Original

Chromatography, Vol.28 No.3 (2007)

Original

Development of a Solvent−Saving Direct−Pumping Recycle Chromatographic
System and Its Application to the Separation of Deuterated Benzenes inLiquid Chromatography
Zarina Aspanut, Yuko Suzuki, Lee Wah Lim and Toyohide Takeuchi*
Department of Chemistry, Faculty of Engineering, Gifu University, 1−1 Yanagido, Gifu 501−1193, Japan
Received for review November 14, 2007. Accepted November 26, 2007

Abstract
A closed−loop direct−pumping recycle chromatography (RC) system was examined for analytical scale to achieve high resolution in high−performance liquid chromatography (HPLC), where a 6−way switching valve was switched after the system had been stabilized, resulting in
no more consumption of additional mobile phase. The extra−column band broadening effect was minimized by using an HPLC pumpequipped with small−discharging−volume plungers, low−dead−volume devices and connecting tubes. The plate number achieved was linear
to the number of cycle, and it increased by more than 10,000 per cycle using a 25−cm separation column packed with 5−µm reversed−phasematerials. The mobile phase conditions were optimized in terms of analysis time, and the present RC system was applied to the separation ofbenzene and deuterated benzene isomers.
Keywords : HPLC, Recycle chromatography, Solvent−saving direct−pumping system, High resolution, Deuterated benzenes

Trone et al. [6] have developed a peak trapping recycle chro-

1. Introduction
When a difficult separation cannot be accomplished by chang-matography (RC) system and optimized it for peak purity assess-ing the column and mobile phase conditions, two alternate methods

ment of active pharmaceutical ingredients. After being analyzed

are available. The most common approach is to increase the col-using a reversed phase analytical column, peaks of interest wereumn length with identical packing material, and the second ap-

trapped and were subsequently introduced to an RC system. They

proach is to recycle the sample through the column. However, thehave achieved over 227,000 theoretical plates for some com-former approach might lead to a limitation to the separation effi-pounds.

ciency due to an increased inlet pressure whereas in the latter case,

Zhang and McConnell [7] have described the development of

the dead volume in the recycle lines causes band broadening anda novel column−switching technique called “simulated moving col-peaks can only be recycled until they begin to overlap. Thus, theumns” (SMC) to quickly achieve complete chiral resolution on col-recycle technique has been limited to preparative chromatographyumns with limited enantioselectivity. SMC helped to achieve chiralwhere larger diameter columns are employed [1−7]. In preparative

resolution by virtually multiplying the column length, thus enhanc-

chromatography, the recycle technique is well−known as a usefuling separation efficiency and resolution, without increasing back-

tool to improve enantioseparation, production rate and recovery

pressure. The results clearly indicated that SMC eliminated the sig-yield.

nificant band broadening that was inevitable in the closed−loop re-Correspondence : Department of Chemistry, Faculty of Engineering, Gifu University, 1−1 Yanagido, Gifu 501−1193, Japan.
Tel : 058−293−2806
Fax：058−293−2806
E−mail : takeuchi@apchem.gifu−u.ac.jp―１３１―Chromatography, Vol.28 No.3 (2007)

and the detector. The only concern of this DP system was severe

cycling techniques currently used in preparative chromatography.
For analytical purposes, RC has been attempted to improveextra−column band broadening that might be caused by the pump

the resolution of analytes of interest [8, 9]. The separation of oxy-and thus it can only be applied to a conventional or preparative LC

gen isotopic compounds was attempted by Tanaka et al. [8] in re-system. This DP recycle LC system was made possible by using aversed−phase LC, based on the small differences in the dissociation

double−plunger type HPLC pump and other low−dead−volume de-constants of organic acids. Oxygen isotopes of benzoic acid as wellvices. The system was then applied to the separation of benzene

as p−chlorobenzoic acid and p−nitrophenol were separated byand deuterated benzenes.

ionization control using two pairs of reversed−phase columns and a
2. Experimental

six−way switching valve [8].

Apparatus

RC is an approach where the sample is being recycled throughthe column(s), thus producing the same separation effect as when a

The DP recycle system, as illustrated in Figure 1, consisted ofrelatively long column is being used. The advantages of RC are

a pump, a 6−way switching valve, an injector, a separation columnthat it allows the separation of compounds with very near retention

and a UV detector. The eluent was first supplied by a double−time, no increase in inlet pressure, and it also solves the problem ofplunger type HPLC pump model LC−20 AD (Shimadzu, Kyoto,

having difficulties in making long columns. On the other hand,Japan). When the system has been stabilized, the 6−way switching

once the analytes are being separated, it would be mixed again dur-valve is switched and the effluent from the detector is directly sup-ing recycle process, and thus peaks can only be recycled until theyplied to the pump and thus avoids substantial consumption of thebegin to overlap.

eluent. The diameter of the separation column was 4.6 mm (L−col-In our previous study, we designed an alternate−pumping

umn ODS, 5 µm ; Chemicals Evaluation and Research Institute,(AP) recycle system for capillary LC using highly permeable

Tokyo, Japan), and sample injection volume was 20 µL. Both themonolithic silica columns [9]. The developed system was proven toinjector and the 6−way switching valve were from Rheodyne (Co-be suitable for the separation of isotopic molecules, either partiallytati, CA, USA) and the connecting tubes between the detector andor completely substituted with deuterium atoms. For difficult iso-pump used were of 1/16” O. D. PEEK tubes. A model UV−970

topic separation that requires long analysis time, the recycle system

UV detector (Jasco, Tokyo, Japan) was used and the analytes wereutilizing capillary columns reduces the consumption of mobile

detected at the wavelength of 210 nm. All data were collected by aphase. However, this AP system requires two columns and theswitching valves needed to be monitored and switched accordingly,
either manually or automatically, so that the samples could be recycled through the columns without being drained away.
The resolution, R s, of two chromatographic peaks is defined
as :

Rs=

α−1
k・ av ・(N )1/2
k av+12(α+1)(1)

where N is the number of theoretical plates, α is the separation factor and kav is the average retention factor of the two peaks. Thus,
the R s is proportional to the square root of N and hence to the column length. So, a 4−fold increase in the length of the column
would double the resolution, and this is accompanied by an increase in the retention time. The recycle technique, which practically increases the length of the column, would theoretically increase the resolution of a separation.
Therefore, the main aim of this work was to develop a direct−
pumping (DP) recycle system in such a way that the eluent was reFigure 1. Schematic diagram of the DP recycle separation system
employed in this work.
1=mobile phase, 2=6−way switching valve, 3=HPLCpump, 4=loop injector, 5=separation column, 6=UV detector, 7=drain.

cycled within the line in a closed−loop, i.e. once the sample is being injected into the system, the valve will be switched and there
will be no additional eluent supplied to the system. This is possibleby placing a 6−way switching valve between the eluent reservoir―１３２―Chromatography, Vol.28 No.3 (2007)

Zarina Aspanut, Yuko Suzuki, Lee Wah Lim and Toyohide Takeuchi

Table 1. Theoretical plate number obtained for thefirst cycle (N1) and average values obtained for the second to the fifth cycle(∆N ).

C−R 7 Ae plus Chromatopac data processor (Shimadzu).
Reagents
Acetonitrile (ACN) and distilled water were of HPLC grade
and obtained from Wako Pure Chemical Industries (Osaka, Japan).

ACN(%)

N1∆N

Special grade benzene was also purchased from Wako Pure Chemi-60
50403013,20015,70017,20015,6006,700
9,100
12,70011,000cal Industry while other deuterated benzenes were obtained fromAldrich Chemical Co. (Milwaukee, WI, USA). Other reagents wereof reagent grade and obtained from Nacalai Tesque (Kyoto, Japan).
The reagents were used as received, unless otherwise noted.

Separation column : L−column ODS (250×4.6 mm I.D.). Eluent :as indicated. Flow−rate : 1.0 mL min−1.Wavelength of UV detection : 210 nm. Analyte : 0.01% benzene. Injection volume : 20 µL.

3. Results and Discussion
N versus number of cycle
The N obtained per cycle was measured by using benzene asthe analyte. When using a 25−cm column packed with 5 µm ODS,N values obtained for the first cycle (N1) depended on the ACN
concentration in the eluent, and N1 values between 13,800 and
17,200 were achieved for 30−60% ACN aqueous solutions as theeluent, as shown in Table 1. It can be seen that the largest N1 isachieved for 40% ACN. Table 1 also shows average additional Nvalues (∆N ) obtained for the second to fifth cycle. The largest ∆Nis also achieved for the 40% ACN. Smaller ∆N values are achievedfor 50 and 60% ACN, which is due to additional extra−column
band broadening.
Figure 2 demonstrates N values obtained for the first eighteen
cycles using 50, 55 and 60% ACN using a 25−cm column packed

Figure 2. Theoretical plate number versus number of cycle in RC.
Separation column : L−column ODS (250×4.6 mm I.
D.). Eluent : as indicated. Flow rate : 1.0 mL min−1.
Wavelength of UV detection : 210 nm. Analyte : 0.01%benzene. Injection volume : 20 µL.

with 5−µm ODS. It can be seen from Figure 2 that the N increases
with increasing number of cycle. It was found that when using
lower concentration of ACN, the increase of N was larger thanthose obtained using higher concentration of ACN although the total analysis time increases with decreasing ACN concentration. In
other words, the larger the retention time, the larger N wasamined, but we have not found any advantage over a single 25−cm

achieved at each cycle. This means that the additional band broad-column. In addition, use of a single 25−cm column packed with 3−

ening due to the recycle cannot be neglected under the conditionsµm ODS did not provide any advantage over the 25−cm column

in Table 1. The discharging volume of each plunger of the pumppacked with 5−µm ODS. Considering these results, a single 25−cm

employed in this work is 10 µL. The tubing between the outlet ofcolumn packed with 5−µm ODS is used as the separation column

the column and the 6−way switching valve as well as between thefor RC in the following examination.

6−way switching valve and the pump was PEEK tubing with 0.25
Separation of benzene and deuterated benzenes

or 0.50 mm ID. The total length of these connecting tubes is ca. 1
m. It is presumed that band broadening caused in the above parts as

Figure 3 demonstrates the separations of benzene and hexa-

well as in the check valves may not be neglected. The cell volume

deuterated benzene (benzene−D6) using different concentration ofof the UV detector can be another potential contribution to addi-

ACN as the eluent, where baseline separations are achieved at dif-tional band broadening. It should be noted that the pumps em-

ferent analysis time, as indicated by the arrows. It can be seen fromployed in this research do not equip any dampener, which could

the figure that faster baseline separation could be achieved for

greatly contribute to extra−column band broadening.

lower ACN concentration. This result can be expected from thedata in Figure 2.

Although 15−cm columns were also examined in this work,
the results were not satisfactory due to the effect of extra−column

Considering the above data, 40% ACN aqueous solution wasband broadening. Two columns connected in tandem were also ex-chosen for the separation of benzene, 1,3,5−trideuterated benzene―１３３―Chromatography, Vol.28 No.3 (2007)

Figure 5. Recycle separation of benzene−d1 and benzene.
Operating conditions are as in Figure 4, except for thesample ; Sample : 0.01% each for benzene (H 6) andbenzene−D 1(D 1).

Figure 3. Recycle separation of benzene and benzene−D6.
Operating conditions are as in Figure 2, except for sample. Sample : 0.01% each for each benzene−D6 (1) andbenzene (2) dissolved in the eluent.

Figure 4. Recycle separation of benzene, benzene−D3 and benzene−D6.
Operating conditions are as in Figure 2, except for theeluent and the sample. Eluent : ACN : water (40 : 60).
Sample : 0.01% each for benzene, benzene−D3 andbenzene−D6 dissolved in the eluent.

Figure 6. Simulation of required analysis time to achieve R s=1
for benzene and benzene−D1.
Simulated conditions are as in Tables 1 and 2.
togram is shown in Figure 5. The upper trace is the enlargement of
the lower trace from 200 to 870 min.
For difficult isotopic separation that requires long analysis(benzene−D3) and benzene−D6. The separation is demonstrated inFigure 4. Baseline separation (i.e., R s=1.5) was achieved after thetime, the closed−line DP−RC system reduces the consumption of

10th cycle and the separation took less than 280 min using a com-mobile phase. This system also provides a relatively simple and in-mercially available conventional column. The calculated N

expensive mean of attaining higher separation efficiency, i.e. any

achieved for the first resolved peak, i.e. benzene−D6, after the 10th

difficult separation could possibly be achieved by recycling thecycle was approximately 111,000.

sample through the column in the closed−line until sufficient reso-The system was also applied for the separation of benzene andlution is attained. This would reduce the turnaround time by opti-

monodeuterated benzene (benzene−D1), which needs much larger

mization of the separation condition on a conventional chroma-

N values. The present closed−line RC system managed to achievetographic system.

a fairly good separation after 800−min operation, and the chroma-―１３４―

Chromatography, Vol.28 No.3 (2007)

Zarina Aspanut, Yuko Suzuki, Lee Wah Lim and Toyohide Takeuchi

Table 2. Retention data of benzene−D1 and benzene for simulation.
The operating conditions are the same as in Table 1 except for the analytes.
[ACN]% (v/v)

tM

kmin

Benzene−D1Benzene

60
50
4030

2.240
2.220
2.269
2.353

2.639
4.558
8.264
16.033

2.646
4.575
8.352
16.294of isotopic compounds. After the system was stabilized, the 6−wayswitching valve was switched and thus resulting in no extra consumption of eluent. The analyte could be recycled in a closed−lineand only minimum amount of eluent that remained in the line issufficient to resolve the sample. It was observed that the N in-α

creased with increasing number of cycle. The optimization of thepresent system in terms of analysis time and resolution is being in-1.0024
1.00361.01061.0163

vestigated.
Acknowledgement
One of the authors, LWL, gratefully acknowledges the Koshiyama Foundation for the financial support.

Simulation of required analysis timeReferencesEquation (1) allows the simulation of analysis time required
for achieving an adequate R s value when we could measure accu-[1]

Yoshida, T. ; Shu, C−K. ; Theimer, E. T. J. Chromatogr.

[2]

Johnson, W. M. P. ; O’ Keefe, D.F. ; Rihs, K. J. Chroma-rate retention factors of analytes and the void volume (tM). Table 2

1977, 137, 461−464.

shows the data obtained for benzene−D1 and benzene. It is seenfrom the table that the separation factor increases with decreasing

togr. 1984, 291, 449−452.

ACN concentration. By using the data shown in Tables 1 and 2, the[3]Okano,T. ; Masuda, S. ; Kusunose, S. ; Komatsu, M. ; Ko-[4]

Crary, J. R. ; Cain−Janicki, K. ; Wijayaratne, R. J. Chroma-analysis. From the simulation in Figure 6, it takes ca. 6 h to achieve[5]Letter, W. S. J. Chromatogr. 1992, 590, 169−173.

R s=1 for benzene−D1 and benzene. In practical, it took more time[6]

required analysis time was calculated for various ACN concentra-bayashi, T. J. Chromatogr. 1984, 294, 460−465.

tions. The result is illustrated in Figure 6. The simulation indicatesthat the lower ACN concentration is more favorable for shorter

togr. 1989, 462, 85−94.

to achieve the separation with R s=1. This may be caused by theTrone, M. D. ; Vaughn, M. S. ; Cole, S. R. J. Chromatogr. A2006, 1133, 104−111.

fact that the effect of extra−column band broadening increases with

[7]

Zhang, Y. ; McConnell, O. J. Chromatogr. A 2004, 1133,[8]Tanaka, N. ; Araki, M. ; Kimata, K. J. Chromatogr. 1986,[9]

Lim, L.W. ; Uzu, H. ; Takeuchi, T. J. Sep. Sci. 2004, 27,

increasing numbers of cycles. A more precise measurement of the227−238.

retention factors is needed in order to improve the accuracy of thesimulation result.

352, 307−312.

4. Conclusion1339−1344.

A DP−RC system was designed and applied to the separation―１３５―