Journal of’chromatography, Elsevier Science Publishers CHROM.
411 (1987) 221 227 B.V., Amsterdam ~ Printed
in The Netherlands
20 039
MULTIPLE DETECTION SYSTEM FOR THE HIGH-RESOLUTION MICROCOLUMN SIZE-EXCLUSION CHROMATOGRAPHIC SEPARATION OF PROTEINS WITH A SLURRY-PACKED CAPILLARY COLUMN
A. HIROSE*
and D. ISHII
Department of Applied Nagoya 464 ! Japan J (Received
September
Chemistry,
Faculty
of Engineering,
Nagoya
University,
Furocho,
Chikusa-ku,
lst, 1987)
SUMMARY
A multiple (triple) detection system with one detector and recorder each was developed for the high-resolution size-exclusion chromatographic separation of proteins with a slurry packed capillary column. Fused-silica capillary columns of length 1, 2.5 and 6 m and I.D. 0.26 mm were connected in this order by joining each end of the shorter column to the top of the longer column through flow-through cells installed side-by-side on a cell block in the detector. By this means it was possible to record the chromatograms three times with one injection. This system is convenient for comparing the chromatograms obtained with different plate numbers and for effecting very high-resolution separations.
INTRODUCTION
When very complex samples, such as fossil fuelslp3 and environmental4 or biologicalS,6 materials, are analysed by liquid chromatography (LC), a very-high resolution technique7-’ g is required. The use of a slurry-packed capillary column of 0.2-0.35 mm I.D. and several metres length using flexible fused-silica tubing as the column materia17-9 seems to be the most promising method so far compared with conventional (4.5 mm I.D.),i”~’ ‘,I3 microbore (l-l.5 mm I.D.),5,6~‘2,13 semiperrneableg,i4~l 5 and slurry-packedg,’ 6 glass capillary and open-tubular capillary columnsg+17~1g.A fused-silica capillary column (1) has sufficient flexibility and hence is convenient to handle, (2) can be prepared more easily than other columns and even such a long column as 5-10 m can be packed by a one-step process with high efficiencyzO, (3) does not need connection with many other columns to obtain very high plate numbers, (4) can be compactly set up in the LC system by coiling, (5) even when very long are available at reasonable cost, (6) can maintain high efficiency (HETP) for many solutes with a variety of retentions (k’) and (7) has an adequate sample capacity. However, when using a long column, a long separation time cannot be avoided and some effective methods or devices for conducting the chromatography~efficiently OO21-9673/87!$03.50
C
1987 Elsevier
Science Publishers
B.V
A. HIROSE,
222
D. ISHII
are therefore required. In this work, a multiple detection system has been developed, which makes it possible to.detect the chromatograms three times with one injection. The system not only gives extremely high-resolution separations, but also makes it easy to compare chromatograms obtained under different column efficiencies. In this work, the system has been applied successfully to the separation of proteins by size exclusion chromatography. EXPERIMENTAL
Apparatus An SPD-2AM spectrophotometric detector and an LC-SA high-pressure pump (Shimazu, Kyoto, Japan) with a maximum pressure of 500 kg/cm2 were used. For multiple detection, a detection cell equipped with three micro flow cells was designed in our laboratory. The details are described later. For injection of samples a laboratory-made injector was used. Materials A TSKgel G3000SW packing (Toyo Soda, Tokyo, Japan) with particle and pore diameters of 10 pm and 1000 A, respectively, was used. Three kinds of flexible fused-silica tubing of 0.05 mm I.D. (0.240.25 mm O:D.), 0.075 mm I.D. (0.28 mm O.D.) (SGE, Ringwood, Australia) and 0.26 mm I.D. (0.6 mm O.D.) (Gasukuro Kogyo, Tokyo, Japan) coated with a polyimide resin were used. The eluent was 0.2 M phosphate buffer (a mixture of 0.2 M KH2P04 and Na2HP04 solutions). Protein samples consisting of molecular weight marker (Oriental Yeast, Tokyo, Japan), aldose, /?-galactosidase and urease (Sigma, St. Louis, MO, U.S.A.) were dissolved in the eluent. All other reagents were of analytical-reagent or LC grade. Column preparation The column material was flexible fused-silica tubing (11 m x 0.26 mm I.D.) in which TSKgel G3000SW was packed by the slurry-packing technique under a final pressure of 500 kg/cm2 using a Shimazu LC-5A pump, methanol-water (1: 1) as the slurry solvent and water as the packing solvent. During the packing of the column, the fused-silica tubing was coiled to a coil diameter of 15-20 cm. The long column obtained was cut to the desired length before use. RESULTS
AND
DISCUSSION
Construction of micro flOw eel/s for multiple detection Three flow cells installed in parallel on the cell block in the detector were used, as shown in Fig. 1. Cells A and B were made of fused-silica tubing (40 cm x 0.075 mm I.D.). Near the centre of the tubing, a length of about 4 mm of the polyimide resin coated on the surface of the tubing was carefully removed by burning it in a small flame from a micro burner. The transparent part obtained can be used as a flow cell and the other parts as connecting tubing. Cell A was connected between the
MULTIPLE
DETECTION
c~lum
A B
C
SYSTEM
FOR
223
SEC OF PROTEINS
To : column
-
I
.-I
Fig. 1. Construction of micro flow cells for multiple detection. 1, Fused-silica tubing of 0.075 mm I.D. (0.28 mm O.D.); 2, fused-silica tubing of 0.05 mm I.D. (0.24-0.25 mm O.D.): 3, fused-silica tubing of 0.26 mm I.D. (0.36 mm O.D.); 4, flow cell A; 5, flow cell B; 6. flow cell C; 7, window; 8, shield; 9, cell block; bpc, back-pressure column.
end of column A of length 1 m and the top of column B of length 2.5 m. In the same manner, cell B was connected between columns B and C of length 6 m. Cell C was made of fused-silica tubing (12 mm x 0.26 mm I.D.) and a length of about 4 mm of the polyimide resin near the centre of the tubing was also burned off and fused-silica tubing (20 cm x 0.05 mm I.D.) was connected to both ends with epoxy glue for use as connecting tubing. Cell C was connected between column C and the back-pressure column connected at the end of the system. This construction of micro flow cells was designed on the basis of the results of a preliminary test on the pressure that each fused-silica tubing could withstand. In this test, the same length of the polyimide resin coated on the tubing was removed as in the flow cells. The maximum pressures measured are given in Table I and were more than 750 kg/cm2 for each tubing except that of 0.26 mm I.D. connected with that of 0.05 mm I.D. by epoxy glue, for which the maximum pressure was only
TABLE
I
MAXIMUM PRESSURE IMIDE RESIN COATINti Fused-silica
(1) (2) (3) (4)
WITHSTOOD REMOVED
tubing
Maximum measured
0.05 mm I.D. (0.24-0.25 mm O.D.) 0.075 mm I.D. (0.28 mm O.D.) 0.26 mm I.D. (0.36 mm O.D.) Connected tubings** of 0.26 and 0.05 mm I.D.
kg/cm2
* The maximum for this test. * See text.
BY THE FUSED-SILICA TO THE SAME LENGTH
pressure
available
TUBING WITH THE POLYAS IN THE FLOW CELL
pressure {kg,fcm2)
> 750* > 750* > 750* loo-170**
with a Shimazu
LC-5A
pump was temporarily
modified
to 750
224
A. HIROSE, D. ISHIT
kg/cm*, as measured by maintaining the pressure for a few hours until the by epoxy glue became disconnected. The pressures on the cells A and B were predicted as follows. When operating the chromatograph at almost the highest pressure of the present pump system, e.g., 400 kg/cm’, the pressure on the second cell B was calculated to be about 250 kg/cm2 when using the present set of three columns as of length 1, 2.5 and 6 m, and for the first cell A it should be much more. Therefore, the tubing of 0.26 mm I.D. connected with that of 0.05 mm I.D. cannot be used for cells A and B. To overcome this problem of low maximum pressure, however, it is not possible to use the 0.26 mm I.D. tubing directly as both connecting tubing and as a flow cell, because the band broadening in the tubing becomes relatively too large and cannot be neglected. These are the reasons why narrow-bore tubing of 0.075 mm I.D. were used for cells A and B. By setting up the cells in parallel on a cell block as in Fig. 1, it becomes to possible to record the chromatograms three times with one detector and recorder each. However, the problem may arise that the separated solutes may pass through the different cells at the same time and be detected simultaneously or, for a similar reason, the elution order of the solute bands may occasionally be reversed on the resulting chromatogram, making the chromatography unsatisfactory. In size-exclusion chromatography, however, such a problem can easily be overcome by arranging the length of three columns appropriately, as in this work. 100-170
part joined
Comparison of detection sensitivity
Although the difference in detection sensitivity can be calculated from the path lengths of cells A (or B) and C, it is recommended that it is measured experimentally, because the sensitivity was sometimes considerably affected by the arrangement of
Fig. 2. Comparison of the detection sensitivities of flow cells A, B and C.
MULTIPLE
DETECTION
~...~.-_--_--J 0
SYSTEM
FOR
225
SEC OF PROTEINS
6
4 Elation time Ch)
Fig. 3. Application of the multiple detection system to the separation of molecular weight Glutamate dehydrogenase (MW = 290000); 2, lactate dehydrogenase (MW = 142000); (MW = 67000); 4, adenylate kinase (MW = 32000); 5, cytochrome c (MW = 12400).
marker. 1, 3, enolase
the cells on the cell block and therefore, even for cells A and B with the same path length, the sensitivities were not identical. On the other hand, as the separated solute bands are increasingly broadened in the columns, it is better to arrange the three cells in order of increasing sensitivity. An example of the measured sensitivities of cells A, B and C is shown in Fig. 2. In this test, the cells were connected with fused-silica tubing of 0.05 mm I.D. without any columns, and it can be seen that cell C gave about a 4-fold higher sensitivity than cell A with respect to peak height. Also, cell A showed slightly lower sensitivity than B; although this difference was small, it could be measured by changing the order of connection of cells A and B and comparing the results obtained. Based on these results, we concluded that the cells should be connected in the order A, B and C, that is, in order of increasing sensitivity. 1 st
I
,
,
2 nd
3 rd
I
1
0
I
I__-_
12
6 Elutna
Fig. 4. Application
detection
time
(h
of the multiple
1 detection
system
to the separation
of aldolase.
A. HIROSE, D. ISHII
226 1 k-
st
2nd ~~
t
0
detection
3 rd __--+_---_-_____-___--_-_-
----
-
------I
15
10
5
Elutlon time
(h)
Fig. 5. Application of the multiple detection system to the separation of
/hgahCtOSidaSe.
Application to the separation of proteins The multiple detection system was applied to separations of molecular weight marker, aldolase, P-galactosidase and urease samples. The results obtained are shown in Figs. 3-6. In each instance, 220 nm was utilized for detection and flow-rates of 0.65-0.85 $/min, which were almost the optimum values, were applied. The flowrates were obtained by applying a pressure of 300400 kg/cm* to the 9.5 m column using 0.2 M phosphate buffer as the eluent. After detection by cell A or B, if the sample is not so complex, as with aldolase in this work, it seems to be unnecessary to apply such a high-resolution technique and the chromatography can be stopped before completing all the steps and a backflushing method can be used to recondition column C quickly. Figs. 5 and 6 show that protein samples generally form very complex mixtures and therefore a very high-resolution separation technique is required and, even with such a long column as 9.5 m as in this work, in some instance it is still not sufficient. Also the present system is convenient for establishing how high a resolution is required for the separation of a complex sample by comparing the chromatograms obtained under different resolutions.
1st
I
O
,
3 t-d detecton
2nd
I
I
5
10 Elution time
-
I
-I-
15
(h)
Fig. 6. Application of the multiple detection system to the separation of urease.
MULTIPLE
DETECTION
SYSTEM
FOR
SEC OF PROTEINS
227
ACKNOWLEDGEMENT
The authors express their thanks to Nomura Japan) for their support of this work.
Kagaku Kogyo (Seto, Aichi,
REFERENCES
10 11 12 13 14 15 16 17 18 19 20
A. Hirose, D. Wiesler and M. Novotny, Chromotogruphia, 18 (1984) 239. M. Novotny, A. Hirose and D. Wiesler, Anal. Chem.. 56 (1984) 1243. H. G. Menet, P. C. Gareil and R. H. Rosset, Anal. Chem., 56 (1984) 1770. A. Hirose and D. Ishii, J. High Resolut. Chromatogr. Chromatogr. Commun., 9 (1986) 533. R. P. W. Scott and P. Kucera, J. Chromatogr., 169 (1979) 51. P. Kucera and G. Manius, J. Chromatogr., 216 (1981) 9. F. J. Yang, J. Chromatogr., 236 (1982) 265. J. C. Gluckman, A. Hirose, V. L. McGuffin and M. Novotny, Chromatographia, 17 (1983) 303. M. Novotny and D. Ishii (Editor), Microcolumn Separations: Columns, Instrumentation and Ancillary Techniques, Elsevier, Amsterdam, 1985. L. R. Snyder, J W. Dolan and S. van der Wal, J. Chromatogr., 203 (1981) 3. I. Halasz and G. Maldener, Anal. Chem., 55 (1983) 1842. R. P. W. Scott, Small Bore Liquid Chromatography Columns: Their Properties and Uses, Wiley, New York, 1984, p. 139. M. Verzele and C. Dewaele, J. High Resolut. Chromatogr. Chromatogr. Commun., 5 (1982) 245. T. Tsuda, I. Tanaka and G. Nakagawa, J. Chromatogr., 239 (1982) 507. V. L. McGuffin and M. Novotny, J. Chromatogr., 255 (1983) 381. Y. Hirata and K. Jinno, J. High Resolut. Chromatogr. Chromatogr. Commun., 6 (1983) 196. M. Krejei, K. Tesaiik and J. Pajurek, J. Chromatogr., 191 (1980) 17. R. Tijssen, J. P. A. Bleumer, A. L. C. Smit and M. E. Van Kreveld, J. Chromatogr., 218 (1981) 137. P. Kucera and G. Guiochon, J. Chromatogr., 283 (1984) 1. A. Hirose and M. Novotny, unpublished work.