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Original Paper
cles to smaller ones in packed columns, from various
efforts made to increase the use of the ancillary tech- thus producing the same separation effect as is obtained
niques in micro LC [1 – 3], and from the recent introduction when a relatively long column is used. The advantages of
and development of a highly permeable monolithic sta- RC are that it allows the separation of compounds with
tionary phase [4]. Generally, two alternative methods are very close retention time, requires no increase in inlet
available when a difficult separation cannot be accom- pressure, and also eliminates any problems involved in
plished by changing the column and mobile phase condi- making long columns. On the other hand, once the ana-
tions. The most common approach is to increase the col- lytes are being separated, they would be mixed again dur-
umn length with identical packing material, but this might ing the recycling process. Thus peaks can only be
lead to a limitation of the separation efficiency due to an recycled until they begin to overlap.
increased inlet pressure. The second approach is to The concept of RC in LC has been studied since the late
recycle the sample through the column, which requires no 1960’s with the advent of HPLC. Basically, there are two
increase in the column inlet pressure. However, the dead operating modes in RC, i. e., direct pumping (DP) [10] and
volume in the recycle lines causes band broadening and alternate pumping (AP) [11 – 14]. In DP-RC, a pump, a
peaks can only be recycled until they begin to overlap. sample injector, a column, and a detector are connected
Thus, the recycling technique has been limited to prepara- in series in a closed loop, as shown in Figure 1 (DP). The
tive chromatography where larger diameter columns are effluent from the detector is fed back to the pump, and the
unresolved sample is recycled within the loop until suffi-
Correspondence: Toyohide Takeuchi, Department of Chemis-
try, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu cient resolution is attained, whereas in AP-RC the pump is
501-1193, Japan. Phone/Fax: +81 58 293 2806. removed from the recycle loop and the sample is recycled
E-mail: takeuchi@apchem.gifu-u.ac.jp. exclusively through the column(s). Figure 1 (AP1) and
J. Sep. Sci. 2004, 27, 1339 – 1344 www.jss-journal.de i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1340 Lim, Uzu, Takeuchi
mode [17 – 19]. Cartoni and Ferretti [17] reported the ear-
liest separation of benzene-deuterobenzene and toluene-
deuterotoluene with reversed-phase LC, and separation
factors higher than those of GC were obtained. For all the
compounds tested in refs. [17 – 19], the deuterated com-
pounds always eluted before their hydrogen analogs.
Baweja [18] suggested that this chromatographic beha-
vior depends fundamentally on the interaction between
the C – H or C – D bonds and the stationary phase. A C – H
bond has a higher oscillation frequency (3300 cm – 1) than
a C – D bond (2334 cm – 1), and it induces greater forces of
attraction (i.e., the van der Waals forces) between itself
and the stationary phase, thus leading to longer retention.
This is supported by the results obtained by Turowski et
al. [19] when they examined the reversed-phase chroma-
tographic separation of 10 pairs of isotopic compounds on
10 different stationary phases having alkyl and aryl groups
bonded to the silica surface.
Sj. van der Wal has studied the isotopic separation of ben-
zene and deuterated benzenes by reversed-phase micro-
bore and recycle HPLC [20] and the limitations of high-
efficiency separation by alternate column RC [21]. By
using a 225 cm60.32 mm fused silica column packed
with 5 lm reversed-phase material, the separation of ben-
zene-d6, monodeuterobenzene, and benzene was
achieved at approximately 680 min with a 15% aqueous
acetonitrile (ACN) eluent under 40 MPa inlet pressure.
This separation was improved with a recycle system using
two conventional 15 cm64.6 mm columns packed with
3 lm material, the separation of monodeuterobenzene
and benzene was achieved after 19 cycles at
approx. 454 min with a 40% aqueous ACN eluent under
Figure 1. Simplified schematic diagrams of the DP and AP 37 MPa inlet pressure [20]. By considering the limiting
recycle designs. I: 4-port valve. II: 6-port valve. III: 8-port effects of retention factors, such as the increased inlet
valve. See text for details (Section 1). pressure, a virtual baseline separation of monodeutero-
benzene and benzene was accomplished by generating
200,000 apparent theoretical plates with two 15-cm col-
(AP2) shows the AP recycle systems that utilize a 6-
umns per cycle after 55 cycles in approximately 12 h
port [13] or an 8-port [14] switching valve, respectively.
under 38 MPa and 20% aqueous ACN elution [21].
The main advantage of the 8-port design over the 6-port
design is that only one detector is needed to monitor the The present paper introduces the use of highly permeable
sample. However, the 8-port design possesses a trivial monolithic silica capillary columns in a newly designed AP
disadvantage, i.e., the direction of flow through the detec- recycle system for microcolumn LC in order to increase
tor is reversed every time the valve is switched. In general, the column efficiency. One of the main advantages of the
there are five main criteria that need to be taken into proposed AP system is that only one detector is used to
account when choosing the mode of operation, i. e., monitor the transfer of analytes from one column to the
(1) dead volume of the pump, (2) cost of the column, other given the same flow direction. Compared to the use
(3) availability of a high-pressure flow cell on the detector, of conventional packed columns in RC reported so far, the
(4) instrumentation control such as the necessity to use of monolithic capillary columns is expected to achieve
actuate the switching valve at regular intervals, and better isotopic separations in terms of the retention time,
(5) solvent consumption. the elution profile, the drawback of too high inlet and back
pressure, and the optimum number of cycles. The system
The separation of partially or completely substituted isoto- was easily assembled with the aid of a commercially avail-
pic molecules with deuterium atoms has been done able low dead-volume 6-port switching valve, and the iso-
mainly by GC [15, 16] and by LC in the reversed-phase topic separation of deuterobenzene, 1,3,5-trideuteroben-
J. Sep. Sci. 2004, 27, 1339 – 1344 www.jss-journal.de i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Separation of benzene and deuterated benzenes by RP and RC LC 1341
J. Sep. Sci. 2004, 27, 1339 – 1344 www.jss-journal.de i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1342 Lim, Uzu, Takeuchi
J. Sep. Sci. 2004, 27, 1339 – 1344 www.jss-journal.de i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Separation of benzene and deuterated benzenes by RP and RC LC 1343
Table 1. The effects of inlet pressure and the theoretical plate number per unit time in recycle chromatography. Operation condi-
tions as in Figure 6.
Inlet pressure Theoretical plate Theoretical plate Linear flow N per unit
[kPa] number [N] height [lm] rate [mm s – 1] time [min – 1]
400 28400 25.0 0.326 403
500 29100 24.4 0.404 510
600 30500 23.3 0.478 635
700 28800 24.7 0.553 692
800 27800 25.6 0.594 715
900 26200 27.1 0.720 819
1,000 25100 28.3 0.795 881
1,500 23100 30.7 1.157 1176
2,000 21200 33.5 1.565 1458
2,500 19600 36.3 1.940 1674
3,000 16900 42.0 2.316 1727
3,500 15300 46.2 2.733 1850
J. Sep. Sci. 2004, 27, 1339 – 1344 www.jss-journal.de i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1344 Lim, Uzu, Takeuchi
Acknowledgement
The authors gratefully acknowledge the Takeda Founda-
tion for financial support.
J. Sep. Sci. 2004, 27, 1339 – 1344 www.jss-journal.de i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim