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On resolution in edge-loading zonal rotors
ANALYTICAL
60, 319-321 (1974)
BIOCHEMISTRY
SHORT COMMUNICATIONS
On
Resolution
in Edge-Loading
Zonal
Rotors’
We earlier reported on passive factors affecting resolution in a variety of zonal rotors (2). Since then “edge-loading” has been presented as a new configuration of zonal rotors (1) with many advantages over the conventional mode of loading and unloading only through the center. Anderson et al. (1) reported that resolution, measured as passive zone broadening, was as good when samples were loaded through the edge as through the center. In the course of some routine separations involving edge unloading in the B-30 rotor, we obtained unexpectedly large zone widths. I decided therefore to investigate passive factors in this and other edge-unloading rotors. The rotors tested were the B-XXIX, identical to that described by Anderson et al. (1)) and the B-30, an edge-loading analog of the B-XIV. The. rotors and .the B-35 and B-60 drive units were all manufactured by the International Equipment Company. The procedures for testing for passive factors affecting resolution were as described previously (2). The only difference here is that sample zones are treated as Gaussian distributions and the zone width expressed as 20, which is the width in cc at E-G of the maximum peak height = 0.606h. This is justified in the case of center unloading, but zones unloaded from the edge are typically skewed towards the center of the rotor. First, I measured the zone width in the B-XXIX and B-30 following loading ‘and unloading from the center of samples of. various sizes (Table 1). It is evident that passive factors are negligible in sample volumes down to 5 ml and that resolution in center unloading is completely comparable to previous measurements by conventional center loading (2). I then unloaded an identical set of samples at the edge and found values of 2~ up to three times larger for the smallest sample volumes (Table 1). The discrepancy between my measurements and those of ‘Supported
in
part
by
a grant
&330026X)
(No.
from
the
National
Sciencf
Foundation..Jouimalseries paper of the New Jersey Agricultural Experiment Station, Department of Biochemistry and Microbiology, New Brunswick, New Jersey, 319 Copyright. All rights
0 1974 by Academic Press,, Inc. of reproduction in any form reserved.
320
SHORT
Resolution
COMMUNICATIONS
TABLE 1 of Various Sample Volumes When Unloaded at the Center and Edge of the B-XXIX and B-30A Rotors0 Recovered zone width
Initial
zone volume (cc) B-XXIX 5 10
20 30 40
B-30A 5 10
20 30 40
(Av
in cc at 0.606 peak height)
Unloaded at center
Unloaded at edge
6.3 9.4 18.3 30.5 42.9
27.8 22.0 45.0 44.9 49.6
4.72 9.01 18.90 26.58 35.43
15.65
16.24 23.62 29.82 35.43
o Samples of 0.2% w/v Blue Dextran were insertsed within a sucrose gradient of 0.2 mg . cc-* and loaded at, the center. The pumping rate was 30 cc. mm-l, and the loading speed was 2000 rpm. The samples were then unloaded immediately elther at the center or at the edge.
Anderson et al. concerns more the results at the center than at the edge. When they loaded and unloaded 20-0~ sample volumes through the center, they observed zone broadening to approximately 40 cc, whereas our zones remained about 20 cc. Upon unloading simple samples from the edge, the volume was found to be about 40 cc, which is about what I found for samples of this size. Two causes contribute to the discrepancy: (a) our system of maintaining a constant tubing diameter of 2 mm yields smaller values of 20 when loading from the center, and (b) the most striking differences we observe between center and edge loading occur with sample volumes that are smaller than those employed by Anderson et al. TABLE 2 Resolution at. Various Pumping Speeds. Recovered zone width Pumping speed (co/mm)
Center
10
6.79 6.35
30 60
7.97
(AV
in cc at 0.606 peak height) Edge 22.74 20.23 17.86
0 Conditions as in Table 1, except that sample volumes were 5 cc throughout loaded and unloaded entirely at, t~he center or entirely at the edge.
and either
SHORT
-
321
COMMUNICATIONS
Loss of Resolution
TABLE 3 at the Edge During Loading
Direction of pumping
Recovered zone width
Compared to Unload(AV
in cc at 0.606 peak height)
(a) renter to edge
15.06 12.25
(b) edge to center
19.93 19.34 17.42
a Conditions as in Table 1, except that 5 cc samples, were either (a) loaded at the center and recovered at the edge or (b) loaded at the edge and recovered at the center. Pumping rate was 60 cc/min.
The problem of the loss of resolution during edge unloading remains. It occurred to me that this loss might be due to the inversion of density that must occur in any liquid column in which a heavy solution is displaced upwards (i.e., centripetally) by a lighter volume. If this was the cause, it should be minimized the more rapidly one unloads. This indeed appears to be the case (Table 2)) since recovered zone width is significantly better at 60 cc/min than at 30 or 10 cc/min. We had inferred from our 1969 studies on resolution that the principal loss of resolution occurs upon loading, not during unloading. That this is also the case for edge unloading is clearly shown by passing samples through the rotor in either direction (Table 3). In Conclusion, the undoubted and in some cases unique advantages of edge unloading must be balanced against substantially poorer resolution of small zones (20 cc and less). Resolution through the edge can, however, be maximized by rapid unloading. ACKNOWLEDGMENT I am grateful for the expert technical assistance of M. Del&raz. REFERENCES 1. ANDERSON,
N. G., NUNLEY,
C.
E., AND RANKIN,
C. T., JR. (1969). Anal.
B&hem.
31, 255. 2. PRICE,
C. A., AND Kovacs,
A. (1969)
Anal.
B&hem.
28, 460.
C. A. Particle
Separation
Facility
Department of Biochemistry Rutgers University New Brunswick, New Jersey Received March $0, 1973;
and
Microbiology
08903 accepted
February
8, 19Y4
PRICE
60, 319-321 (1974)
BIOCHEMISTRY
SHORT COMMUNICATIONS
On
Resolution
in Edge-Loading
Zonal
Rotors’
We earlier reported on passive factors affecting resolution in a variety of zonal rotors (2). Since then “edge-loading” has been presented as a new configuration of zonal rotors (1) with many advantages over the conventional mode of loading and unloading only through the center. Anderson et al. (1) reported that resolution, measured as passive zone broadening, was as good when samples were loaded through the edge as through the center. In the course of some routine separations involving edge unloading in the B-30 rotor, we obtained unexpectedly large zone widths. I decided therefore to investigate passive factors in this and other edge-unloading rotors. The rotors tested were the B-XXIX, identical to that described by Anderson et al. (1)) and the B-30, an edge-loading analog of the B-XIV. The. rotors and .the B-35 and B-60 drive units were all manufactured by the International Equipment Company. The procedures for testing for passive factors affecting resolution were as described previously (2). The only difference here is that sample zones are treated as Gaussian distributions and the zone width expressed as 20, which is the width in cc at E-G of the maximum peak height = 0.606h. This is justified in the case of center unloading, but zones unloaded from the edge are typically skewed towards the center of the rotor. First, I measured the zone width in the B-XXIX and B-30 following loading ‘and unloading from the center of samples of. various sizes (Table 1). It is evident that passive factors are negligible in sample volumes down to 5 ml and that resolution in center unloading is completely comparable to previous measurements by conventional center loading (2). I then unloaded an identical set of samples at the edge and found values of 2~ up to three times larger for the smallest sample volumes (Table 1). The discrepancy between my measurements and those of ‘Supported
in
part
by
a grant
&330026X)
(No.
from
the
National
Sciencf
Foundation..Jouimalseries paper of the New Jersey Agricultural Experiment Station, Department of Biochemistry and Microbiology, New Brunswick, New Jersey, 319 Copyright. All rights
0 1974 by Academic Press,, Inc. of reproduction in any form reserved.
320
SHORT
Resolution
COMMUNICATIONS
TABLE 1 of Various Sample Volumes When Unloaded at the Center and Edge of the B-XXIX and B-30A Rotors0 Recovered zone width
Initial
zone volume (cc) B-XXIX 5 10
20 30 40
B-30A 5 10
20 30 40
(Av
in cc at 0.606 peak height)
Unloaded at center
Unloaded at edge
6.3 9.4 18.3 30.5 42.9
27.8 22.0 45.0 44.9 49.6
4.72 9.01 18.90 26.58 35.43
15.65
16.24 23.62 29.82 35.43
o Samples of 0.2% w/v Blue Dextran were insertsed within a sucrose gradient of 0.2 mg . cc-* and loaded at, the center. The pumping rate was 30 cc. mm-l, and the loading speed was 2000 rpm. The samples were then unloaded immediately elther at the center or at the edge.
Anderson et al. concerns more the results at the center than at the edge. When they loaded and unloaded 20-0~ sample volumes through the center, they observed zone broadening to approximately 40 cc, whereas our zones remained about 20 cc. Upon unloading simple samples from the edge, the volume was found to be about 40 cc, which is about what I found for samples of this size. Two causes contribute to the discrepancy: (a) our system of maintaining a constant tubing diameter of 2 mm yields smaller values of 20 when loading from the center, and (b) the most striking differences we observe between center and edge loading occur with sample volumes that are smaller than those employed by Anderson et al. TABLE 2 Resolution at. Various Pumping Speeds. Recovered zone width Pumping speed (co/mm)
Center
10
6.79 6.35
30 60
7.97
(AV
in cc at 0.606 peak height) Edge 22.74 20.23 17.86
0 Conditions as in Table 1, except that sample volumes were 5 cc throughout loaded and unloaded entirely at, t~he center or entirely at the edge.
and either
SHORT
-
321
COMMUNICATIONS
Loss of Resolution
TABLE 3 at the Edge During Loading
Direction of pumping
Recovered zone width
Compared to Unload(AV
in cc at 0.606 peak height)
(a) renter to edge
15.06 12.25
(b) edge to center
19.93 19.34 17.42
a Conditions as in Table 1, except that 5 cc samples, were either (a) loaded at the center and recovered at the edge or (b) loaded at the edge and recovered at the center. Pumping rate was 60 cc/min.
The problem of the loss of resolution during edge unloading remains. It occurred to me that this loss might be due to the inversion of density that must occur in any liquid column in which a heavy solution is displaced upwards (i.e., centripetally) by a lighter volume. If this was the cause, it should be minimized the more rapidly one unloads. This indeed appears to be the case (Table 2)) since recovered zone width is significantly better at 60 cc/min than at 30 or 10 cc/min. We had inferred from our 1969 studies on resolution that the principal loss of resolution occurs upon loading, not during unloading. That this is also the case for edge unloading is clearly shown by passing samples through the rotor in either direction (Table 3). In Conclusion, the undoubted and in some cases unique advantages of edge unloading must be balanced against substantially poorer resolution of small zones (20 cc and less). Resolution through the edge can, however, be maximized by rapid unloading. ACKNOWLEDGMENT I am grateful for the expert technical assistance of M. Del&raz. REFERENCES 1. ANDERSON,
N. G., NUNLEY,
C.
E., AND RANKIN,
C. T., JR. (1969). Anal.
B&hem.
31, 255. 2. PRICE,
C. A., AND Kovacs,
A. (1969)
Anal.
B&hem.
28, 460.
C. A. Particle
Separation
Facility
Department of Biochemistry Rutgers University New Brunswick, New Jersey Received March $0, 1973;
and
Microbiology
08903 accepted
February
8, 19Y4
PRICE