Anal. Chem. 1980, 52, 1548-1549
1548
known to be completely in the thione form (5). Our XPS spectra also showed only one S(2p) doublet, which is consistent with t h e presence of only one tautomeric form.
CONCLUSIONS For dithizone and its related compounds, there is no broadening of the S(2p) XPS peaks, which suggests that only one tautomeric form is present. T h e binding energies for nitrogen in these compounds suggest that none are doubly bound to carbon, and therefore the single tautomer is of the thione form. Dithizone and its related compounds all show shake-up peaks which are due to transitions of the same energy (2.7 eV) as that of their corresponding optical spectra. These shake-up peaks are probably due to excitation of K molecular groups, similar to the mechanism orbitals in the -N=N-
proposed by others (3, 4 ) for doubly bound carbon.
LITERATURE CITED (1) Irving, H. M. N. H. "Dithizone", Analytical Sciences Monographs; The Chemical Society: London, 1977; p 9. (2) Katrib, A.; Baddar, F. G.; El-Rayyes, N. R.; ACHajjar, F. H. J. Mol. Stnrct.; Proceedings of the XIV Eurowan Congress on Molecular SDectroscoDy. .. Frankfurt,-1979, in press. . (3) Carison, T. A.: Dress, W. B.: Grim. F. A,: Haggerty, J. S. J. Nectron Soectrosc. Relat. Phsnom. 1977. IO. 147. (4) C k , D. T.; Adarns, D. B. J. Electron Spectrosc. ReLst. Phenom. 1975, 7 , 401. (5) Hardlng, M., Adarns, M. J.; Alsop, P. A,, Irving, H. M. N. H. Anal. Chin?. Acta 1973, 6 7 , 204.
RECEIVED for review November 27,1979. Accepted April 18, 1980.
Rotating Flow Mixing Device for Post Column Reaction in High Performance Liquid Chromatography Shin-ichiro Kobayashi and Kazuhiro Imal" Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo 7-3- 1, Bunkyo-ku, Tokyo 113, Japan
High performance liquid chromatography (HPLC) has been widely used in many fields of science. In order to increase the sensitivity or enhance the selectivity of detection in HPLC, post column reaction or derivatization has often been performed (1). For such purposes, the thorough mixing of eluate from a column with reagent solution(s) is an important factor to obtain a stable base line and reproducible peak heights, and the short duration of the mixing time is also important to restrain peak broadening. Several mixing devices have been proposed (2-5). In this work, a mixing device with a small vessel in which two flowing solutions are caused to rotate was developed and compared with the known mixing devices in view of the signal-to-noise (S/N) ratio of the glucose peak eluted from a column and peak broadening.
Table I. Effects of Mixing Devices on the Mixing of Two Solutions with the Same Flow Rates and on Peak Broadeninga
EXPERIMENTAL
Experimental conditions are the same as in Figure 2. The S/N ratio (12.1) of the peak obtained with A1 was regarded as one unit.
Mixing Device. The mixing devices used in this study, which were manufactured with diflon by Kyowa Seimitsu Co., Tokyo, are divided into three types, A, B, and C, as shown in Figure 1. Precise measurements for them are listed in Table I. Type A is a conventional mixing device which simply combines two flowing solutions, and type B is an improved one by Frei and co-workers ( 2 , 3 ) . Type C, a newly developed one from our laboratory, has a cylindrical vessel (2.5 mm i.d. X 5 mm, inner dimension, about 25 pL) where two flowing solutions are rotated in the same direction, two inlets which enter the bottom of the vessel at opposite sides, and an outlet in the center of the top of the vessel. Apparatus a n d Procedure. Sample solution (a few mg of glucose in 20 p L of solution I) was injected with a model 1720 syringe injector (Rheodyne Inc., Berkeley, Calif.) onto a column of p-Bondapak CI8(Waters Associates, Milford, Mass.). Elution was performed with solution I (0.05 M Tris-hydrochloric acid buffer, pH 7.7) delivered with a reciprocating pump (KHD type, Kyowa Seimitsu Co., Tokyo) and the flow line was connected to the inlet of the mixing device. Acetonitrile (solution II), a presumed reagent solution, was delivered by the second reciprocating pump via a damper (DAM type, Umetani Seiki Co., Kyoto) to the other inlet of the mixing device. Stainless steel tubing (0.8 mm i.d. X 100 mm) connected the outlet of the device to a re0003-2700/80/0352-1548$01 .OO/O
inner diameter, mm inlet mixing solution solution device I II outlet A1 A2 B1
0.8 0.8 0.8
0.8
B2
0.8
0.3 0.5 0.8 0.5 0.3
c1
0.5
c2
c3
0.8 0.8
c4
0.8
0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8
0.3 0.8
relative S/N ratiob
half width,
1 0.9 1.0 0.9 1.6 1.5
22
s
2 1
23 2 2 2 2
22 24 24
1.8 1.9
a
Table 11. Effects of Mixing Devices on the Mixing of Two Solutions with Different Flow Ratesa and on Peak Broadening mixing device
relative
S/Nratio
A1 A2 B1
B2 c3 c4
1 1.0 1.1 1.0
1.4 1.7
half width, S
4 4 4 4 4 4
1 1 0 1 1 1
a Flow rates of solutions I and I1 were 0.25 mL/min and 1.0 mL/min, respective1 and 3 mg of glucose were injected onto the column. {'The S / N ratio (11.9) of the peak obtained with A1 was regarded as one unit.
fractometer (Refract Detector, Toyo Soda Co., Tokyo) for monitoring. Experiments were performed at 25 "C. All chemicals used were reagent grade. 0 1980 American Chemical Society
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.
1549
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
