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HETP values of large-scale columns for liquid chromatography
[j. Ferment. Technol., Vol. 65, No. 1,111-113. 1987]
Note
HETP Values of Large-Scale Columns for LiquidChromatography EIZO SADA, SHIGEO KATOH, AKIHIKO KONDO, a n d SEIjI ISHIDA* Chemical Engineering Department, Kyoto University, Kyoto 606, dapan ; *Snow Brand Milk Products Co. Ltd., Tochigi, Japan
Two columns of 100 mm and 200 mm in diameter for bench-scale liquid chromatography were designed to obtain a small dead volume and an appropriately uniform distribution of liquid, and the values of the height equivalent to a theoretical plate were measured for three kinds of packing beads, Sepharose 4B, Cellulofine GC-700 m and Silica. A suitable pressure drop through a distribution plate and/or a packed bed helps to distribute liquid flow uniformly over the column diameter, and reasonable HETP values were obtained.
With the development of bioindustries, chromatographic methods have become w i d e l y used in l a r g e - s c a l e p u r i f i c a t i o n for a variety of biological materials. In large columns, a nonuniform distribution of liquid fl0w, w h i c h m i g h t d e c r e a s e t h e d e g r e e o f resolution, should be avoided through the suitable design of column end-pieces. An efficient d i s t r i b u t i o n o f l i q u i d f l o w o v e r t h e column diameter and a small dead volume of l i q u i d a t t h e i n l e t a r e c r i t i c a l factors. T a k i n g i n t o a c c o u n t these factors, w e d e s i g n e d t w o c o l u m n s o f 100 m m a n d 200 m m i n d i a m e t e r a n d d e t e r m i n e d t h e h e i g h t e q u i v a l e n t to a t h e o r e t i c a l p l a t e ( H E T P ) o f these c o l u m n s p a c k e d w i t h t h r e e k i n d s o f b e a d s (soft gel to h a r d silica) b y t h e i m p u l s e m e t h o d w i t h NaCI and albumin.
Experimental Methods C o l u n m design Figure 1 shows a vertical section of the columns used. Liquid flow is radially distributed through six openings of 1.0 mm in diameter on the wall of a dead-end feeder pipe, which interrupts the axial momentum of liquid flow. A distribution plate, made ofsintered glass plate (mesh no. 2 and no. 4, 7 mm thick, average pore size 40-50 #m and 5-10/~m, respectively), is mounted in a tapered end-plate fixed to the feeder pipe. The height of the distribution plate
can be adjusted to 2-3 mm above the packed bed to reduce the liquid dead volume at the inlet. A small pipe fitted with a stopcock passes through this end-plate to drain liquid. A bottom plate, made of sintered glass plate no. 2, was mounted in another tapered end-plate of similar construction. Materials Three kinds of packing beads were used. Sepharose 4B (Pharmacia Fine Chemicals, diameter 60-140 #m) is a soft, deformable gel which is widely used as an affinity chromatography medium. The rate of liquid flow through a Sepharose bed is limited by deformation of the gel beads caused by the pressure drop through the bed and reaches a plateau at about 0.2 kg/cm~, x) Silica (Merck, average size 63-200/~m, nonspherical) is non-deformable, and the pressure drop through the bed is low. Cellulofine GC-700 m (Chisso Co., diameter 45-105 #m) is made of cellulose and used for gel chromatography. The pressure drop through the bed is relatively high but shows a linear relationship to the liquid flow rate over a wide range. Its properties are intermediate between those of Sepharose and Silica. Chemicals used were of reagent grade. Methods A slurry of packing beads suspended in a buffer solution (20 mM phosphate + 150mM NaCI, pH 7.6) was introduced into the column under mild stirring. After sedimentation the distribution plate was lowered to 2-3 mm above the bed at a constant liquid flow rate. For a deformable gel, this has to be readjusted for different flow rates. An impulse of NaC1 solution (2 M) or albumin solution (2 mg/cm a)
112
SADAet al.
I
I
feeder
[J. Ferment. Technol., O.E
i
[ ~ OA
U
? u
I-"' 0.~ "r-
i,i
l_ Ihl II1
--//sOll--o 2'0
superficial
oO-~--"o-/-+0 60 liquid velocity ( c m l h )
Fig. 2. HETP of Cellulofine GC-700 m packed in columns (100 mm and 200 mm i.d.). Fig. 1. Vertical section of column (diameter, 100 or 200 mm). was introduced into the column, and the response was measured by use of a conductivity meter (M & S Iustru. Co., CD-35 MII) or a spectrophotometer (Shimadzu UV-140). On the basis of the plate theory, 2) the HETP was calculated from the bandwidth W of the impulse-response curve a t a height of the peak height multiplied by e-x/s.
L
HETP-4(tR/W)~ where tR is the retention time and L is the height of the
bed. All experiments were carried out at room temperature (23±3°C). Results
and Discussion
Figure 2 shows the H E T P values of Cellulofine GC-700 m packed in the 100 m m i.d. column (bed height 12.5 cm) a n d the 200 m m i.d. c o l u m n (bed height 5.2 era) with the no. 2 distribution plate. T h e H E T P values for a l b u m i n increased linearly with the superficial liquid velocity u, showing a relationship similar to the van D e e m t e r equation without molecular diffusion effect.m HETP=A+Bu T h e values for N a C I showed no appreciable change for different superficial liquid velocities because the molecular diffusion o f NaC1 is two orders of m a g n i t u d e higher. T h e
column NaCl albumin o zx 1oo ¢ 4, (bed* 30ram liq.) 200 • •
value of the intercept is nearly equal to twice of the average diameter of the beads, which is the ideal value for packed beds.4) This indicates a uniform flow distribution over the column diameter and no appreciable dispersion at the inlet. W h e n a 30-mm liquid layer was kept on the top of the bed by elevating the distribution plate, the H E T P values increased because of dispersion in this layer, indicating that a small dead volume at the inlet is critical. T h e H E T P values for the 2 0 0 m m i.d. column were equal to those for the 100 m m i.d. column. T h e liquid flow is well distributed also in this large column. W i t h the soft gel (Sepharose 4B, bed height ca. 15.5 cm), the H E T P values for NaC1 were independent of the superficial liquid velocity and equal to the ideal value. But the H E T P values of Silica (bed height 14.7 cm) packed in the 100 m m i.d. column with the no. 2 distribution plate were larger, as shown in Fig. 3. This implies n o n u n i f o r m distribution of liquid, although the slightly larger diameter and irregular shape of the beads m i g h t have some effect. T h e pressure
Vol. 65, 1987]
HETP Values of Large-Scale Column Chromatography
113
2
112--.
"E iJ v v
A
E
u v
,',
A
0.1
I-ILl "1"
A
A A
• 0.1
A
A •
• 0
~
~ 0 -'~
0
O []
0
O
A
A
20
Superficial
liquid
-~-Q-I
I
40
60
80
0
velocity (cmlh)
Fig. 3. HETP of Sepharose 4B and Silica for NaC1 and pressure drop through distribution plate no. 4 (average pore size 5-10 Fro). column [] A © •
Sepharose 4]3 Silica Silica Silica
drop through this bed is low (less than 0.03 kg/cm2 at 60 cm/h) compared with other two. To compensate for this low pressure drop the no. 4 distribution plate, the pressure drop of which is shown in Fig. 3, was substituted for the no. 2 plate, which shows no appreciable pressure drop. The H E T P values of Silica in the 100-mm column (bed height 14.9 cm) and the 200-mm column (bed height 4.7 cm) were improved. The pressure drop through the plate helps to distribute liquid uniformly over the column in a similar manner to the pressure drop of the soft gel bed. A suitable pressure drop through the distributor and/or the bed is also critical for the uniform distribution of liquid. Thus the design of the inlet to minimize the dead volume of liquid and to distribute liquid uniformly allows chromatographic columns to be scaled up to 2 0 0 m m i.d.
100 100 I00 200
sinteredglass no. no. no. no.
2 2 4 4
without additional spreading of chromatographic bands. Acknowledgment
The present research was supported by a Grant-inAid for Developmental Scientific Research by the Ministry of Education, Science and Culture of Japan. References
1) Sada, E., Katoh, S., Shiozawa, S.: Biotechnol. Bioeng., 24, 2279 (1982). 2) Hamilton, P.B., Bogue, D. C., Anderson, R.A.: Anal. Chem., 32, 1782 (1960). 3) van Deemter, J.J., Zuiderweg, F.J., Klinkenberg, A.: Chem.Eng. Sci.,5,271 (1956). 4) Nakanishi, K., Yamamoto, S., Matsuno, R., Kamikubo, T.: Agric. Biol. Chem., 41, 1465 (1977). (Received August 28, 1986)
Note
HETP Values of Large-Scale Columns for LiquidChromatography EIZO SADA, SHIGEO KATOH, AKIHIKO KONDO, a n d SEIjI ISHIDA* Chemical Engineering Department, Kyoto University, Kyoto 606, dapan ; *Snow Brand Milk Products Co. Ltd., Tochigi, Japan
Two columns of 100 mm and 200 mm in diameter for bench-scale liquid chromatography were designed to obtain a small dead volume and an appropriately uniform distribution of liquid, and the values of the height equivalent to a theoretical plate were measured for three kinds of packing beads, Sepharose 4B, Cellulofine GC-700 m and Silica. A suitable pressure drop through a distribution plate and/or a packed bed helps to distribute liquid flow uniformly over the column diameter, and reasonable HETP values were obtained.
With the development of bioindustries, chromatographic methods have become w i d e l y used in l a r g e - s c a l e p u r i f i c a t i o n for a variety of biological materials. In large columns, a nonuniform distribution of liquid fl0w, w h i c h m i g h t d e c r e a s e t h e d e g r e e o f resolution, should be avoided through the suitable design of column end-pieces. An efficient d i s t r i b u t i o n o f l i q u i d f l o w o v e r t h e column diameter and a small dead volume of l i q u i d a t t h e i n l e t a r e c r i t i c a l factors. T a k i n g i n t o a c c o u n t these factors, w e d e s i g n e d t w o c o l u m n s o f 100 m m a n d 200 m m i n d i a m e t e r a n d d e t e r m i n e d t h e h e i g h t e q u i v a l e n t to a t h e o r e t i c a l p l a t e ( H E T P ) o f these c o l u m n s p a c k e d w i t h t h r e e k i n d s o f b e a d s (soft gel to h a r d silica) b y t h e i m p u l s e m e t h o d w i t h NaCI and albumin.
Experimental Methods C o l u n m design Figure 1 shows a vertical section of the columns used. Liquid flow is radially distributed through six openings of 1.0 mm in diameter on the wall of a dead-end feeder pipe, which interrupts the axial momentum of liquid flow. A distribution plate, made ofsintered glass plate (mesh no. 2 and no. 4, 7 mm thick, average pore size 40-50 #m and 5-10/~m, respectively), is mounted in a tapered end-plate fixed to the feeder pipe. The height of the distribution plate
can be adjusted to 2-3 mm above the packed bed to reduce the liquid dead volume at the inlet. A small pipe fitted with a stopcock passes through this end-plate to drain liquid. A bottom plate, made of sintered glass plate no. 2, was mounted in another tapered end-plate of similar construction. Materials Three kinds of packing beads were used. Sepharose 4B (Pharmacia Fine Chemicals, diameter 60-140 #m) is a soft, deformable gel which is widely used as an affinity chromatography medium. The rate of liquid flow through a Sepharose bed is limited by deformation of the gel beads caused by the pressure drop through the bed and reaches a plateau at about 0.2 kg/cm~, x) Silica (Merck, average size 63-200/~m, nonspherical) is non-deformable, and the pressure drop through the bed is low. Cellulofine GC-700 m (Chisso Co., diameter 45-105 #m) is made of cellulose and used for gel chromatography. The pressure drop through the bed is relatively high but shows a linear relationship to the liquid flow rate over a wide range. Its properties are intermediate between those of Sepharose and Silica. Chemicals used were of reagent grade. Methods A slurry of packing beads suspended in a buffer solution (20 mM phosphate + 150mM NaCI, pH 7.6) was introduced into the column under mild stirring. After sedimentation the distribution plate was lowered to 2-3 mm above the bed at a constant liquid flow rate. For a deformable gel, this has to be readjusted for different flow rates. An impulse of NaC1 solution (2 M) or albumin solution (2 mg/cm a)
112
SADAet al.
I
I
feeder
[J. Ferment. Technol., O.E
i
[ ~ OA
U
? u
I-"' 0.~ "r-
i,i
l_ Ihl II1
--//sOll--o 2'0
superficial
oO-~--"o-/-+0 60 liquid velocity ( c m l h )
Fig. 2. HETP of Cellulofine GC-700 m packed in columns (100 mm and 200 mm i.d.). Fig. 1. Vertical section of column (diameter, 100 or 200 mm). was introduced into the column, and the response was measured by use of a conductivity meter (M & S Iustru. Co., CD-35 MII) or a spectrophotometer (Shimadzu UV-140). On the basis of the plate theory, 2) the HETP was calculated from the bandwidth W of the impulse-response curve a t a height of the peak height multiplied by e-x/s.
L
HETP-4(tR/W)~ where tR is the retention time and L is the height of the
bed. All experiments were carried out at room temperature (23±3°C). Results
and Discussion
Figure 2 shows the H E T P values of Cellulofine GC-700 m packed in the 100 m m i.d. column (bed height 12.5 cm) a n d the 200 m m i.d. c o l u m n (bed height 5.2 era) with the no. 2 distribution plate. T h e H E T P values for a l b u m i n increased linearly with the superficial liquid velocity u, showing a relationship similar to the van D e e m t e r equation without molecular diffusion effect.m HETP=A+Bu T h e values for N a C I showed no appreciable change for different superficial liquid velocities because the molecular diffusion o f NaC1 is two orders of m a g n i t u d e higher. T h e
column NaCl albumin o zx 1oo ¢ 4, (bed* 30ram liq.) 200 • •
value of the intercept is nearly equal to twice of the average diameter of the beads, which is the ideal value for packed beds.4) This indicates a uniform flow distribution over the column diameter and no appreciable dispersion at the inlet. W h e n a 30-mm liquid layer was kept on the top of the bed by elevating the distribution plate, the H E T P values increased because of dispersion in this layer, indicating that a small dead volume at the inlet is critical. T h e H E T P values for the 2 0 0 m m i.d. column were equal to those for the 100 m m i.d. column. T h e liquid flow is well distributed also in this large column. W i t h the soft gel (Sepharose 4B, bed height ca. 15.5 cm), the H E T P values for NaC1 were independent of the superficial liquid velocity and equal to the ideal value. But the H E T P values of Silica (bed height 14.7 cm) packed in the 100 m m i.d. column with the no. 2 distribution plate were larger, as shown in Fig. 3. This implies n o n u n i f o r m distribution of liquid, although the slightly larger diameter and irregular shape of the beads m i g h t have some effect. T h e pressure
Vol. 65, 1987]
HETP Values of Large-Scale Column Chromatography
113
2
112--.
"E iJ v v
A
E
u v
,',
A
0.1
I-ILl "1"
A
A A
• 0.1
A
A •
• 0
~
~ 0 -'~
0
O []
0
O
A
A
20
Superficial
liquid
-~-Q-I
I
40
60
80
0
velocity (cmlh)
Fig. 3. HETP of Sepharose 4B and Silica for NaC1 and pressure drop through distribution plate no. 4 (average pore size 5-10 Fro). column [] A © •
Sepharose 4]3 Silica Silica Silica
drop through this bed is low (less than 0.03 kg/cm2 at 60 cm/h) compared with other two. To compensate for this low pressure drop the no. 4 distribution plate, the pressure drop of which is shown in Fig. 3, was substituted for the no. 2 plate, which shows no appreciable pressure drop. The H E T P values of Silica in the 100-mm column (bed height 14.9 cm) and the 200-mm column (bed height 4.7 cm) were improved. The pressure drop through the plate helps to distribute liquid uniformly over the column in a similar manner to the pressure drop of the soft gel bed. A suitable pressure drop through the distributor and/or the bed is also critical for the uniform distribution of liquid. Thus the design of the inlet to minimize the dead volume of liquid and to distribute liquid uniformly allows chromatographic columns to be scaled up to 2 0 0 m m i.d.
100 100 I00 200
sinteredglass no. no. no. no.
2 2 4 4
without additional spreading of chromatographic bands. Acknowledgment
The present research was supported by a Grant-inAid for Developmental Scientific Research by the Ministry of Education, Science and Culture of Japan. References
1) Sada, E., Katoh, S., Shiozawa, S.: Biotechnol. Bioeng., 24, 2279 (1982). 2) Hamilton, P.B., Bogue, D. C., Anderson, R.A.: Anal. Chem., 32, 1782 (1960). 3) van Deemter, J.J., Zuiderweg, F.J., Klinkenberg, A.: Chem.Eng. Sci.,5,271 (1956). 4) Nakanishi, K., Yamamoto, S., Matsuno, R., Kamikubo, T.: Agric. Biol. Chem., 41, 1465 (1977). (Received August 28, 1986)