Anal. Chem. 1087, 59, 85-90
LITERATURE CITED (1) De$, 2.;Macek, K.; Janlk, J. I n Liquid Column Chromatography: A Survey of M e m Technlques and AppIicatiOns; Elsevier: Amsterdam, 1975. (2) Snyder, L.; Kirkland, J. I n An Introduction to Modern Liquid Chromatography; Wlley: New York, 1981. (3) POOb, C. F.: Schuette, S. A. I n Confempofary PTactice of Chromafcgraphy; Elsevier: Amsterdam, 1984; pp 213-345. (4) Sorei, R. H. A.; Hulshoff, A. I n Advances in Chromatography; Gddings, J. C., Grushka, E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1983; Vol. 21, pp 87-129. (5) Hearn, M. T. W. I n Advances In Chromatography; G i i n g , J. C.; Grushka, E.; Cazes, J.; Brown, P. R., Eds.; Marcel Dekker: New York, 1980; Vol. 18, pp 59-100. (6) "Ion Pair Chromatography" I n Chromatographic Science Series ; Hearn, M. T. W., Ed.; Marcel Dekker: New York, 1985; Voi. 31. (7) Karger, B. L.; Le Page, J. N.; Tanaka, N. I n High-Performance Liquid Chromafography; Horvath, Cs., Ed.; Academic: New York, 1980; Vol. 1, pp 113-206. (8) Berry, V. V.; Shansky, R. W. J. Chromatogr. 1984, 284, 303-318. (9) Berry. V. V. J. Chromatogr. 1084, 290, 143-181. (10) Berry, V. V. J. Chfomafogr. 1985, 321, 33-43. (11) Huber, J. F. K.; Meijers, C. A. M.; Hulsman, J. A. R. I n Advances in Chromatography 1971: Zlatkis, A., Ed.; University of Houston: Houston, TX, 1971; pp 230-235. (12) Snyder, L. R.; Kirkland, J. J. I n Advances in Chromatography 1971; Ziatkis, A., Ed.; Unlverslty of Houston: Houston, TX, 1971, p 328. (13) Berry, V. V.; Engeihardt, H. J. Chromatogr. 1974. 95, 27-38. (14) Crommen, C. J.; Fransson, B.; Schlll, G. J. Chromafogr. 1977, 142, 283-297. (15) Euston, C. B.; Baker, D. R. Am. Lab. (Fairfiekj, Conn.) 1979, 7 1 , 91-92, 94, 98, ?8-100. (16) Slais, K.; Krejci, M.; Kourilovl, D. J. Chromatogr. 1988, 352, 179-197. (17) Nllsson, L. B.; Westerlund, D. Anal. Chem. 1985, 5 7 , 1835-1840. (18) Davis, J. M.; Glddlngs, J. C. Anal. Chem. 1983, 5 5 , 418-424.
(22) (23) (24) (25) . . (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (38)
85
Council Dkectlve of 27 July 1976 on the Approximation of the Laws of the Member States relating to Cosmetic Products (76/768/EEC), Official Journal No. L262, 27.9.1976, pp 189-200. Modifled by the Ammendment Directive of 17 May 1982 (82/288/EEC), Official Journal NO. L167, 15.6.1982, pp 1-32. Bailey, J. E., Jr. J. Chromafogr. 1985, 347, 163-172. Wittmer, D. P.; Nuessle, N. 0.; Haney, W. G., Jr. Anal. Chem. 1975, 47, 1422-1423. Gloor, R.; Johnson, E. L. J. Chromatogr. Sci. 1977, 15, 413-423. Jandera, P.; Engelhardt, H. Chromatograph& 1980, 13, 18-23. Jandera, P.; Churicek, J.; Bartosovl, J. Chromatograph& 1980, 73, 485-492. Wesener, J. W. N. Vriie Universheit. Amsterdam, 1983; unDublished data. DiBussolo, J. M.; Gant, J. R. J. Chromafogr. 1985, AC8605293 327, 67-78. Slais, K.; Frei, R. W., Anal. Chem. (In press). Knox, J. H.; Laird, G. R. J. Chromatogr. 1978, 722, 17-34. Handbook of Chemistry and phvsics; CRC: Boca Raton, FL, 1981; VOl. 62, p D-130. Van de Venne, J. L. M.; Hendrlx, J. L. H. M.; Deelder, R. S. J. Chromatogr. 1978, 767, 1-16, Bartha, A.; Vigh, Gy.; Billlet, H.; De Galan, L. Chromatographia 1985, 2 0 , 587-590. Bartha, A.; Vlgh, Gy.; Biiilet, H. A. H.; De Galan, L. J. Chromafogr. 1084, 303, 29-38. Bldllngmeyer, B. A.; Deming, S. N.; Prlce, W. P., Jr.; Sachok, B.; Petrusek, M. J. Chromatogr. 1979, 786, 419-434. Shelly, D.; Gluckman, J. C.; Novotny, M. V. Anal. Chem. 1984, 56, 2990-2992. Guthrle, E.; Jorgenson, J. Anal. Chem. 1084, 5 6 , 483-486. hlderloos, D. 0.; Rowlen, K. L.; Blrks, J. W.; Avery, J. P.; Enke, C. 0. Anal. Chem. 1088, 5 8 , 900-903.
RECEIVEDfor review June 9, 1986. Accepted September 8, 1986.
Performance of Annular Membrane and Screen-Tee Reactors for Postcolumn-Reaction Detection of Metal Ions Separated by Liquid Chromatography R. M. Cassidy* and S. Elchuk General Chemistry Branch, Chalk River Nuclear Laboratories, Atomic Energy of Canada Limited, Chalk River, Ontario, Canada KOJ 1JO
Purnendu K. Dasgupta Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-4260 The experlmentai factors that contrlbute to base ilne nolse In the postcolumn-reactiondetectlon of metal ions eluted from hlgh-perfonnancedynamic ion exchangers have been evaluated and compared for a screen-tee reactor (- 1 pL internal volume) and three annular membrane reactlons having Internal volumes of 1.5, 5.3, and 9.6 pL. Measurements of mixing homogeneity, performed with pulseless gas-pressure pumping, showed that both reactor deslgns gave a mixing homogeneity that was 99.983% of that theoretically possible (perfect mixing) for two solutions differing In absorbance by 2.38 units. For normal operation with high-performance reciprocating pumps for eluent delivery, pump pulsations were responsible for 90-100% of the observed peak-tcqeak noise. Column efficiency measurements with a series of ianthanlde metal Ions gave similar curves for both designs (HETP values of 0.01-0.04 mm). Both reactors gave reproducible peak areas, had good peak shapes, and operated rellabty. With the membrane reactors some leakage of eluent into the reagent solution occurred at high eluent or reagent flow rates, but thls was not a problem for normal operating conditlons.
Postcolumn-reaction (PCR) systems have played an im-
portant role in the extension of liquid chromatographic techniques to the determination of a wide range of analytes that are otherwise difficult to monitor with common detector systems. One of the earliest reported dedicated liquid chromatographs, an amino acid analyzer, relied on a PCR system (I), and the continued attractiveness of such approaches is reflected in the growing commercial availability of PCR systems. Extensive information on PCR design, operation, and chemistry is available in a recently published monograph (21, and reviews of different aspects of PCR systems appear on a frequent basis in the literature. Advances in PCR design have been particularly important to the development of inorganic high-performance liquid chromotography (HPLC). Recent comparisons of HPLC-PCR and isotope-dilution mass spectrometry for the determination of metal ions has shown that the HPLC-PCR can offer significant advantagesfor some metal-ion determinations,even when accuracies and precisions of 1% are required (3, 4). The PCR system used in the above studies ( 3 , 4 ) involved the reaction of a colorimetric reagent with the eluted metal ions and subsequent detection with a variable-wavelength UV-visible detector. Similar PCR chemistry has been used by many workers and necessary equipment is now offered commercially by a number of manufacturers. While the
0003-2700/87/0359-0085$01.50/00 1986 American Chemical Society
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ANALYTICAL CHEMISTRY. VOL. 59, NO. 1. JANUARY 1987
sensitivitiesof such systems can be quite good (detection limits of 0.1-1 ng are not uncommon), the reported values of peak-bpeak base line noise (BLN) are >l(rAU (absorbance units), which is -10-fold larger than the noise specifications of many modern absorption detectors. Consequently, improvements in PCR systems are required if the full capabilities of detectors are to he realized. Several reviews are available that discuss the design of different typea of reaction chambers (2,5-7), hut most of these pertain to the determination of organics, which often require long reaction times. Consequently the focus of such reviews is frequently on the fabrication of large volume reaction chambers that minimize hand broadening. The PCR detection of metal ions often involves fast reactions, and peak delay systems (coils or beds) are not usually required. The base line noise that is ohserved with PCR system that use UV-visible absorbance detectors for the determination of metal ions arises from two sources: (a) imperfect mixing of two liquids with different optical properties: (h) changes in the flow rates, resulting primarily from pump pulsations, of the reagent and eluent solutions. These factors cause short-term fluctuations in the composition of the combined eluent-reagent solutions and, thus, contribute to the observed noise. Mixing homogeneity attainable in low-volume reactors has rarely been quantitatively described, although a large number of reactor designs have been proposed in the literature. Recently a sophisticated commercial mixer has been introduced that contains 36 clockwise and anticlockwise spin chambers with a total volume of 10 & ( v i e t Micro Mixer, the Lee Co.). Pump pulsations can also he a significant contributor to the BLN, hut once again limited quantitative data are available. Porous membranes have heen used for reagent addition to flowing streams (8,9)and for pbase separators in PCR system (IO),and a hollow fiber membrane has been described for reagent addition in PCR systems (11). Recently, a filament-filled ion exchange fiber reactor, in which flow occurs through the annulus, has been shown to he an efficient suppressor in anion chromatography (12,13).Similar devices, but made of inert microporous membrane tubes, have heen shown to be useful for reagent introduction in flow-injection analysis (24,15). Because of the low internal volume that can be attained in the annular configuration, this design is also of potential utility in a HPLC-PCR system. The puipose of the studies reported here was to compare quantitatively the mixing noise obtained with annular membrane reactors and a low volume screen-tee mixture and to assess the overall performance of these two types of systems for the PCR detection of metal ions.
REACfNl IMEl
B
ELUfNl
SCREEN
-
DETECTOR
MAN
PLUG
10
ELUENT
DETEFO FERRULE
diagram of membrane (A) and screen-tee (6) mixers. Construction details are glven in the Experimental Section. Flgura 1. Schematic
t
Fleue 2. phctosaph of membrane reactor used fattm FCR detectan of metal ions.
and all eluents were filtered through a 0.45-pm filter. Postcolumn Reactors. Schematica of the screen and the membrane reactors are shown in Figure 1. Figure 2 shows a photograph of the membrane unit used in this work. The screen mixer was made by the insertion of squarecut 'f ,6 in. o.d., 0.007 in. i.d. tubing into a bored-out tee. One or two stainleas steel Bereen disks (120 mesh) were placed between the eluent inlet tuhe and the outlet tube to the detector (Figure 1). The PCR reagent solution flowed along the outside of the eluent inlet tube and then entered and mixed with the eluent within the screen. The resulting solution then flowed into the outlet tube. This design was easier to fabricate and resulted in better performance compared to an earlier tee-mixer design reported by Cassidy and Elchuk (17).
The membrane reactors were made by inserting monofilament nylon fishing lines (280,305,and 350 pm 0.d.; 8 Ib, 10 Ih, and 12 Ib test, respectively) inside Celgard X-20 (Celanese Corp., EXPERIMENTAL SECTION Charlotte, NC) microporous hollow fibers (400-pm bore, 25-pm Apparatus and Reagents. The HPLC componentsconsisted wall, 0.03-pm mean pore size, 40% porosity, burst presaure rated of a single piston Spectra Physics M8700 pump, a dual piston at 1.5 MPa (220 psig)). A small piece (4 mm) of 0.38 mm 0.d. Waters Associates M6000A pump, an ISCO M314 syringe pump, stainless steel syringe needle tubing was inserted at each end of a Rheodyne 7125 sample valve, a Waters Associates 440 detector the filament-filled membrane tuhe. The tube was then coiled (658 nm and 436 nm), and a Nelson Analytical IBM-3000 data (eight turns for 100 mm length) on a 3 mm 0.d. glass rod. The system. The Nehn Analytical system was used for the calculation membrane was secured in position with PTFE tape and then of column efficienciesby the moment method based on integral immersed in boiling water for a few minutes. On cooling and summations (16). The columns were 15-cm and IO-cm Supelco removal of the tape, the filament-filled membrane retained the LC18 columns packed with 5-pM and 3-pM C,, reversed-phase helical configuration. The ends were then inserted into PTFE packings. For some studies a 2-em guard column was also used, tubing (1.6 mm 0.d.. 0.28 mm bore, end flaredwith a suitable tool) Freshly distilled-deionized water (Milli-Q sptem) was used for to a depth of 6 mm. A nichrome wire crimp was placed at the all eluents and solutions. Stmk solutionsof a-hydroxyisobutyric ends of the PTFE tubing to secure the inserted membrane, and acid (HIBA), sodium n-octanesulfonate (C6SO;), and 2J-bisthe wire crimp was then covered with PTFE tape. The assembly [~o-arsonophenyl~azo]-1,8-dihydroxynaphthalene~3,6-disulfonic was enclosed in a PTFE tube (100 mm X 6.4 mm o.d.1, and sealed acid (Arsenazo 111) were purified by passing them through a to compression-typetee fittings with Vespel ferrules (Figure 1). column packed with a strong cation exchange resin: H+form for Before initial use, the membrane was wetted with methanol HIBA and Arsenazo I11 and NHl+ form for CBS03-. The PCR Three main configurationsof the above membrane reactors were reagent was 1.5 X lo4 mol.L-' in Arsenazo 111, 0.1 mol.L-' in used in the present work a 5-cm membrane tube filled with a "OS, and 0.01 mol.L-' in urea (used to stabilize the Arsenazo 350-rm filament with calculated internal volume (V,)of 1.5 p L 111). The reagent solution was filtered through a 0.2-pm filter and a dculated effective hydraulic radius (q,) of 97 pm; a IO-cm
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
87
Table I. Base Line Noise for Membrane Reactor"
reagent
eluent
flow,
flow,
wavelength,
mL.min-'
mL-min-'
nm
0.0
0.0 0.0
436 436 436 658 658 658 658 658
0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.5
0.0 0.5 0.5 1.0
2.0
background absorbance, AU
base line noise,
mixing
homogeneity
AU x 103
2.38 2.38 1.19
0.19 0.24 0.42 0.024 0.048
0.12 0.06 0.06 0.04 0.024
pumping systemsb
0.999 83 0.999 64 0.999 10 0.999 48 0.999 48
0.11 0.065 0.065
"Data for screen-tee reactor was essentially the same as that shown here for a 5 pL volume membrane reactor. *Pumping systems, G, S, and W refer to gas pressure, syring, and Waters reciprocating-pistonpumps, respectively. The subscripts R and E refer to reagent and eluent. membrane tube filled with a 305-pm filament (VI = 5.3 p L , r h = 129 pm); a 15-cm membrane tube filled with a 280-pm filament (VI = 9.6 pL, r h = 143 pm). The reagent solution was delivered to the membrane and screen reactors from a helium-pressurized reservoir (capacity 500 mL) or by a syringe pump (ISCO, Model M314).
RESULTS AND DISCUSSION Mixing Noise. The peak-to-peak base line noise (BLN) observed with UV-visible detectors and PCR detection can be approximated by (BLN)2 = (DN)2
+ (CN)2+ (MN)2 + (FN)2
(1)
where DN is detector noise, CN is cell noise, MN is mixing noise, and FN is noise from flow pulsations. Detector noise, DN, is that observed in the absence of a liquid in the cell or in the presence of a nonabsorbing liquid. AU at The value of DN measured in this work was 2 X 658 nm, which is close to the manufacturers specification of 5 X low5AU at 254 nm. For a homogeneous liquid of finite background absorbance (BA) flowing through the detector, the BLN will be greater than that for a nonabsorbing liquid. This contribution to noise can be termed cell noise (CN) and is usually a result of thermal effects. For a typical draft-free air-conditioned laboratory environment and a detector with an internal heat exchanger, this cell noise generally amounts to 5 X to 2 X lo4 times BA (It?), provided that BA is not so high that the detector is light starved and DN becomes the dominant term in eq 1. Consequently it is convenient to define the term relative cell noise (RCN) where RCN = CN/BA. Noise levels for a homogeneous liquid of BA = 2.38 AU are shown in Figure 3 (curves A and B) for a static and a pulseless flow (0.5 mlernin-') condition. Values of base line noise obtained for these experimental conditions were measured to be 1.9 X and 2.4 X AU, respectively (Table I), corresponding to RCN values of 8.0 X and 1.0 X (DN < CN under these conditions). A RCN value of 1 X 10"' was used to calculate the value of CN for the variety of BA values used in these studies. In most PCR systems the BLN is dominated by the inhomogeneity of the liquid entering the detector, irrespective of whether the inhomogeneityis caused by flow pulsations (FN) or mixing inefficiency (MN) of the reactor. The mixing efficiency of a particular reactor system can be quantitatively evaluated if flow pulsations are eliminated through the use of pulseless reagent/eluent delivery systems. Curves C and D in Figure 3 show typical noise levels observed when an absorbing liquid (2.38 AU) was mixed with a nonabsorbing liquid at equal flow rates (0.5 mL-min-') through the membrane reactor. These BLN studies were done at a high reagent absorbance (2.38 AU) to maximize the mixing noise. Some measurements were also made at an absorbance typical of normal PCR operation (0.12 AU, 658 nm). The
I
0
I
I
2
I
I
I
4
I
6
I
I
I
I
8
TIME (mid
Flgure 3. Base line noise for membrane reactors with gas-pressure pumping for eluent and reagents: curve A, stopped flow noise for reagent; curve 8,noise for reagent at 0.5 mL.min-'; curve C, noise for mixing reagent and eluent at 0.5 mL-min-' with no back pressure; curve D, same as C but with 344 kPa (50 psig) back pressure on cell to eliminate bubble formation. Experimental conditions were as follows: reagent absorbance, 2.38 AU; eluent absorbance, 0. values of BN observed for some different systems studied are summarized in Table I. The results for the membrane and screen-tee reactors were essentially the same for all operating conditions studied, and thus only the data for the membrane reactor are included in Table I. When bubble formation problems were adequately removed by application of back pressure to the detector cell (curve D), the observed BLN was 4.2 X lo4 AU for (Table I). From eq 1,the actual contribution of mixing inhomogeneity (MN) to the observed gLN in curve D is 4 X lo4 AU. We can define relative mixing noise (RMN) as RMN = MN/[BA2 - BAJ (2) where BA2 and BA1 are the background absorbances of the two liquids being mixed. In this case BA1 is zero and RMN is therefore 1.7 X lo4. The RMN calculated from data obtained at 658 nm (Table I) was 3.6 X UT4, which is in reasonable agreement with the 436-nm measurement, considering the low values of BA and BN a t 658 nm. If a mixing homogeneity of unity implies perfect mixing with zero MN or RMN, we can define mixing homogeneity (MH) as MH = 1 - RMN (3) where MH = 1 corresponds to perfect mixing. The value of
88
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
A
B
i
C
~SX~O-~AU
FLOW ImL min-']
0
1
2
Figure 5. Extracolumn variance for membrane and screen-tee reactors, without column: internal volume of membrane reactor, 5 yL; reagent flow, 0.5 rnL-min-'; curve A, membrane reactor; curve B, screen-tee reactor.
TIME (minl
Figure 4. Detector noise caused by pump pulsations: curve A, 0.1 ml-min-' reagent and eluent and back pressure of 689 kPa (100 psig); curve B, 0.5 mL-min-' reagent and eluent and back pressure of 4.8 MPa (700 pstg); curve C,0.1 rnL.-min-' reagent and eluent and back presure of 20.7 MPa (3000 psig); curve D, 0.5 mL.min-' reagent and eluent and back pressure of 20.7 MPa. Experimental conditions were as follows: waters pump for eluent and gas pressure for Arsenazo 111; eluent, 0.28 mol.L-' HIBA at pH 3.9; reagent, 120 yg.mL-' Arsenazo 111, 0.1 mo1.L-l HNO,, 0.01 mo1.L-l urea. Columns were not present: back presswe was controlled with an adjustable pressure relief valve.
MH for the screen-tee and membrane reactors was 0.999 83. Both gas-pressurized and syringe delivery of reagent and eluent gave similar values of BN, but longer term (4 min) oscillations, measuring 2 X AU in amplitude at 658 nm, were observed with the syringe pump. Although the attainable limit of detection (LOD) is not significantly impaired by the longer term oscillation, a pneumatic reagent delivery system is attractive in that it costs a fraction of the price of a highpressure syringe pump. The data in Table I and above demonstrate that both the membrane reactor and the screen-tee mixer yield excellent mixing homogeneity. It is clear that for many practical cases, the use of a design as complex as that employed in the Lee mixer will be largely superfluous. Table I also includes data for more typical chromatographic situations where a reciprocating pump is used for eluent delivery. The mixing homogeneity decreased markedly, compared to pulseless pumping, indicating clearly that the dominant contributor to BLN, at least with the reactors/mixer described in this work, was not reactor mixing efficiency but pump pulsations. Pump Noise. Typical PCR noise levels observed for flow pulsations produced by a high-performancedual-piston pump are shown in Figure 4; an adjustable pressure relief valve was used in place of a column for these measurements to permit the application of a variety of back pressures at any given flow rate. At low back pressure and low flow rates, modern reciprocating-pistonpumps produce relatively large fluctuations in the base line, even when equipped with spring-loaded check valves, as shown in curve A of Figure 4. At higher back pressures (curves C and D, Figure 4), BLN was reduced considerably. For normal separation conditions (Table I),the pump noise was responsible for 90-100% of the total BLN observed. During these studies a variety of experimental conditionswere used for the membrane and screen mixer, and both gave the same response to pump pulsations. There was no evidence for any dampening of pump pulsations by any of the membrane reactors; however, it should be noted that these reactors are of low holdup volume compared to those
in use in commercial PCR systems (19). It is clear that if no additional measures are taken to dampen the pump pulsations, it will remain the dominant source of noise. To reduce the noise originating from pump pulsations to a level comparable to that originating in the reactor, the pump pulsations in the present case must be reduced approximately by a factor of 5. With less expensive single piston pumps, the amplitude of pulsations is nearly an order of magnitude greater and the effectiveness of any pulse dampening system employed must be proportionately greater. The removal of small pump pulsations under the highpressure conditions present at the front of the column is difficult, and other solutions to this problem are desirable. An easily implemented approach is the use of a packed-bed mixer. Studies with a variety of 15-cmpacked bed mixers (1-4 mm i.d.) showed that the BLN for a single piston pump could be reduced from 11 X lo-' to 1.7 x AU. The mixers were packed with glass beads ranging in size from 20 to 100 mesh, and the optimum mixer was 3 mm x 15 cm packed with 20-25 mesh beads. The change in column efficiency was 25% for a peak with k' = 2.8 and HETP = 0.06 mm. Another technique that should prove useful for the removal of pump noise is dual-wavelength coincidence correction. This involves simultaneous monitoring of the eluate at two wavelengths (Wl, W2) followed by a postrun base line correction. One wavelength, W,, is used to monitor the absorbance of the metal chelates formed in the reactor, and the other wavelength, W2, is used to monitor some other major species (the reagent in these studies). The variation of the absorbance at W2 can be used as a measure of the variations in the mixing of reagent and eluent, and after appropriate normalization this base line can be subtracted from the chromatogram obtained at W, to yield a less noisy chromatogram. The potential of this approach was examined with two detector cells in series, but the response to each cell to pump pulsations was not identical and the base line noise was only reduced 2- to &fold. Such approaches should work effectively with true simultaneous dual wavelength detection in one cell. Theoretically, i-emoval of all types of mixing noise should be achieved with no decrease in column efficiency. Extracolumn Band Broadening. Aside from mixing efficiency, another important parameter that must be considered for PCR reactors is extracolumn band broadening. The total contribution to peak variance (u2 in pL2) observed when the column was removed is summarized in Figure 5 for both reactor designs. The data in Figure 5 represent the total variance from injector (5-pL injection), reactor, and detector cell (8 pL). the variance expected for a ByL cell is in the range of 100-200 yL2 (20), depending on the effect of turbulence.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
I
89
I 1.5
3.0
LINEAR VELOCITY ( m m . i ' )
Figure 6. Column efficiency of screen-tee and membrane reactor having a 5-pL internal volume: column, 3-pm Supeicosil LC18 sample, 5 pL of a solution containing 150 ng of each lanthanide; eluent, 0.28 mobL-' HIBA at pH 3.9 with 0.014 moi.L-' CBS03-;detection at 658 nm after reaction wlth Arsenazo 111; reagent flow, 0.5 mL.min-'. Capacity factors, k', for Gd,Sm, Nd, Ce, and La were 0.3, 0.7, 1.8, 3.1, and 5.2, respectively. Solid symbols are for screen-tee data and open symbols for membrane data.
Consequently a portion of the variances in Figure 5 came from band-broadening processes outside of the readors. The larger variances observed for the membrane reactor at intermediate flow rates (Figure 5) are likely due to its slightly larger volume (5 pL compared to 1 pL for screen reactor), but flow patterns and diffusion of sample through the membrane (see below) might also have an effect. When used with a 10-cm column packed with 3-pm particles, both reactors gave essentially the same column efficiency for the separation of lanthanide metal ions (Figure 6). Total extracolumn band broadening was appreciable for some peaks. The largest contribution to total variance from extracolumn band broadening occurred at a flow rate of 0.5 mL-min-' and was 4 , 6 , 1 3 , 3 0 , and 39% for La, Ce, Nd, Sm, and Gd, respectively (It' = 5.3 to 0.3, see Figure 6). Membrane mixers with an internal volume of 1.5 pL and 9.6 pL gave an 8% decrease and a 21 % increase in total extracolumn variance, respectively, relative to the 5-pL membrane reactor when measured for Gd at 0.5 mL-min-'. It should be noted that the band dispersion observed with even the largest volume reactor used in this work is much smaller than for the membrane reactors reported by Davis and Peterson (1I ) . Membrane Performance. Decreases in extracolumn band broadening through reductions in the internal volume (increased diameter of the filament) of the membrane reactors were limited by increased pressure drops and subsequent leakage of the eluent through the membrane into the reagent solution. As the annular gap in the membrane reactors decreased, the pressure differential necessary to maintain the desired rate of flow between the inlet and the outlet of the membrane reactor, proportional to the inverse fourth power of the hydraulic radius, rh, became larger than the applied pressure on the reagent side. Consequently,the eluent stream began to leak out through the membrane immediately after entering the reactor and reentered the inner stream through the membrane, along with the reagent, at the other end of the device. This caused a significant sample loss (up to 25% was observed) and an erratic base line, because the reagent-eluent mixture that diffuses back into the inner stream at the other end is inhomogeneous. The eluent leakage problem cannot be solved by indefinitely increasing the pressure on the reagent side. Aside from bubble formation problems in the detector for a pneumatic delivery system, the membranes are compressible and attempts to achieve a high radial differential pressure gradient resulted in compression of the membrane,
1
SO
100
1
150
ZOO
HELIUM PRESSURE lpslgl
Figure 7. Reagent introduction rates as a function of applied reagent pressure and eluent flow rate: curve A, no eluent flow; curve B, eluent flow 1.0 ml-min-l; curve C, eluent flow 2.0 ml-min-l. No back pressure was on the cell; the same membrane reactor as in Figure 6 was used.
decreased rh,and increased internal pressure. The net result was that at high applied reagent pressures, the radial differential pressure became constant and independent of the applied reagent pressure. Consequently, the reagent introduction rate under such conditions remained constant, as shown in Figure 7. The slope of the linear portion in curve A is 1.3 pL.min-'-kPa-' (9.2 pL.min-'.psig-') or 1.1 pL.cm-2. min-'.kPa-' for a membrane diameter of 424 pm (log mean diameter) and active length of 8.8 cm. This may be compared with the nominal specification of the membrane manufacturer for its water permeability which is 1.7 pL.cm-2.min-1.kPa-1. Measurements made with water in place of the reagent solution (no eluent flow) gave a permeability of 1.2 pL.cm-2min-l-kPa-'. If the linear portion of curve B is extrapolated, the X intercept, 128 kPa (23 psi), may be regarded as the pressure drop between the membrane inlet and outlet, which must be overcome before the reagent can be introduced. For a 10 cm flow path, this pressure drop for the stated flow rate for a liquid of viscosity 1.0 CPimplies, according to the Hagen-Poiseuille equation (ZI),a hydraulic radius of 128 pm, in excellent agreement with the nominal rh value of 129 pM. The threshold pressure for reagent introduction for a eluent flow rate of 2 ml-min-' (curve C), 462 kPa (67 psi), is significantly higher than twice the threshold pressure for curve B, indicating that at the minimum reagent pressures used to generate curve C, compressional decrease of rh has already begun. That such compression was not significant in the linear portion of curve B is inferred from the essentially identical slopes of the linear portions of curves A and B. Further, it is also clear from Figure 7 that the onset of the self-regulation of the differential pressure begins at lower applied reagent pressures for higher eluent flow rates, and the pressure-independent reagent introduction rate attained at the plateau also decreases. If problems associated with eluent leakage are to be avoided, then total eluent plus reagent flow should not exceed the pressure-independent reagent introduction rate without eluent flow (plateau value of curve A). It is possible, however, to operate at acceptably low noise levels with eluent leakage, if the membrane is immersed in a large stirred reservoir of the reagent (22),but this alternative was not explored in this work. Greater total flow rates may most conveniently be obtained by selecting a different membrane. The Accurel PP hollow fiber (BASF Fibers, Enka, NC), for example, is made of the same inert basanaterial as Celgard but due to considerably different characteristics (300 pm bore, 0.2 pm mean pore size, 75% surface porosity) allows greater radial flow at the same pressure differential. Further, because of considerably thicker walls (150 pm), these fibers resist compressional collapse to a greater degree, permitting higher
90
Anal. Chem. 1987, 59, 90-94
applied pressures on the reagent side without reaching the plateau level. In practice, both the membrane reactors and the screen-tee mixer gave reproducible peak areas and good peak shapes and operated reliably over eluent flow rates of 0.5-2.0 mlsrnin-l.
LITERATURE CITED (1) Spackman, D. H.; Stein, W. H.; Moore, S. Anal. Chem. 1958, 30,
1190- 1206. (2) Reaction Defectors in Liquid Chromatography; Krull, I. S., Ed.; Academic: New York, in press. (3) Knight, C. H.; Cassidy, R. M.; Recoskie, 8. M.; Green, L. W Anal. Chem. 1984, 56, 474-478. (4) Cassidy, e. M.; Elchuk, S.; Elliot, N. L.; Green, L. W.; Knight, C. H.; Recoskii, 0. M. Anal. Chem. 1986, 58, 1181-1186. (5) Frei, R. W. Chfomtrographla 1082, 75, 161-166. (6)Jansen, H.; Brinkman, U. A. Th.; Frei, R . W. J. Chromatogr Sci. 1985, 23, 279-284. (7) Huber. J. F. K.; Jonker, K. M.; Poppe, H. Anal. Chem. 1980, 52, 2-9. (8) Malavoltl, N. L.; Piiosof, D.; Neiman, T. A. Anal. Chem. 1984, 56, 2 191-2 195. (9) Pllosof, D.; Neiman, T. A. Anal. Chem. 1982, 54, 1698-1701. (10) Apffel, J. A.; Brinkman, U. A. T.; Frei, R. W. Chromafographia 1984, 78, 5-10. (11) Davis, J. C.; Peterson, D. P. Anal. Chem. 1985, 57, 768-771.
(12) Dasgupta, P. K. Anal. Chem. 1984, 56, 103-105. (13) Dasgupta, P. K. Anal. Chem. 1984. 56, 769-772. (14) Dasgupta, P. K.; Gupta, V. K. Environ. Sci. Techno/. 1988, 20, 524-526. (15) Hwang, H.; Dasgupta, P. K. Anal. Chem. 1986, 58, 1521-1524. (16) Grushka, Eli Anal. Chem. 1972, 44, 1733-1738. (17) Elchuk, S.; Cassidy, R. M. Anal. Chem. 1979, 51, 1434-1438. (18) Dasgupta, P. K., unpublished results. (19) Riviello, J. M.; Pohl, C. A. 35th Pittsburgh Conference on Analytical Chemlstry and Applied Spectroscopy, Atlanta City, NJ, March 1984; paper 506. (20) Kirkhnd, J. J.; Yu, W. W.; Stoklosa, H. J.; Dilks, C. H., Jr. J. Chromatogr. Sci. 1977, 75,303-316. (21) Dasgupta, P. K. J. Liq. Chromafogr. 1984, 7, 2367-2382. (22) Dasgupta, P. K.; Yang, H. C. Anal. Chem. 1986, 58, 2839-2844.
RECEIVED for review May 14, 1986. Accepted September 16, 1986. The membrane reactor work at Texas Tech University is supported by the State of Texas Advanced Technology Research Program and by the U.S. Department of Energy, Office of Basic Energy Sciences, through Grant No. DEFG05-ER-13281. However, this report has not been subjected to review by the agency and no endorsements should be inferred.
Operational Variables for the Separation of Styrene-Methyl Methacrylate Copolymers According to Chemical Composition by Liquid Adsorption Chromatography Sadao Mori* and Yoshitaka Uno Department of Industrial Chemistry, Faculty of Engineering, Mie University, Tsu, Mie 514, Japan
The copolymers of a large range of cmposnlon were separated wlth a mlxture of chloroform (or 1,P-dkhloroethane (DCE)) and ethanol on a sillca gel column by linear gradient elutlon. Chloroform (and DCE) wlthout ethanol retatned the copolymers in the column. By the addltlon of ethanol to chloroform, copolymers having less methyl methacrylate (MMA) started to elute, and wlth lncreaslng ethanol content In chlorofann, those having more MMA could be eluted. The copolymers tend to adsorb on the column at hlgher column temperature, and thaw having more MMA require a lower column temperature for elutlon. Ethanol content or column temperature dld not affect peak retentlon volume for the copolymers. The effects of both ethanol concentratlon and column temperature were attrlbuted to the change of populatlon of free sUanol groups on the surface of sillca gel, because the hydrogen bonding of carbonyl groups in the copolymers to the sllanol groups was the main mechanlsm of thls separation.
The accurate determination of the chemical composition distribution (CCD) for copolymers is very important for the characterization of copolymers. Among several techniques to measure CCD, high-performance liquid chromatography (HPLC) holds great promise because of its high efficiency. There are a number of published papers in this area, e.g., separations of styrene-methyl acrylate copolymers on a silica gel column ( I ) , styrene-acrylonitrile copolymers by precipitation liquid chromatography ( 2 ) , styrene-butadiene copolymers on a polyacrylonitrile gel column (3),styrenemethyl
methacrylate copolymers on a silica gel column ( 4 ) ,styrenemethyl methacrylate block copolymers by column adsorption chromatography using a 50-mm-i.d. cylindrical column ( 5 ) , and styrene-n-butyl methacrylate copolymers by orthogonal chromatography (6). In a previous paper (7), separation of styrene-methyl methacrylate random copolymers (P(S-MMA)) according to chemical composition by liquid adsorption chromatography (LAC) was reported. Silica gel was used as an adsorbent. The copolymers were separated by stepwise gradient elution using chloroform and 1,2-dichloroethane (DCE) as mobile phases. When DCE was used as the mobile phase, the copolymers adsorbed on the surface of silica gel, though polystyrene (PS) eluted from the column. When chloroform was used as the mobile phase instead of DCE, the copolymers having a methyl methacrylate (MMA) component less than 45% could be eluted as well as PS. With increasing chloroform content in a mixture of chloroform and DCE, the copolymers have been separated as a function of their compositions. In the paper, the effects of ethanol content in the mobile phase on the elution behavior of these copolymers were suggested. In the present work, the role of ethanol in the mobile phase in the separation of the copolymers was investigated first in addition to column temperature effects. Then, optimal separation conditions of copolymer components were investigated by linear gradient elution.
EXPERIMENTAL SECTION Apparatus. LAC measurements were performed on a Jasco TRIROTAR-VI high-performance liquid chromatograph (Japan Spectroscopic Co., Ltd., Hachioji, Tokyo 192, Japan) with a
variable-wavelength ultraviolet absorption detector Model
0003-2700/87/0359-0090$01.50/00 1986 American Chemical Society
