Anal. Chem. 1980, 52, 1549-1551
Figure 1. Types of mixing devices
$1
P
'I
I1
I!
Ti me (min) Figure 2. Chromatograms obtained with A1 and C4. Flow rate of solutions I and I1 was 0.5 mL/min, and 1.5 mg of glucose were injected onto the column. S/N ratios with A1 and C4 were 12.1 and 22.6,
respectively
RESULTS AND DISCUSSION T h e signal-to-noise (S/N) ratio of the peak for a certain quantity of glucose was measured as the index of the mixing, and half of the peak width was used as the index of the peak broadening (Figure 2). Experiment with the Same Flow Rates. The same flow rate (0.5 mL/min) was used for both solutions I and 11. By inference from the S / N ratios shown in Table I, the devices of type C were more effective for mixing than types A and B. Of the type C mixers, C3 and C4 gave better results. Little difference in peak broadening was observed among the mixing devices. Although A2 has inlets of different diameters, no difference in the S/Nratios or the peak widths was observed regardless of the inlet used for solution I. Similar results were obtained with B2, C3, and C4.
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Experiment with Different Flow Rates. The flow rates, 0.25 mL/min for solution I and 1.0 mL/min for solution 11, were adopted. As shown in Table 11, the best mixing effect was obtained with C4 and no differences in peak broadenings were observed among the devices. With A2, there was no difference in the mixing effect regardless of which inlet was used for solution I. T h e same result was observed with B2. On the other hand, with C3 and C4, the better mixing effect was observed when the flow line of solution I1 (the faster flow rate) was connected to the inlet with the smaller diameter. These results suggested that a larger difference between the flow rates of the two solutions might give a better mixing effect. Then, the ratio of the flow rates between solutions I and I1 was changed to 1:9; 0.2 mL/min (solution I) and 1.8 mL/min (solution 11). As was expected, C4 gave the best mixing effect when the line of solution I1 was connected to the inlet with the smaller diameter (S/N ratios with A l , B1, and C4 were 1, 2.7, and 4.4, respectively). However, probably due to the disturbance of flow in the vessel, C4 gave a slightly broader peak (80 s) than either A1 (76 s) or B1 (60 s). One can use a larger or smaller vessel, or add more inlets to the vessel according to the purposes of the experiment. The diameters of the inlets should be modified according to the flow rates of the solutions and their properties, such as viscosity and miscibility. An application of the device to chemiluminescence detection in HPLC has recently been reported (6). ACKNOWLEDGMENT The authors express their thanks to Zenzo Tamura of this University for his valuable discussions and support. Thanks are also due to Hachiro Nagata of Kyowa Seimitsu Co. for manufacturing the mixing devices. LITERATURE CITED (1) J. F. Lawrence and R. W. Frei, "Chemical Derivatization in Liquid Chromatography", Elsevier, New York. 1976. (2) R. W. Frei, L. Michel, and W . Santi, J . Chromafogr., 125, 665 (1976). (3) R. W. Frei, L. Michei, and W . Santi, J . Chromafogr., 142, 261 (1977). (4) S. Katz, W. W. Pitt. Jr., and G. Jones, Jr., Clin. Chem., 19. 817 (1973). (5) S. Katz and W. W . Pitt, Jr.. Anal. Lett., 5 , 177 (1972). (6) S. Kobayashi and K. Imai, Anal. Chem., 52, 424 (1980).
RECEIVED for review February 1, 1980. Accepted April 15, 1980.
Liquid Chromatography Electrochemical Detector with a Porous Membrane Separator Kenneth A. Rubinson," 1. William Gilbert, and Harry B. Mark, Jr. Department of Chemistty, University of Cincinnati, Cincinnati, Ohio 4522 1
Detection of liquid chromatography eluents by constantvoltage amperometry has become one of the standard methods in the field (I). A wide range of flow-through electrochemical cells has been created for this purpose (2-9). However, they all have some limitations either regarding difficulty of construction or inability to be used in a wide range of solvents. These problems have been noted by the respective workers (2-9). the Of reports ( I o , II), a simp1e, effective electrochemical liquid-chromatography detector can be constructed which circumvents the above Problems. The active solution thickness of t h e cell is a t most only a few hundered microns. With a highly conducting electrolyte 0003-2700/80/0352-1549$01 .OO/O
outside the permselective (dialysis) boundary of the cell, this is also the effective conducting path length. The lower limit of electrolyte needed in the mobile phase is anticipated to be lower than other cell designs. As a result, the solvent range for electrochemical detection can be extended.
EXPERIMENTAL A schematic diagram of the detector is shown in Figure 1. The construction is straightforward. The 0.5-mm gold wire (W) of about 5-7 cm length is inserted through a short length of glass
melting-point-capillary tube (c). Inserted in the outlet side is a small bore (0.58 mm) polyethylene tube (B) used t o direct the effluent to a fraction collector if desired. A length of porous polymer tubing (dashed 1ines)see below-with i.d. 650-750 wm t 3 1980 American
Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 9, AUGUST 1980
Ref
Aux
Flgure 1. Schematic diagram in cross section of the electrochemical detector. The dashed line represents the Nafion or the hydrolyzed cellulose acetate (dialysis)membrane. The inlet at the right (A) is a Teflon tube into which the membrane is force fit. The hatched area (W) is the 0.5-mm wire working electrode. At the left is the working electrode and outlet tube (B) cemented wffh epoxy (stippled region) into the glass capillary connector (C). The detector is immersed in a small reservoir of electrolyte solution (D) in which are placed the reference and auxiliary electrodes, Ref and Aux, respectively is slid over the wire and into the glass connecting tube. Both ends of the glass tube are then sealed with fast-setting epoxy. The porous polymer tube extends just over the end of the wire and is force fit into a standard 0.8-mm i.d. Teflon tube (A). This connects with the chromatography column using standard screw connectors. This inlet connection has proved adequate if construction is done after all solvent swelling has occurred. The completed detector is then cemented into a small reservoir used to contain an external electrolyte solution (D) in which are placed the reference and auxiliary electrodes, here a calomel reference electrode and platinum wire auxiliary. A Bioanalytical Systems model CV-1A potentiostat was used to control this three-electrode system. The output was plotted on a Houston Instruments Omniscribe chart recorder. A small variable-potential dc power supply supplied an offset to the output when needed. Because of the short electrolyte pathway in the chromatography mobile phase and the small Faradaic current levels, only low supporting electrolyte concentrations are needed. The lowest limits have not yet been tested. In this preliminary work, methanol-water with 1mM Na2S04and acetonitrile with 0.1 mM tetraethylammonium perchlorate have been used. Water with KCl was used as the external electrolyte for all solvent systems. Two different membranes have been employed: Nafion (DuPont) (IO, 11) and base-hydrolyzed cellulose acetate (12). Identical results were obtained for both membranes in water and methanol-water solvents. Many solvents react with cellulose acetate and in them it cannot be compared with Nafion, which retains its integrity. Both membranes are tubes of approximately the same diameter. The cellulose acetate membrane is 30 pm thick in the dry, untreated state. This is rendered porous by hydrolysis on both sides simultaneously for 1 2 h at ambient temperature in 0.2 N KOH. Both ends are left untreated to aid mounting since the hydrolyzed membrane lacks stiffness. Both membrane materials are affected slightly by solvent swelling and the inside diameters are variable. For a 6-cm long wire (electrode area 0.92 em2),the possible dead volumes range from about 5 to about 15 pL. The chromatography was done using an Altex model 100 pump controlled by a model 420 computer. The sample was injected through a Rheodyne rotary valve incorporating a 20-pL sample loop. A 250 X 4.6 mm Knauer column was filled with Lichrosorb RP18, 10-pm packing. This was preceded by a 70 X 2 mm precolumn filled with Co-Pel1 ODS. The water was distilled, deionized, and deaerated. Various purified alkane phosphonates were the generous gift of D. Francis. The mobile phases and detector potentials used for the three samples tested are listed in Table I.
RESULTS Quantitation of samples of 9,lO-diphenylanthracene with constant conditions (see the figure legend) over two decades of concentration is shown in Figure 2 . Each point is the average of peak currents for three samples. In a separate series of experiments on the same compound, the reproducibility of the results was tested with 20 repetitive injections of 0.8 pg (2.5 nmol) samples. With a flow rate of 0.8 mL/min a t +1.4 V vs. SCE, the peak current value obtained was 1584 f 48 nA. T h e relative standard deviation *.r
Table I. Chromatographic Samples and Conditions detector potential, V
compound
mobile phase
9,lO-diphenylanthracene
acetonitrile + 0.1 mM tetraethylammonium perchlorate 1:lmethanol:water + 1 0 mM acetate buffer, pH 4.0 water + 25 mM acetate buffer, pH 4.9 + alkyl phosphonate ion pairing reagent
catechol vanadate, Vv
I
I
I
I
I I I I I
I
+ 1.40 +0.70
-0.51
I
I
l
l
1
Sample S i z e h g ) Flgure 2. Log-log plot of the peak current vs. sample size of 9,lOdiphenylanthracene. Each point is the average of three determinations. In addition to the chromatographic conditions stated in t h e section of experimental details, the flow rate was 0.8 mL/min. The electrode used is 5.8 cm long and surrounded with a Nafion membrane is, thus, 3.0%. T h e active length of the gold electrode was 5.8 cm. The value of k', the capacity factor for the peak, was 3.1. Similar results are obtainable for catechol in watermethanol with either a Nafion or hydrolyzed cellulose acetate membrane. I t was also found that, with all else being constant, the current peak height is proportional to the gold working electrode length. I n principle, there is nothing prohibiting the detection of nonplatable metals with electrochemical detection. Among these are the elements forming refractory oxides-just those most difficult to handle with spectroscopic analytical techniques. In preliminary experiments, clear peaks were measured for samples of 20 ng (0.4 nmol) of vanadate, Vv. Since there are no sharp edges with the coaxial geometry, any bubbles present in the mobile phase are simply swept through to the outlet. I t was found that any gas bubbles that do arise cause only a short term change of the output current. T h e length of time this change occurs depends on the dead volume and flow rate. For a flow rate of 1.0 mL/min and dead volume of 5 kL, the bubble will be in the active area of the detector for about 0.3 s. Since both types of membrane are relatively pliable, detectors do not have perfect, regular coaxial geometry. At some points, the membrane appears t o touch the wire, while on the opposite side the liquid layer will be double the average annulus thickness. I t is not obvious what overall effect this irregularity will have on peak currents. The variability could prove beneficial by enhancing vortex mixing and so reduce the thickness of the unstirred layer. This may enhance the response. Bending or forming the wire into different shapes
Anal. Chem. 1980, 52, 1551-1552
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peak approximately a 0.1 to 0.5 mM solution of the material is in the detector, the flow could be stopped and cyclic voltammetry could be carried out on such species. With more selective dialysis membranes, such experiments could be done on even lower mass species. As mentioned above, the cell functions well with aqueous KCl as the external electrolyte contacting the membrane with either water-methanol or acetonitrile as the internal, mobile phase. Of course, the only limitation on the working electrode composition is whether the material can be fabricated in the appropriate shape.
may enhance the response. These subjects will be investigated in the future. If the Nafion were acting as an ion-exchange membrane, complex electrochemical effects might be expected since different solvents each with different solutes (both charged a n d uncharged) reside on opposite sides of the membrane. T h e hydrolyzed cellulose is neutral, and it may be surprising t h a t no difference was seen in results using these two types of membrane. However, a simple test suggests the reason. With 2 M NaCl inside the Nafion tube immersed in distilled water, within 15 min osmotic effects caused the volume of the liquid inside to double. At the same time, a significant amount of chloride ion was seen to have flowed out through the Ndion membrane. This was detected by precipitation with Ag+. The Nafon membrane a d s as if it has large (on the molecular level) holes allowing a t least a fraction of it to appear as a simple neutral barrier. These osmotic effects will be negligible with the mobile phase flowing through the detector. T h e total amount of sample lost by diffusion through both types of membrane is small owing to the short periods of relatively high concentrations of solutes that occur a t chromatographic peaks. The effect of this effusion on the current response curve will also be small because the solution a t the electrode surface is being replenished faster than once a second in normal use. When the cellulose acetate membrane is hydrolyzed as described above, it will retain molecules with molecular masses greater than about 300. If chromatography is carried out on larger amounts of a sample such that a t the chromatographic
LITERATURE CITED (1) Kissinger, P. T. Anal. Cbem. 1977, 4 9 , 447A-456A. (2) Kissinger, P. T.; Refshange, C.: Dreiling. R.: Adams, R. N. Anal. Lett. 1973. 6 . 465-477. (3) Fleet,' B.; Little, C. J. J. Cbromatogr. Scl. 1974, 12, 747-752. (4) Taylor, L. R.; Johnson, D. C. Anal. Cbem. 1974, 46, 262-263. (5) Daveport, R. J.: Johnson, D. C. Anal. Cbem. 1971, 46, 1971-1972. (6) Lankalma, J.; Poppe, H. J. Cbromatogr. 1976, 725, 375-388. (7) Blaedel, W. J.; Dinwiddie, D. E. Anal. Cbem. 1975, 4 7 , 1070-1075. (8) Blaedel, W. J.; Ylm, 2 . Anal. Cbem. 1978, 50, 1722-1724. (9) Oosterhuis, B.; Brunt, K.; Westerink, B. H. C.; Doornbos, D. A. Anal. Cbem. 1980, 52, 203-205. 1328-1329. J.; Czerwinski, A.; Mark, H. E., Jr. Anal. Cbem. 1979, 57, (10) Caja, (11) Caja, J.; Czerwinski, A,; Rubinson, K. A,; Heineman, W. R.; Mark, H. B., Jr. Anal. Cbem. 1980, 52, 1010-1013. (12) Rubinson, K. A.; Baker. P. F. Roc. R . SOC.London, Ser. B 1979, 205, 323-345.
RECEIVED for review April 7, 1980. Accepted May 16, 1980. The chromatographic equipment used in this work was purchased through NSF Grant No. CHE77-15219 to T.W.G.
Determination of Low Levels of Copper by Atomic Absorption Spectrometry with a Simplified Extraction Technique W. Orville Calhoun' and I?. B. Hurley Badische Corporation, P. 0. Drawer D, Wiiiiamsburg, Virginia 23 185
T h e sensitivity of atomic absorption spectrophotometry for copper is approximately 90 pg/L for 1% absorption ( I ) . The sensitivity is less for viscous or concentrated salt solutions. Accurate measurement of low levels of copper,
