. J. Harkiss, J . Chromatogr., 89, 96-98 (1974). P. I.A. Szilagyi, D. E. Schmidt, and J. P. Green, Anal. Chem., 40, 2009-2013 (1968). D. J. Jenden, R. A. Booth, and M. Roch, Anal. Chem., 44, 1879-1881 (1972). D. E. Schmidt and R. C. Speth, Anal. Biochem., 67, 353-357 (1975).
1333
(13) J. L. W. Pohlmann and S. L. k h a n , J. Chromatcgr., 131, 297-301 (1977). (14) 1. Hanin and R. F. Skinner, Anal. Biochem., 66, 568-583 (1975). (15) C. G. Hammar, I.Hanin. B. Holmstedt. R J. Kitz. D. J. Jenden, and B. Karien, Nature (London). 220, 915-917 (1968). (16) S. Eksborg and G. Schiil, Anal. Chem., 45, 2092-2100 (1973). (17) J. S. Hayes, M. A. Alizade, and K. Brendel, Anal. Chim. Acta, 80, 361-367 (19751. (16) D. Speed and M. Richardson. J . Chrorrtatogr , 35, 497-505 (1968). (19) F. Chastellain and P. Hirsbrunner, Fresenius' 2. Anal. Chem., 278, 207-208 (1976). (20) W. K. Gnrley, C. D. Haas, and S. Bakermarl, Anal. Biochem., 19, 197-200 (19671. (21) T. W.'Gilberi and R. A. Dobbs, Anal. Chem., 45, 1390-1393 (1973). (22) Larry C. Hansen, Ph.D. Thesis, University of Cincinnati, Cincinnati, Ohio, 1973. (23) M. D. Arguello and J. S. Fritz, Anal. Chem., 49, 1595-1598 (1977). (24) W. K. W. Chen, R. B. Mesrobian, D. S. Baihntine, D. J. Metz, and A. Glines, J . Poiym, Sci., 23, 903-913 (1957) (25) Richard A. Dobbs, Ph.D. Thesis, University of Cincinnati, Cincinnati, Ohio, 1973. (26) D. J. Jenden and L. B. Campbell, in "Methods of Biochemical Analysis, Supplemental Volume", D. Glick, Ed., Interscience, New York, N.Y. 1971. (27) I. A. Fowliss and R. P. W. Scott, J . Chromatogr., 11, 1 (1963).
RECEIVED for review December 19, 1977. Accepted May 8, 1978. J.G.D. gratefully acknowledges the University of Cincinnati Research Council for support from a Summer Fellowship. M.S.D. gratefully acknowledges the University of Cincinnati for support through a Twitchell Fellowship. Partial support of this work in the form of a Frederick Gardner Cottrell Grant from the Research Corporation is also gratefully acknowledged.
Evaluation of a Computer-Controlled Stopped-Flow System for Fundamental Kinetic Studies Glen E. Mieling and Harry L. Pardue" Department of Chemistry, Purdue University, West Lafayetfe, Indiana
This paper describes the application of a computer-controlled stopped-flow system for a kinetic study of the Fe(II1)thiocyanate reaction. Kinetic equations are developed for a proposed mechanism involving two parallel pathways, and results of some 456 experiments run under computer control are Interpreted and processed to provide expliclt values for forward and reverse rate constants for the proposed mechanism as well as activation and thermodynamic parameters. Of the 456 experiments run, three were rejected as being inconsistent with other data and only nine were lost completely because of an inadequate supply of stock reagent. The imprecision was about 0.03 s-' for rate constants in the range of 1.8 to 5 s-'.
The stopped-flow mixing method represents one of the most useful techniques for kinetic studies involving reactions with half-lives in the range from a few milliseconds to a few seconds. Because the method generates data a t a relatively rapid rate, some investigators have used small laboratory computers on-line with stopped-flow instrumentation (1-3). While these innovations have greatly improved the convenience and reliability of stopped-flow studies, most of them do not take full advantage of the capabilities of the computers because they are used only for data acquisition and processing. Coupled with automatic reagent and sample handling equipment, a
47907
small computer can also control and execute most of the operations required to carry out major phases of stopped-flow kinetic studies. An earlier report from this laboratory described a unique sampling/mixing system that permitted control of the sampling and mixing steps via electrical switches ( 4 ) . A more recent report described a reagent preparation system that could mix up to five solutions and one diluent in a wide range of proportions and deliver many such solutions in sequence to the sampling/mixing system ( 5 ) . The total integrated system is controlled by a computer system so that after stock reagents are supplied and the conditions for several experiments are entered into the computer via Teletype, then all steps required to perform the desired experiments and process the data are carried out automatically under computer control and without operator intervention. Although data were presented to illustrate some features of the quantitative performance of the system, no data were presented to evaluate performance for a detailed kinetic study and that is the subject of this paper. In this work, the Fe(II1)-SCN- reaction is used as a model system to further evaluate the performance of the computer controlled stopped-flow instrument. Although the thermodynamic and spectral properties of' the monothiocyanato complex of Fe(II1) in acid solution have been well characterized (6-8), there are significant discrepancies among kinetic rate constants reported for the system (9-23). Therefore, in
0003-2700/78/0350-1333$01.00/0 0 1978 American Chemical Society
