Anal. Chem. 1907, 59. 2221-2224 MUes, P. J . ASSOC.Off. A M . Chem. 1978, 6 1 , 1388. Hurst, W. J.; Snyder, K. P., Martin, R. A., Jr. J. Liq. Chromatogr. 1983, 6 , 2067. Thompson, D.;Lee, S.; Allen, R. J. Assoc. Off. Anal. Chem. 1983, 6 6 , 1380. Melvano, R.; Buzzlgoll, G.; Scarlatlni, M.; Cenderelli, G.; Gondolfi, C.; Grasso, P. Anal. Chem. 1972, 201. Allegrini, M.; Boyer, K. W.; Tanner, J. T. J. Assoc. Off. Anal. Chem. 1881. 6 4 . 1111. Heckman; M. M. J. Assoc. Off. Anal. Chem. 1979, 62, 1045. Fisher, P. W. F.; L’Abbe, R. J. Assoc. Off. Anal. Chem. 1981, 6 4 , 71.
2221
Ahmad, N. Aust. J. Dairy Techno/. 1977. 32, 102. Johansen, 0.; Stelnnes, E. Analyst (London) 1978, 707, 455. Propst, R. C. Anal. Chem. 1977, 49, 1198. Gorsuch, T. T. The Destruction of Organic Matter; Pergamon: New York, 1970; pp 137-138. (14) Colovos, G.;Wilson, G. S.; Moyers, J. L. Anal. Chem. 1974, 46, 1045. (10) (1 1) (12) (13)
for review December 22* lg8&Accepted May 7,
1987.
Construction and Comparison of Open Tubular Reactors for Postcolumn Reaction Detection in Liquid Chromatography Carl M. Selavka, Kai-Sheng Jiao, and Ira S. Krull* Barnett Institute of Chemical Analysis and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115 There has been a great deal of interest in the past 10 years surrounding the use of derivatization in high-performance liquid chromatography (LC) for improving detection selectivity or sensitivity. On-line, postcolumn derivatization offers a number of advantages over other possible derivatization approaches, but the possibility of band broadening in the postcolumn reactor (PCR) must always be a concern of the analyst. The choice of PCR design is generally dictated by the reaction time (trx) required: segmented flow systems are best for t,, > 5 min, packed bed reactors are useful for 1 min < t,, < 5 min, and knitted open tubular (KOT) reactors are best incorporated for 0 min < t,, < 5 min ( I ) . There have been a number of reports regarding the theoretical basis for minimizing band broadening when open tubular reactors are geometrically deformed by helically coiling the tubing (2-5). Recently, this concept has been extended to, and the hydrodynamic equations explained and solved for, capillary supercritical fluid chromatography (6). The principles for improvements in radial mixing in coiled tubes have been exploited through the introduction of the KOT, also known as a three-dimensional coiled tube ( I , 7, 8), but the equations for these reactors have not been solved to date. Our interest in the development of a postcolumn on-line photochemical reactor for improved electrochemical (EC) detection in LC first led us to the incorporation of coiled Teflon tubing as the PCR, in which the tubing was wound about a Hg lamp (9). While this PCR design enabled the demonstration of the detection principle, there were problems with band broadening, resulting in loss of resolution and poor detection limits. T o improve upon the initial LC-photolysis-EC (LC-hv-EC) system, we wished to incorporate a KOT PCR, but the only literature report that described the actual construction process for such a reactor (7) utilized a “French knitting” children’s toy (called “Strickliesel” in Germany), which we were unable to locate in area stores. Only after consultation (10) with one of the authors of the previous work were we able to satisfactorily construct our own KOT. Shortly after we performed the work described in this paper, a report appeared regarding the construction of KOTs using crocheting tools and techniques (11). The crocheting techniques appeared relatively straightforward, except for the authors’ admonition to fill the tubing with water before construction, in order to avoid kinking of the tubing during the construction process. This kinking would reduce the lifetime of the finished KOT by increasing its back pressure. The methods we describe do not involve the use of tools of any kind. The impetus for this paper is the large number of requests we have received for instruction in constructing a KOT, as
a result of some publications on LC-hv-EC which incorporated KOT PCRs (12). In this short report, the design of three different KOT reactors is described. The performances of these reactors are then compared to coiled and straight open tubes, using plate height vs. linear velocity (h vs. u) plots and calculations of relative band broadening. The effect of tubing diameter on the performance of the PCRs is also discussed. Finally, the criteria for the use of these KOT designs in chemical and photochemical postcolumn reaction detection schemes are briefly delineated.
EXPERIMENTAL SECTION Apparatus. The flow injection analysis (FIA) system used has been described elsewhere (12) and consisted of a pump, injector, and UV (214 nm) detector. The time constant on the UV detector was set at 0.5 s. The PCRs were constructed from Teflon tubing (Rainin Instrument Co., Woburn, MA) having 0.8-, 0.5or 0.3-mm i.d., in lengths chosen to give constant reactor volumes of 1.7 mL (3.38, 8.66, and 24.05 m, respectively). Reagents. HPLC-grade methanol (MeOH) and water used in the mobile phase were obtained as the Omnisolv grade (MCB, Cherry Hill, NJ) and were degassed before use. The test analyte, sodium benzoate, was obtained as the food grade (J. T. Baker Chemical Co., Phillipsburg, NJ) and was used as received. Procedure. In general, the best performance from a KOT will be realized if the tubing is turned in alternating 180° arcs in three dimensions, keeping the diameter of the arc as small as possible without pinching the cross section of the tube into an elliptical geometry. This maximizes the aspect ratio (A = d / D , where d is the internal diameter of the tube and D is the diameter of the arc through which the tubing is turned), which yields better radial mixing in a KOT. Pinching the tubing leads to an undue amount of back pressure (increases AP),which shortens the lifetime of the KOT PCR and limits the range of flow rates available for a separation. Three distinct KOT geometries were studied, labeled KOT1, KOT2, and KOT3 for reference. The construction of KOT1, which was designed by Uwe Neue of Waters Chromatography Division, Millipore Corp., is shown in Figure 1. The analytical tubing is knitted around a backbone tube which, depending on the eventual use of the PCR, can be made of steel or Teflon. However, it should be noted that the use of a Teflon backbone tube having a larger internal diameter than the analytical tube tends to limit the chance of deforming the cross section of the analytical tube, because the backbone deforms first if too much tension is put on the analytical tube during knitting. In the design of KOT1, the analytical tube turns back against itself on each loop, which repeatedly changes the direction of flow in three dimensions. The construction of KOT2, which was designed by one of the authors (C.M.S.) in our laboratory, is demonstrated in Figures 2 and 3. Once again, a backbone tube is used in the design of
0003-2700/87/0359-2221$01.50/0 0 1987 American Chemical Society
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ANALYTICAL CKMISTRY, VOL. 59. NO. 17, SEPTEMBER 1. 1987
Flgure 1. Construction of KOT1. m e analytical tubing is passed around the backbone. making a loop. It IS then turned against nself. Isavding beMnd and under the backbone tube. to lam the seomd kcp.
Flgure 4. Appearance 01 KOT3. This KOT is consbucted by making wmtend knots. of ahmaling direction. in the analyncal lube. The KOT is anached to a spwl having a diameter 01 21 mm.
Table I. Summary of Physical Properties of PCRS geometry
i.d., mm
straight coil coil KOTl KOT2 KOT2 KOT2 KOT3
0.5 0.8 0.5 0.5 0.8
0.5 0.3 0.5
100Ao
AP (pi), at 2 mL/min
red. facto$
420 340
3.8 2.4 7.7 8.9 6.3 3.3
530 550 1730
5.51 7.51 9.@1 8.61
7.1
600
4.21
450
690
' A = asp& ratio = (analytical tube i.d.)/(diameter of knitted arc). 'Reduction factor: reduction in the length of the PCR following knitting.
npue 2. Consrmctbn 01 KOT2 The analytral Ming s directed war and under lhe backbone, then lhrough the preceding length of h a analytical tube to form the t 1st loop It IS men turned against Rsell. waveis wder the backbone. and is pushed though me fm a o p to t a m the second imp.
Figure 3. Final step in the construction of KOT2. While the end of lha analytical tube is pulled. the second loop is pulled back over the first. Slight tension will keep the tube in the proper orientation until lh9 next knot is completed.
thisKOT. The procedures in Figure 2, delineating the formation of the knot using two loops around the backbone, is straightforward; Figure 3 demonstrates the technique for pulling the second Imp of analytical tubing over the first, which makes a tighter KOT. This technique also helps reduce the overall length of the KOT, which may be important for some applications (uida infra). KOT3 is constructed from a single length of tubing (the analytid tubing), by tying overhand knots of ahmating direction
in the tube. As shown in Figure 4, KOT3 has a somewhat more disordered appearancethan KOT1 or KOT2, but it in quite simple to construct. It should be noted, however, that it is easier to stretch the tube composing KOT3, or deform its crms section, during the knitting proeess, than for KOTl or KOT2. KOTl and KOT3 were constructed with 0.5 mm i.d. tubing, while three examples of KOT2 were constructed by using 0.3-, 0.5-, and O.Bmm tubing. FoUowing construction,the KOTS were attached to a strip chart paper spool having a diameter of 21 mm, which approximated the dimensions of the UV lamp used in LC-hrEC (22),by tying the free ends of the backbone tubing to the spool. In addition, PCRS having a 'coil" geometry were prepared by simply wrapping appropriate lengths of 0.5- and O.Smm tubing around the spools. Finally, a "straight" piece of 0.5 mm Teflon tubing was included in the study; this tube traversed the laboratory in a circle having a diameter of -2 m. To facilitate evaluation, each of the PCRs was connected between the injector and the UV detector. and five replicate injections of a 1ppm sodium benzoate solution (prepared in the 50/50 MeOH/H20 mobile phase) were performed. The injection procedure was repeated for each flow rate between 0.25 and 3.00 mL/min, a t 0.25 mL/min intervals, and the response from the UV detector was collected hy using a recorder chart speed of 20 cmlmin. Data reduction involved measurements and calculations of Wo,,(peak width a t 10% peak height [seconds]), W, (width a t base line for tangents drawn to sides of the peak [seconds]), to(retention time at peak centroid [seconds]),BIA (asymmetry faetor [dimensionless]). u: (peakvariance [s2]),K (relativevarianm [dimensionless]), and h (plate height [centimeters]), using the following formulas (4, 13):
h = LW,.z/16toz u? =
Wo,,z/([1.764(B/A)z] - [ll.l5(B/A)]
K = (u?[KOT])/(u?[straight])
(1)
+ 28)
(2)
(3)
ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987 3.5
2223
Table 11. Comparison of Variance Contributions from 0.5 mm i.d. PCRs
3.0 2.5 h
+
ut?; s2
Kb
straight
712.5 f 25.5 203.4 f 2.7 40.7 f 0.8 38.6 f 1.6 34.8 f 1.3
1.000 0.285 0.057 0.054 0.049
coil KOTl KOT2 KOT3
l a 1 2.0
geometry
O.S-rnrn
1.5
ut2 = peak variance. Values are mean f standard deviation (n = 5), measured at 1 mL/min. K is relative variance, normalized
I .o
0
5
IO
15 u
20
25
to the straight PCR eeometrv.
30
[cm/acl
Figure 5. h vs. u curves for KOT2, prepared from tubes having three different internal diameters.
20 ..
Ll
15 ..
4
KOTI
9
KO12
9
4
KO11
9
KO12
* KOTS
* KO13
h (a)
IO
.'
5
..
0.
I O1 00
I
I
05
15 20 Flow Rsts (rnl/rninl
IO
hvs. fcurves for KOT1, 0.5-mm tubing.
Figure 7.
25
SO
KOT2, and KOT3 constructed using
It also was desirable to compare the relative efficiency of radial mixing for each of the PCR designs. As shown in Figure 6, simple coiling of the analytical tubing did not sufficiently generate secondary flow even when the mobile phase flow rate was adjusted to elevated values (since the PCRs were all constructed with 0.5-mm tubing, F and u are equivalent for this study). On the other hand, even at low flow rates all of the KOTs demonstrated radial mixing which was much better developed than for the coil, giving h values between 1.2 and 2.6 cm throughout the flow rate range studied. The variance contributions from these reactors were also calculated (for F = 1.0 mL/min) and compared, and the results of these calculations are presented in Table 11. It should be noted that the h values for the KOTs were higher than expected (values for efficient KOTs typically range between 1 and 2 cm (14)). The cause of these elevated values was the use of straight pieces of tube before and after the KOTs, to connect the reactors to the injector and detector. The lengths of these straight pieces were relatively long (each about 12 cm) in order to mimic the conditions needed in the use of the KOTs with a photochemical reactor. This is not the ideal situation, and in general one should minimize the length of the straight connecting pieces to retain optimum performance. Figure 7 provides a more detailed comparison of the three KOTs; it is clear that all of these geometries provided similar performance. One interesting and unexpected result of the comparison of these PCRs was the excellent performance of KOT3. The design of this KOT does not entail the symmetrical deformations that KOTl and KOT2 incorporate, yet the radial mixing is essentially complete at low flow rates and the reactor exhibits good band-broadening characteristics. When the merits of performance are coupled to the ease of construction, it appears that KOT3 may be the reactor of choice for a PCR system where final reactor dimensions are not important. When finished reactor length is important, as in LC-hv-EC, KOT2 offers an alternative that will retain analytical resolution and peak shape, while fulfilling the physical requirements of the apparatus. If the finished PCR length is not an issue, and if long reaction times are required,
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ANALYTICAL CHEMISTRY, VOL. 59,
NO. 17, SEPTEMBER
KOTl offers the best alternative; in this situation, it is easier to pinch the 0.8-mm tubing during the construction of KOT3, and KOTl is easier to prepare than KOT2. Even though the discussion of Figure 5 suggested that a 0.8-mm KOT2 will give poor performance, it should be emphasized that h vs. u curves are very sensitive to changes in the convective mixing efficiency of a reactor. The curves are useful for quantitatively comparing KOTs, but may overemphasize the differences one might expect when it comes to practical use. For example, a KOT2 PCR was constructed with sufficient 0.8-mm tubing to give an internal volume of 6 mL and was incorporated in the photochemical reactor for improving thermal energy analysis (TEA) detection (25). In this case, the variance (for a peak having k’ = 2) only increased about 7% over that for a 2-mL KOT2 constructed with 0.5mm tubing. Teflon PCRs offer a great deal of flexibility in design, as well as the obvious advantage of inertness and resistance to even the most aggressive aqueous reagents (e.g., concentrated acids or bases). However, it has been our experience that under certain conditions, the use of organic solvents other than methanol, ethanol, or acetonitrile may cause swelling, leakage, or rupture of Teflon tubing, especially when it is used in a P is elevated). In addition, when KOT PCRs KOT (where i are used in a photochemical apparatus, fluoride is liberated from the tubing (polyfluoroethylene) during irradiation with UV light ( I @ , and this causes the tubing to turn brittle and eventually rupture after about 700 h of use. The effect is exacerbated if proper cooling is not provided for the lamp and KOT assembly. For these and other reasons, there may be some applications which are better served by the use of glass or quartz capillary tubes. Unfortunately, these materials are easily broken, difficult to connect to fittings, and can be quite expensive. If the PCR involves the use of an on-line, liquid/liquid extractor with on-line phase separation, the vigorous radial mixing in a KOT produces very small droplets of organic solvent in the aqueous phase, which makes phase separation difficult unless a membrane phase separator is used (17). In such cases, it may be best to incorporate coiled tubing as the PCR. As a final note, the three KOTs described here by no means represent the “best” designs that can be imagined. Rather, these geometries are easy to construct, without the use of tools, and offer attractive performance under a wide range of tube diameters and flow rate conditions. Any other
1, 1987
design will work as well as, or better than, these KOTs, as long as the design incorporates three-dimensional coiling of the tube, with maximized aspect ratios.
ACKNOWLEDGMENT We thank Uwe Neue at Waters Chromatography Division, Millipore Corp., for technical assistance and comments on the paper. The UV detector was donated by Bioanalytical Systems, Inc., and we thank P. Kissinger and R. Shoup for their technical support and interest in this work. We also thank B. D. Karcher, C.-X. Gao, Y. Widener, and H. H. Stuting for their technical assistance in the preparation of the paper.
LITERATURE CITED (1) Lillig, 8; Engelhardt, H. I n Reaction Defection in Liquid Chromatography; Krull, 1. S., Ed.; Marcel Dekker: New York, 1986; Chapter 1. (2) Deelder, R. S.; Kroll, M. G. F.; Beeren, A. J. B.; Van Den Berg, J. H. M. J . Chromatogr. 1978, 149, 669-682. (3) Tijssen, R. S e p . Sci. Techno/. 1978, 73, 681-722. (4) Hofmann, K.; Halisz, I.J . Chromatogr. 1979, 173, 211-228. (5) Tljssen, R. Anal. Chim. Acta 1980, 774, 71-89. (6) Springston, S. R.; Novotny, M. Anal. Chem. 1986, 58, 2699-2704. (7) Engelhardt, H.; Neue, U. Chromatographia 1982, 75, 403-408. (8) Uihleln, M.; Schwab, E. Chromfographia 1982, 75, 140-146. (9) Krull, I.S.; Ding, X.-D.; Selavka, C. M.; Bratin, K.; Forcier, G. J . Forensic Sci. 1984, 29, 449-463. IO) Neue, U., Waters Chromatography Division, Millipore Corp.. personal communication, 29 November 1983. 11) Poulsen, J. R.;Birks, K. S.; Gandelman, M. S.; Birks, J. W. Chromatographia 1986, 22, 231. 12) Selavka, C. M.; Krull, I. S.; Lurie, I.S. J . Chromatogr. Sci. 1985, 23, 499. 13) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 55, 730-737. 14) Neue, U. D. Ph.D. Thesls, Saarbrucken University, Saarbrucken, 1978. 15) Selavka, C. M.; Tontarski, R. E.; Strobel, R. A. J . Forensic Sci. 1987, 3213).941 - . . (16) Batley, G. E. Anal. Chem. 1984, 56, 2261-2262. (1 7) Karcher, B. D., Northeastern University, personal communication, 25 January 1987.
--.-,.
RECEIVED for review February 17, 1987. Accepted May 18, 1987. This work was supported by a Phase 1 NIH-SBIR (lR43ES040576-01) grant, an NIH Biomedical Research grant (RR07143), and a grant from Pfizer, Inc. (Groton, CT). Support for C.M.S. was provided by a National Institute of Justice, U S . Department of Justice, Graduate Research Fellowship (86-IJ-CX-0058). Points of view or opinions stated in this document are those of the authors and do not necessarily represent the official position or policies of the U S . Department of Justice. This is contribution number 312 from the Barnett Institute of Chemical Analvsis and Materials Science at Northeastern University. .
