CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
PARALLEL PRODUCTION OF GRADIENTS
tl
IN VERTICAL ROTORS
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Norbert Karl Herzog
August, 1979
The Thesis of Norbert Karl Herzog is approved:
St~ven B. Oppenheimer, Ph.D.
Advisor
Phillip Sheeler, Ph.D.
Committee Chairman
California State University, Northridge
ii
ACKNOWLEDGMENTS
I wish to express my deepest appreciation to my
committee chairman, Dr. Phillip Sheeler.
His generosity
in giving of his time, knowledge and support has made
this thesis possible.
I feel very fortunate to have had
the opportunity to work under such an excellent teacher.
I would like to thank Dr. Kenneth C. Jones and
Dr. Steven B. Oppenheimer for serving on my graduate
committee.
I also would like to thank Harry R. White,
whose technical skills and advice were invaluable.
A
special thanks is extended to Mark Doolittle for his
help and friendship.
I would like to thank my parents for all their
support and faith, without which it would all have been
so much more difficult.
Finally, to Jean Talmage, my fiancee, a special
thanks for typing this thesis and for all her love and
encouragement.
iii
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS
iii
LIST OF FIGURES
vii
ABSTRACT
I.
ix
INTRODUCTION
1
History of Centrifugation
2
First uses of centrifugation
2
Early preparative rotors
4
Differential and density
gradient centrifugation
7
Reorienting gradients
8
Zonal rotors
11
Vertical tube rotors
13
Density Gradients:
Theory and Production
13
Step gradients
13
Continuous gradients
14
Linear gradients
17
Exponential gradients
21
Complex gradients . .
23
iv
Page
Multiple gradients
24
25
Factors Affecting Resolution
Wall effects
25
Convection
26
Swirling
.
26
'
Gradient recovery
II.
'
.
ANALYSIS OF SEVERAL METHODS
FOR THE PRODUCTION OF
MULTIPLE DENSITY GRADIENTS
27
.
..
Gradients Produced Individually
.
29
29
Gradient Maker A
29
Gradient Maker B
30
Simultaneous Production of
Multiple Gradients
III.
.
30
Static manifold .
30
Rotating manifold
30
ROTOR DESCRIPTION, OPERATION
AND PERFORMANCE . . .
40
Lucite Vertical Rotor
40
..
Description
40
Operation •
43
Performance
50
Tests using erythrocytes
50
Tests using Percell density beads
51
Gradient reorientation
56
Modification I of the SV-288
Vertical Tube Rotor . . . .
v
59
Page
Description .
59
Performance
60
Modification II of the
SV-288 Vertical Tube Rotor
IV,
69
Description . . .
69
Operation without tubes
74
Operation with
modified centrifuge tubes
79
Performance . ,
79
Static tests
79
Dynamic tests
82
82
CONCLUSIONS
99
REFERENCES
vi
LIST OF FIGURES
Figure
1.
Page
Reorientation in the
lucite vertical rotor
10
2.
Step gradients
16
3.
Gradient makers
19
4.
Results of gradients
produced individually I . . . . . . . . . .
32
Results of gradients
produced individually II
34
5.
•
•
•
"!
"'
•
•
•
•
6'
Gradients produced using a
static distributor . . .
36
7.
Gradients produced using a
rotating distributor
39
Assembled lucite vertical rotor
and loading apparatus . . .
42
Components of lucite vertical rotor
and loading apparatus . . . . .
45
10.
Components of the lucite vertical rotor
47
11.
Assembled lucite vertical rotor .
49
12.
Sample zones containing
two populations of erythrocytes
53
Separated populations of erythrocytes
following centrifugation . . . . .
55
Isopycnic banding of
Percell density beads . .
59
8,
9.
13.
14,
vii
Figure
Page
Loading and sealing caps for the
modified SV-288 rotor . . . . .
62
Cross section of modified SV-288 rotor
and plunger system
. . . . . . .
64
Modified SV-288 rotor
with loading caps .
66
Modified SV-288 rotor
with cap assemblies
68
Redesigned cap assemblies
and other components
71
20.
Cross section of the SV-288 rotor
and redesigned loading apparatus
73
21.
Loading assembly for the
modified SV-288 rotor .
76
22,
Fully assembled apparatus
78
23~
Modified centrifuge tubes
81
24.
Gradients produced in the
modified SV-288 rotor .
15.
16.
17.
18.
1q,
25,
26.
•
•
~
•
"!
~
84
•
Recovery of gradients and sample zones
formed in the modified SV-288 rotor
Distribution of hemoglobin
as a function of fraction number
86
. .
.
27 ·.
Distribution of hemoglobin
as a function of sucrose concentration
28 ..
Modified SV-288 rotor
showing various modes of use
viii
. .
. .
89
91
. .
97
ABSTRACT
PARALLEL PRODUCTION OF GRADIENTS
IN VERTICAL ROTORS
by
Norbert Karl Herzog
Master of Science in Biology
Despite the popularity of tube-format density
gradient centrifugation and the increase in the numbers of
tubes that may be accommodated by the newer rotors available, the problem still persists of efficiently producing
identical density gradients for each tube position.
This
problem has been compounded with the emergence of "vertical
tube rotors" - the most recent development and innovation
in centrifugal methodology.
A number of solutions have
been proposed to attack the problem of multiple gradient
production and several of these are examined and evaluated
in this work.
Most successful and particularly useful is
an approach in which a system of plungers incorporated
into the design of a vertical tube rotor is used to appor-
ix
tiona single stream of gradient among the rotor's chambers.
A similar approach is taken to empty (either in
concert or in sequence) the gradient (and separated particles) when centrifugation is completed.
The apparatus
produces gradients in short order and with exceptional
uniformity.
Rotor performance was analyzed using bio-
logical and model test systems.
X
I
INTRODUCTION
The physiology of the cell cannot be fully
understood unless we succeed in determining the
constitution of its parts, and the relation
which undoubtedly exists between its morphology
and the distribution of its biochemical function
(Claude, 1946).
Biologists attempting to study the structural
entities which comprise a living cell were only able to
speculate as to their functions and properties before the
advent of fractionation techniques.
What better way to
study the highly organized machinery of a cell than to
progressively disassemble it as one would a machine and
study the components individually (Bernard, 1865).
Only
when the structures and functions of these individual
elements of a cell are understood can we then hope to
understand the cell as a unit.
However, to dismantle a
cell and not alter or destroy the internal structures is
a most difficult task.
Great care must be exercised to
maintain the structures as close to the living state as
possible.
Procedures to dismantle a tissue or cells have
been extensively reviewed (Claude, 1946a; Dounce et al.,
1
2
1955; Greenbaum et al., 1960; DeDuve, 1964).
The product
of a dismantling procedure is a mixture of particles which
can yield little more information than could the intact
cell.
The ability to separate cellular components or
other complex mixtures of particles from one another
depends upon a difference in some physical properties
between the particles.
Separation techniques exploit
differences in the size, shape, density, electrical charge,
or solvent affinity of particles (Sheeler, in preparation).
However, the requirement of maintaining the particle
structure or properties severely limits which separation
technique can be applied to a given problem
Of all the
various separation techniques developed, centrifugation
is probably the most widely applicable, permitting the
separation of particles ranging from proteins to whole
cells,
A.
History of Centrifugation
1.
First uses of centrifugation.
The application
of centrifugation in biological investigations can be
traced as far back as 1806, when T.A. Knight published his
work on the effects of gravitational forces on germinating
seedlings.
However, during the early 1800's centrifuga-
tion was most commonly employed by chemists in the collection of precipitates.
Advances leading to improved optics
3
for light microscopes occurred around 1865
1
permitting
cytologists to observe the internal structure of cells in
greater detail than ever before.
Efforts to characterize
the cellular inclusions included attempts to determine
their densities by subjecting whole cells to a centrifugal
force
1
thereby stratifying the inclusions within the cell
(Mottier, 1899).
However, the 19th Century did not wit-
ness the use of centrifugal techniques in the isolation or
separation of cellular inclusions (Sheeler, in preparation).
In 1902, A.E. Wright employed centrifugal methods
in the separation of white blood cells from the more predominant red blood cells.
This probably represents the
first separation of cells by centrifugal means.
Such
techniques, in which particles are separated from one
another and/or are collected to facilitate their study,
are now termed preparative techniques.
While this is the
primary purpose behind these preparative techniques, they
may also provide some information about the particles.
Despite the interest in cells and their inclusions,
much of the impetus for the continued development of centrifugal technology stemmed from biochemical research on
proteins or colloids.
A controversy surrounded the ques-
tion of the polydispersity of a single protein species.
Attempts were made to resolve this question by subjecting
colloidal solutions to a centrifugal force and observing
the sedimenting particles, thereby allowing the calcula-
4
tion of various physical properties of these particles.
Such centrifugal techniques and the equipment involved in
their use are termed analytical.
Early experiments apply-
ing analytical centrifugation proved unsuccessful due to
the unrecognized phenomenon of convection, related to the
design of the equipment.
Among those scientists involved in colloidal
research was The Svedberg, who in 1923 together with
H. Rinde built the first ultracentrifuge in which convectional artifacts were eliminated.
This technological
advance allowed Svedberg to do extensive pioneering work
on colloids, and in 1926 he was awarded a Nobel Prize.
Svedberg continued to work with analytical centrifuges
until 1940, developing drive systems using electric motors
and oil turbines.
2.
Early preparative rotors.
A number of other
research groups were involved in the development of centrifugation equipment.
In 1925, Henriot and Huguenard intro-
duced an air-turbine drive for the ultracentrifuge.
Though only small volumes of material could be processed,
very high speeds could be attained (Beams, 1938), permitting the study of particles having fairly small size
and mass.
These air driven ultracentrifuges did not in-
clude evacuated chambers surrounding the rotor.
Air
friction limited the speed of the rotor and made it dif-
5
ficult to maintain a constant temperature within the rotor.
Temperature fluctuations induce thermal convection within
the contents of the rotor, decreasing the potential resolution (Beams, 1938),
Though the phenomenon of thermal
convection was recognized at that time, it was only
qualitatively understood.
In the mid-1930's, the air-driven, vacuum-type
centrifuge was introduced (Beams and Pickels, 1935; Bauer
and Pickels, 1936; Beams et al., 1938); a vacuum chamber
surrounded the rotor, permitting greater rotational
spee.d.
In these centrifuges, larger rotors could be
employed, allowing larger samples and a greater variety
of applications.
The first fixed angle rotors were built
by J.H. Bauer and E.G. Pickels for processing largeamounts
of material in the air driven, vacuum type centrifuge.
The sample compartments of a fixed angle rotor, into
which the tubes are placed, are inclined at a given angle
with respect to the axis of rotation.
The angle at which
the sample compartments are inclined represents a compromise between mass and efficiency.
R. Wyckoff and
J. Lagsdin built rotors with compartments at various
angles, concluding that the smaller the angle of inclination, the greater the efficiency of the rotor.
However,
diminishing angles of inclination are accompanied by an
increase in the weight of the rotor, requiring more energy
for acceleration.
In
addition, the Bauer and Pickels
6
method of sealing their fixed angle rotors required the
use of inserts to prevent collapse of the unsupported
regions of the centrifuge tubes at high speeds.
The
smaller the angle of inclination, the greater the region
of the tube requiring support.
Consequently, a consider-
able amount of the tube capacity became unavailable
(Masket, 1941).
The majority of the fixed angle rotors
were built with compartments at angles between 20° and
40°.
A unique rotor was designed and built by
A.V. Masket in 1941.
The rotor had compartments that were
inclinced at only 10°, taking advantage of the increased
....,
efficiency possible at such a small angle of inclination
(Masket, 1941).
The rotor employed a novel method of
capping each tube individually with a small insert to
both seal and support the top of the tube.
This rotor
design overcame the limitations of the Bauer and Pickels
design, making it possible to use rotors with tubes inclined at only 10° without their collapsing or leaking.
The rotor was successfully
employed in the study of a
calcium-protein relationship (Chanutin, 1942; Ludwig, 1942;
Masket, 1942).
Yet, in spite of this success, fixed an-
gle rotors of such reduced angles of inclination were not
produced commercially and a rotor of a similar design was
not to appear again for 36 years.
7
3.
gation.
Differential and density gradient centrifu-
The product of a procedure to disrupt cells or
a tissue is a heterogeneous mixture of particles varying
in size, shape and density.
This mixture of particles
may be fractionated if it is subjected to several centrifugations of successively increasing force.
Increasing
the centrifugal force each time removes from the suspen-.
sion classes of particles decreasing in size and/or density (Claude, 1946b).
This technique, called differ-
ential centrifugation, grew in popularity through the
1930's and 1940's largely through the efforts of
Albert Claude.
Density gradient centrifugation utilizes a column
of liquid of increasing density onto which the sample is
layered,
When subjected to a centrifugal force, the par-
ticles sediment through the gradient on the basis of
their size and/or density.
Gradients can be designed so
that upon centrifugation the particles arrive at different
levels within the gradient.
The separated particles are
then collected as fractions by any one of a number of
means,
Credit for the development of this technique is
most often given to M.L. Brakke (Brakke, 1951).
However,
in 1938, M. Behrens separated particles of a tissue
extract on a continuous benzene-carbon tetrachloride
density gradient which he made using a gradient engine
(Behrens, 1938).
His work also represents the first sepa-
8
ration of particles on the basis of density, an approach
now known as isopycnic density gradient centrifugation.
Until 1949, density gradients were not utilized to separate particles in the conventional sense.
Instead, they
were used to stabilize a sedimenting boundary of particles
against convection.
In 1949, the first aqueous density
gradients were employed in the centrifugal fractionation
of liver tissue homogenate (Anderson, 1955).
In 1951,
the potato yellow dwarf virus was successfully isolated
on a sucrose density gradient and may have been the first
separation of particles by velocity sedimentation (Brakke,
1951) ..
4.
Reorienting gradients.
Density gradient
centrifugation using a fixed angle rotor represents the
first application of the reorienting gradient principle
(Lindgren et al., 1950; Turner et al., 1951).
A gradient
in a centrifuge tube can be thought of as an infinite
series of isodense planes.
With the sample layered onto
the surface of the gradient, the tubes are loaded into
the fixed angle rotor and the rotor is accelerated.
Dur-
ing acceleration, the isodense planes of the gradient,
as well as the particles on top of or entrained within
the gradient, will reorient themselves.
Under the influ-
ence of both gravity and centrifugal force, the planes
become a series of paraboloids (Fig. 1).
As the centri-
9
0
Figure 1
Reorientation in the lucite vertical rotor
(A) rotor at rest
(B) 105 r p m
(C) 215 r P m
(D) 505 r P m
(E) 1300 r p m
(F) 2500 r p m
'
11
fugal force increases, the focal points of the paraboloids
travel vertically downward until the isodense planes are
completely reoriented to a vertical position (Sheeler,
in preparation).
Among the first to recognize and employ
gradient reorientation were F.T. Lindgren (Lindgren et al.,
1950) and.R.H. Turner (Turner et al., 1951).
5.
Zonal rotors.
In 1962, the Zonal Centrifuge
Development Program was begun at the Oak Ridge National
Laboratory (Oak Ridge, Tennessee).
It was. the purpose
of this program to design rotors which would eliminate
the restrictions of sample size and loss of resolution
due to artifacts inherent in the designs of fixed-angle
and swinging bucket rotors.
A zonal rotor eliminates
tubes entirely, confining the gradient and sample instead
to a hollow bowl.
The bowl is divided into sectors by
septa; however, the septa do not seal the compartments
off from one another, insuring that all compartments fill
equally.
Since the septa project radially from the core
of the rotor, wall effects and the resulting convection
are eliminated.
Zonal rotors are filled with one gradient
and a single, large sample can be processed.
Of the many
rotors designed and built by N.G. Anderson at Oak Ridge,
the majority employed rotating seals to allow dynamic
loading and unloading of the rotor (Anderson, 1962;
Anderson, 1966; Anderson et al., 1964a; Anderson et al.,
12
1966; Anderson et al., 1967).
Some of Anderson's rotors
are presently available commercially.
Also begun at Oak Ridge was the development of
zonal rotors that utilized the reorienting gradient principle (Anderson et al., 1962a,b; and 1964b).
The develop-
ment of these rotors was begun in an effort to obtain an
alternative to rotating seals.
The rotating seal has a
tendency to "cross-leak," as well as being quite costly.
Reograd zonal rotors are unloaded statically but may be
loaded either statically or dynamically,
During decelera-
tion, the gradient reorients from the vertical to the
horizontal.
In 1964, the first reorienting zonal rotor,
the A-VII, was built by N.G. Anderson (Anderson, 1964b).
Not a great deal of emphasis was placed on the development of.this type of rotor.
The A-XVI reorienting gra-
dient zonal rotor was constructed to isolate nuclei
(~lrod
et al., 1969) and represented one of the final
products of this development.
The development of a widely
applicable reorienting gradient zonal rotor culminated
instead in the laboratory of P. Sheeler at California
State University (Northridge, California) with the design
of the SZ-14 rotor (Sheeler and Wells, 1969; Sheeler
et al., 1970; Wells et al., 1970). The success of the
SZ-14 and later the TZ-28 reorienting gradient zonal
rotors built by Dupont/Sorvall led to the development of
another reorienting gradient zonal rotor, the JCF-Z by the
13
Special Instruments Division of Beckman Instruments, Inc.
6.
Vertical tube rotors.
Recently, Dupont/Sorvall
Instruments introduced rotors in which the long axis of
the centrifuge tube remains perpendicular to the direction
of the centrifugal force.
"vertical rotors."
Such rotors have been te·rmed
The design of these rotors bears a
remarkable similarity to the 10°-angle fixed angle rotor
built by A.V. Masket in 1941 (see above).
Density gra-
dients in a vertical tube rotor must reorient during
acceleration and deceleration (Fig. 1).
In the reoriented
gradient, the distance a particle must travel to a given
·point within that gradient is much reduced over that distance it must travel in conventional tube-type rotors.
Since the entire tube is close to the edge of the rotor,
the particles in the tube are subjected to a higher and
more uniform centrifugal force throughout the gradient.
These two factors reduce the time required for many
separations, making the latest rotor to employ the reorienting gradient principle also among the most efficient
rotors presently available.
B.
Density Gradients: Theory and Production
1,
Step gradients.
Step gradients can be pre-
pared by layering a dense solution in the bottom of a
centrifuge tube, followed by layers of solutions decreas-
14
ing in density (Fig. 2).
The sample is then layered onto
the surface of the gradient, often followed by an overlay
consisting of liquid less dense than the sample.
Ideally,
particles in the sample should be aligned in one plane
to maximize the resolution.
An overlay is employed to
eliminate the meniscus of the sample zone, thereby more
closely approaching the ideal situation.
The sharp inter-
faces created by the solutions of differing densities
will in time break down by diffusion so that the gradient
profile will more closely resemble the dashed line in
Fig. 2.
These gradients are simple to prepare, requiring
little more than a pipette.
They should be employed with
some caution since sharp interfaces in a gradient may
produce artificial banding of the sedimenting particles.
Sedimenting particles suddenly encountering an increase
in density and/or viscosity in the medium decelerate,
and accumulate at the interfaces of the gradient.
Par-
ticles of different sedimentation rates may then contaminate the band formed as they join the particles at
the interface.
Other factors that affect resolution of
such gradients will be discussed below.
2.
Continuous gradients.
As the name implies,
the density in continuous gradients changes in a continuous, smooth manner.
The simplest but most time con-
suming way of creating such a gradient is to allow dif-
15
Figure 2
Step gradient preparation by layering of
solutions decreasing in density into a centrifuge
tube.
The graph illustrates the profile of a step
gradient and the profile of the gradient after
diffusion (dashed line).
~
0
tt-
1',
0
...
...
-m
-
'
''
'
\
\
'
...
...
.
'
\
...
'\
''
',
''
l
l
I
I
I
...
I
I
I
I
t
I
t
:::::>
...J
0
>
'
Al.ISN30
,: •
•I
:E
''
' ',
I
w
J.N310\fM9 d3.LS--
0..
0
1-
17
fusion to alter a step gradient.
Brakke used this method
to make the gradients he employed in the separation of the
potato yellow dwarf virus (Brakke, 1951).
The formation
of gradients by diffusion is limited to solutes which
have a fairly high diffusion coefficient, such as sucrose.
Most experimenters now prefer to use mechanical devices
to produce continuous gradients.
In 1938, Behrens employed an elegant device to
produce continuous non-aqueous gradients, (Behrens, 1938).
Over the years, a variety of devices termed gradient
engines or gradient makers have been described in the
literature.
These devices are designed to produce linear
gradients (Britten and Roberts, 1960; Choules, 1962;
Davis, 1965; Lei£, 1968a and b), exponential gradients
(Hinton and Dobrota, 1969; Gordon and Ramjoue, 1977), or
gradients of other shapes (Bock and Ling, 1954; Margolis,
1969; Michov, 1978; Sartory and Halsall, 1978).
a,
linear gradients.
The most common, yet
one of the most difficult types of gradients to produce,
is the linear gradient.
The density of a linear gradient
will change at a constant rate with respect to the volume.
A device to produce such a gradient consists of two interconnected chambers of identical geometries (Fig. 3a).
Equal volumes of the limits or endpoints of the gradient
are added to the chambers.
One limit is allowed to flow
into the chamber containing the other limit at a given
18
Figure 3
(A)
A linear gradient maker proposed by Britten
and Roberts, 1960.
(B)
A modification of a gradient maker introduced
by Schumaker in 1967.
By varying either the
number of syringes filled with air or gradient
limit, gradients of different characteristics
can be produced.
(C)
An illustration of the GF-2 gradient maker
available from Dupont/Sorvall Co.
Both
linear and exponential gradients can be produced using this device.
(D)
An exponential gradient maker with a variable
capacity mixing chamber.
(E)
A device designed to produce gradients of
variable shapes as proposed by Bock and Ling
in 1954.
The shape of the partition deter-
mines the shape of the gradient produced.
B
A
STIRRER
P£RtSTAL TtC
PUMP
c
D
~
~~
d
~~
•
1I
LIMIT
A
::lJ::JT
= ?
Jl
•
i
- .I
:=--:J
LIMIT
8
MIX I~
~·
MAGNETIC
STIRR£R
E
l LIMIT
-~---~~-~~~-~--
--
20
rate, and the two are mixed.
Simultaneously, liquid is
removed from the mixing chamber at twice the entry rate.
A useful equation which permits the calculation of the
concentration (Ct) of the effluent from such a gradient
maker at any given time (t) is:
c t = cm + (Cr - cm) (vt/2V.)
~
Cr is the concentration in the resevoir (limit A in
Fig. 3a); Cm is the concentration in the mixing chamber
(limit Bin Fig. 3a); Vt is the volume of gradient removed
at time, t; and
v.
~
is the initial volume in each chamber.
Though seeming quite simple,
the production of
gradients that are actually linear is quite difficult.
Not only must the chambers of the·gradient maker be of
the same geometry, but the volume of liquid in each must
decrease at the same rate, both chambers must always contain equal volumes, and the two liquids must be quickly
and thoroughly mixed (Sheeler, in preparation). The latter
becomes difficult when very dense or viscous solutions are
employed.
Other gradient makers of varying complexities
have been designed to fulfill or eliminate these requirements.
Two such gradient makers are illustrated in
Fig. 3b and Fig. 3c.
The device depicted in Fig. 3c is a
modification of the two chambered device previously
described.
The pistons apply a downward force onto the
surface of the two limits, insuring that equal volumes
21
are maintained in both chambers and that the volumes in
each chamber decrease equally.
Such a device is commer-
cially available for zonal centrifugation from Dupont/
Sorvall Instruments.
The device depicted in Fig. 3c is a modification
of one presented by Schumaker (Schumaker, 1967).
One
gradient limit is placed in the mixing chamber, an identical amount of the second limit into the syringe whose
line leads to the bottom of that chamber.
A second syr-
inge is filled with a similar amount of air.
The contents
of the two syringes are simultaneously forced into the
mixing chamber, expelling a stream of gradient from the
exit port.
The gradient produced under these given con-
ditions is linear.
A
useful expression for such a system
is:
ct is the concentration of the effluent at time, t.
cb
and Vb are the concentration and volume of the limit in
the syringe(s).
C
a
and V are the concentration and
a
volume of the limit in the mixing chamber and Vt is the
volume withdrawn from the mixing chamber at time, t
(Sheeler, in preparation).
b.
exponential gradients.
A second form of
22
density gradient, in which the density changes logarithmically, is referred to as an exponential gradient.
Expo-
nential gradients are not as popular as linear gradients,
but are considerably easier to make.
Two simple devices
that produce exponential gradients are illustrated in
Figs. 3c and 3d.
The mixing chamber initially contains
one of the gradient limits and the volume of liquid in
this chamber is kept constant throughout the production
of the gradient.
As liquid is removed from the mixing
chamber (e.g., by a peristaltic pump) the reservoir limit
is simultaneously drawn into the mixing chamber.
The
reservoir volume decreases and represents the total
volume of gradient that can be produced.
If the mixing
chamber initially contains the light limit, the density
of the gradient produced will increase exponentially
will have a convex shape.
and
A concave gradient is produced
when the dense limit is placed into the mixing chamber
and the density of the gradient decreases exponentially.
The degree of concavity or convexity of an exponential
gradient is determined by the mixing chamber volume.
Therefore, the design of an exponential gradient maker
should include a mixing chamber of variable capacity
(Figs 3c and 3d).
The device illustrated in Fig. 3c
is depicted in the mode that produces a linear gradient.
The gradient maker may be used to produce exponential
gradients by removing the piston from the chamber on the
23
right, converting it to a reservoir and locking the remaining piston at the height to accommodate the desired fixed
mixing chamber volume.
An expression permitting the cal-
culation of the various parameters of an exponential
gradient is:
Ct is the concentration of the effluent at time, t.
Cr
is the concentration of the reservoir limit; Cm is the
initial concentration of the mixing chamber; Vt is the
volume of the gradient withdrawn at time, t; and Vm is
the mixing chamber volume.
c.
complex gradients.
Under some conditions
it may be desirable to produce gradients of a complex
shape,· combining linear and exponential regions or forming
gradients of other geometric shapes.
Combining gradient
shapes from two sources is awkward and must be precise to
prevent the formation of a step which may induce artificial
banding.
Several gradient makers have been employed to
produce gradients of variable shapes.
The device illus-
trated in Fig. 3e is one in which the gradient shape is
determined by the shape of the baffle or partition which
can be varied.
Gradient makers which employ cam-guided
proportioning pumps to produce continuous gradients of
any desired shape are commercially available from Beckman
24
Instruments and L.K.B, Instruments.
d.
multiple gradients.
Each of the gradient
makers described thus far produces a single gradient
leaving a major problem unsolved:
The tube format of
centrifugation often requires the production of multiple
gradients.
Very few gradient makers have been described
that are capable of producing multiple, identical gradients
and none have gained wide acceptance.
One device pro-
posed by Leif (1968) employs a complex series of peristaltic pumps.
Others utilize a spinning
distribute~
to
apportion a stream of gradient into the tubes or buckets
that are also spinning (Albright and Anderson, 1957;
.....
Siakotos and Wirth, 1967i Candler et al., 1967).
Altemate
methods used to produce multiple gradients include rotating manifolds designed to distribute a stream of gradient
liquid equally among a number of lines leading to stationary centrifuge tubes, static manifolds designed to
divert the gradient stream equally into the tubes, and
multiple exit ports at the mixing side of the gradient
maker which in conjunction with multiple head peristaltic
pumps draw equal volumes of gradient into each tube.
Per-
haps the most common method of making many identical gradients is to produce them individually and consecutively.
Recently, a relatively simple and inexpensive device that
allows the simultaneous production of up to 8 identical
gradients was described by our laboratory (Sheeler et al.,
25
1978).
This device employs modified centrifuge tubes
that accommodate molded plastic inserts fitted with
o-rings.
The inserts in each tube are attached to a ser-
ies of plunger rods which in turn attach to a piston.
In order to load the tubes with identical gradients, each
tube is fitted with a conical cap connected by way of a
distributor to the gradient source.
The plungers are
raised by the piston to the fully elevated position and
the gradient is then drawn into each tube by lowering the
piston.
The inserts descending in concert insure that the
gradient is distributed equally among the tubes.
The
entire operation requires only 5 to 10 minutes.
C.
Factors Affecting Resolution
1.
Wall effects.
One of the artifacts present
in tube-type centrifuge rotors is wall effects (Anderson,
1955).
The walls of centrifuge tubes: are generally paral-
lel to one another.
Du~ing
centrifugation, particles
sediment radially and strike the walls of the centrifuge
tube.
Consequently, there is an increase in the particle
concentration at the circumference of the tube and a
decrease in the particle concentration at the center of
the tube.
The localized increase of the particle concen-
tration will establish a convection current within the
tube.
Particles will move in bulk along the walls of the
tube tuward the bottom, forcing the inner core of liquid
26
upward (Schumaker, 19.67).
2.
Convection.
Convection increases the pellet-
ing efficiency of rotors (especially of fixed angle
rotors).
However, convection decreases the resolution of
density gradient centrifugation.
Particles striking the
walls of the centrifuge tube may in fact adhere to the
wall.
The increase in concentration of particles may in-
duce particle agglutination.
Convection and these other
effects that are a result of the parallel nature of the
walls of a centrifuge tube will broaden zones within a
density gradient and may cross-contaminate zones with
particles of other zones, .
3.
Swirling.
An artifact termed "swirling" by
Anderson (1955) may more precisely be defined as the
Coriolis effect.
Coriolis force is a small inertial force
directed perpendicular to both the axis of rotation and
the direction of the centrifugal force (Sheeler, in preparation).
This force deflects particles in a direction
opposite to the direction of the spinning rotor.
The
influence of this force is minor as long as the rotor is
spinning at a constant speed.
Changes in the speed of the
rotor, i.e., acceleration or deceleration, dramatically
increase the effect of the Coriolis force.
Acceleration
and deceleration must therefore be carefully controlled,
so that changes in rotor rpm are gradual and smooth.
27
4.
Gradient recovery.
An important factor
affecting the success of a given particle separation is
the efficiency with which the recovery of the gradient
and the entrained particle zones from the centrifuge tube
or rotor is achieved.
The goal of all collection methods
is the recovery of the gradient and separated particles
zones as distinct fractions with as little cross-contamination, broadening or mixing as possible.
The walls of the tube or rotor chamber leading to
the collection port should converge upon that point.
Converging walls will condense the zones within a gradient,
increasing their thickness as well as that of the regions
between the particle zones.
Converging walls insure that
zones within a gradient may be completely removed before
contamination by particles of any other zones.
The length
and diameter of the tubing leading from the collection
port should be minimized to eliminate mixing due to
laminar flow.
A common method of collecting gradients is to
puncture the bottom of the centrifuge tube and collect
fractions as the gradient flows out.
Another popular
method is to draw out the gradient through a canula lowered to the bottom of the tube.
Both of these methods
take advantage of the converging walls formed by the hemispherical shape of the bottom of the centrifuge tube.
Another collection method employs a dense liquid
28
pumped into the bottom of the tube to displace the gradient upward and out through a conical cap.
Having the
option to collect gradients from either the top or the
bottom is advantageous.
Resolution may be improved by
collecting a particle zone from the closest end of the
centrifuge tube.
Since our laboratory has long been involved in the
design of centrifugal equipment and methodology, the
parallel chambers that characterize the recently developed
vertical tube rotor provided the ideal rotor configuration
for both solving and simplifying multiple gradient production.
A vertical tube rotor was constructed incorporating
an integrated system of plungers which fill the rotor
chambers (or tubes if desired) with identical gradients,
samples and overlays.
The design also provides an effi-
cient means of collecting the gradient upon conclusion of
centrifugation.
This thesis will describe the evolution
of this approach, ultimately leading to the modification
of a commercial vertical rotor.
The performance of the
rotors is analyzed using both biological and non-biological
model test systems.
In addition, this thesis will examine
some of the phenomena associated with the use of vertical
rotors in density gradient centrifugation and their effects
upon the performance of such systems.
II
ANALYSIS OF SEVERAL METHODS FOR THE PRODUCTION
OF MULTIPLE DENSITY GRADIENTS
A.
Gradients Produced Individually
1.
Gradient Maker A.
The gradient maker seen in
Fig. 3a was used to produce six gradients.
Sufficient
volumes of the gradient limits were prepared for six graclients of equal volumes.
The solutions were equilibrated
to room temperature and all trials were done at room
temperature.
As each gradient was completed, it was set
aside until all gradients were made.
The time required
to make each gradient was between 15 and 20 minutes, a
total of 90 to 120 minutes for all six.
The gradients
were then collected as a series of 2 ml fractions in the
same order in which they were prepared.
Therefore, for
each gradient the time between the production and collection was approximately equal.
A canula was lowered through
each gradient to the bottom of the tube and the gradients
withdrawn dense end first.
Each fraction was agitated
and its sucrose concentration determined by refractometry
using a Baush and Lomb Abbe 3-L Refractometer.
29
Results
30
are expressed as refractive index and % (w/w) sucrose
(Fig. 4).
The curve represents mean values for all six
gradients and standard deviations represented by the
vertical bars.
It can be seen that in addition to being
time consuming, the profiles of the six gradients varied
considerably.
2.
The values varied an average of 41%.
Gradient Maker B.
A second gradient maker
used to produce six individual gradients is illustrated
in Fig. 3b.
The basic procedure used was the same as
described above.
Though the gradients produced closely
approximate the predicted curve, there was still considerable variance among the individual gradients
(Fig. 5).
The average variance between the values was
16%.
B.
Simultaneous Production of Multiple Gradients
1.
Static manifold.
A method of producing mul-
tiple gradients from a single source employed a radially
symmetric static distributor.
The source of the gradient
may be any of the gradient makers in Fig. 3
having suf-
ficient capacity for the total volume of limits required
for six gradients.
Profiles of gradients produced by this
method (Fig. 6) revealed variation in both slope and total
apportioned volume of the gradients.
2.
Rotating manifold.
Another method of pro-
31
Figure 4
Six density gradients produced individually
with linear gradient maker illustrated in Fig. 3a.
The curve is the mean values for the sucrose concentration in corresponding fractions of the
gradients.
~ix
Standard deviations are represented
by vertical bars through each mean.
1.3700
1
1.'3650
20
1.3600
'j.
X
1.&.1
~
0
z
1.&.1
(/)
0
1.&.1
>
1(.)
15
1.'3550
a::
(.)
:::>
(/)
<t
0::
~
.....
1.&.1
a::
1.3500
10
1.3450
FRACTION NUMBER
33
Figure 5
Six gradients produced individually with
the gradient maker illustrated in Fig. 3b.
The
curve is the mean values for the sucrose concentrations in corresponding fractions of the six
gradients.
Standard deviations are represented
by vertical bars through each mean.
30
1.3800
1.3750
25
1.3700
"i
!
X
w
0
~
1.3650
w
-20
>
i=
~
Ill
0
0:
0
:::>
<II
0:
~
I.L
w
0:
1.3600
15
1.3550
1.3500
2
3
4
5
6
7
8
FRACTION
9
NUMBER
10
II
12
13
14
15
35
Figure 6
Profiles of six gradients simultaneousty
produced from a single source using a static manifold to distribute the gradient to tubes.
/'
1.3700
1.36!50
20
X
Ul
0
-
1.3600
~
~
~
Ul
Ul
f/)
2::
0
a::
10
0
<
a::
IG
u. 1.3!5!50
Ul
a::
;:)
f/)
~
1.3!500
10
1.34!50
1.3410
2
3
4
7
6
9
10
FRACTION NUMBER
II
12
13
14
1!5
37
clueing multiple gradients from a single source which employs a rotating manifold was tested.
The rotating mani-
fold makes transient contact with six channels, apportioning the gradient stream among six centrifuge tubes.
It may be seen in Fig. 7 that the variations are greater
than those obtained with the static distributor.
Clearly
this use of a rotating manifold distributor does not improve upon the results obtained with the static distributor.
38
Figure 7
Profiles of six gradients simultaneously
produced from a single gradient source using a
rotating manifold to distribute the gradient liquid
to six tubes.
1.3600-
15
1.3550 -
1.3500-
X
11.1
0
10
~
>
i=
~
11.1
11.1
(/)
1.3450
0
0::
~
1.)
:::>
0::
(/)
IJ..
11.1
0::
~
5
1.3400
1.3350
1.3300
~--~----.-----,----,----,----,----,----.----.----,-----,----,-~
2
3
4
5
6
7
FRACTION NUMBER
8
9
II
12
13
III
ROTOR DESCRIPTION, OPERATION
AND PERFORMANCE
Tube density gradients produced by any of the
methods previously discussed must still be loaded into an
appropriate rotor, then mounted on the centrifuge drive.
The recent introduction of vertical tube rotors has made
the production of several identical gradients directly
within a rotor feasible, employing an approach recently
demonstrated to be both simple and efficient.
The gradient
is simultaneously drawn into tubes by a system of interconnected plungers.
The plungers lower in concert, appor-
tioning the gradient equally among the tubes, forming up
to eight identical gradients (Sheeler, 1978).
A lucite
vertical rotor was constructed in order to examine the
feasibility of incorporating the approach directly into
the design of the rotor itself.
A.
Lucite Vertical Rotor
1.
Description.
The lucite vertical rotor and
plunger system are shown in Fig. 8.
40
Individual components
41
Figure 8
Fully assembled lucite vertical rotor and
loading apparatus.
(A) knurled sealing knob;
(B) Luer-lock fittings and chamber inlets; (C) lid;
(Pl
chambers;
(~)
chamber plungers; (F) plunger
rods; (G) horizontal plate; (H) axial support
shaft; (I) base.
c
D
F
G
I
43
can be seen in Figs. 9, 10, and 11.
porates two vertical chambers.
The rotor body incor-
A vertical channel drilled
into the floor of each chamber accommodated the plunger
rod.
The chamber inserts (plungers) are fitted with
three o-rings to effect a seal with the chamber wall and
floor.
The capacity of each rotor chamber with the insert
in place is 23 ml.
The undersides of the inserts were
axially threaded to accommodate the ends of the plunger
rods.
The rods were connected to a floating plate made
to rise (or fall) along the vertical supporting shaft.
The underside of the rotor lid contains two conicai recesses leading to the exit ports, which were threaded
to accept Luer-lock valves.
Annular o-rings around the
lid recesses served to seal the rotor chambers.
A large
bolt passing through the center of the rotor and the lid
and the knurled knob seal all rotor parts together.
2.
Operation.
The rotor body is placed onto the
supporting shaft and the inserts pushed to the floor of
the chambers.
The plunger rods are threaded from below
into each insert and engaged with the floating plate by
two nuts.
The plate is manually elevated to raise both
inserts to the top of the chambers and lines leading from
a gradient maker connected to the Luer-lock valves.
A
small amount of cushion is pumped into and drawn out of
the conical recesses in order to expel all air.
The de-
44
Figure 9
Disassembled components of the lucite
vertical rotor and loading apparatus.
(A) under-
side of rotor lid; (B) horizontal plate, axial
shaft and base; (C) knurled sealing knob; (D) rotor
body; (E) plunger rods with plungers attached.
'
Q
•
w
46
Figure 10
Components of the lucite vertical rotor
including, (A) the rotor lid with (B) the conical ,
recesses and chamber inlets, (C) the rotor body,
(D) the knurled sealing knob, and (E) the chamber
inserts.
()
w
0
- i
48
Figure 11
The assembled lucite vertical rotor with
chamber inserts as they appear in the fully loaded
position.
50
sired amount of cushion is left in the tubes and is followed by the gradient, sample and overlay.
The plungers
descend simultaneously as the two chambers are filled.
The rotor chambers were sealed in either of two
ways:
(1) simply closing the Luer-lock valves was suffi-
cient for centrifugation at low speeds, or (2) replacing
the valves with sealed Luer-lock tips.
The latter was
found to be a more secure method for sealing the rotor
chambers for higher speed runs.
The plungers were dis-
engaged and the rotor transferred from the support shaft
to the centrifuge.
The entire loading operation required
15 minutes.
Following centrifugation, the rotor was returned
to the supporting shaft and the plungers re-engaged.
Gra-
dients were collected individually using a peristaltic
pump to withdraw the gradient from the chambers.
The
negative pressure created by the pump caused the inserts
to rise in parallel in the chamber, displacing the gradient and its contents upward into and through the conical
recesses in the rotor lid.
3.
Performance.
a.
tests using erythrocytes.
The rotor was
used to separate the two populations of erythrocytes in a
mixture of human and mouse blood.
Since human erythro-
cytes have an average size of 100 ~m 3 and mouse erythro-
51
cytes 50 pm 3 , the two cell populations were readily separable on the basis of differences in sedimentation rates.
A 4.5 ml cushion of 45% sucrose in isotonic saline
was loaded into each chamber followed by 22 ml of a
10-30% sucrose in isotonic saline exponential gradient.
All concentrations are expressed as % (w/w) sucrose.
This
is followed by the sample: 2 ml at a final concentration
of 5 x 10 6 cells/ml in 5% sucrose in isotonic saline.
The sample was followed by sufficient overlay (isotonic
saline) to complete the filling of both chambers.
Centrifugation was carried out at a speed of
1150 rpm for 6.l minutes in a Dupont/Sorvall OTD-50
centrifuge.
An automatic rate controller was employed to
provide the necessary slow acceleration.
The rotor was
decelerated using the "reograd 0 program of the centrifuge.
Results are shown in Figs. 12 and 13.
The cells were
sedimented to equivalent gradient positions in the
rotor's chambers.
b.
tests using Percoll density beads.
Percoll is a low viscosity, low osmolarity and non-toxic
suspension of colloidal silica particles coated with
polyvinylpyrrolidone (PVP).
A variety of colored beads
of differ'ent densities are available for use as density
markers in Percoll gradients.
A mixture of three bead
types prepared in 3% (w/w) Percoll was separated on a
linear 10-100% Percoll gradient using the lucite rotor.
52
Figure 12
Sample zones containing equal concentrations of mouse and human erythrocytes suspended
in 5% (w/w) sucrose.
The sample was drawn onto
a 10-30% (w/w) sucrose exponential gradient and
cushion of 45% (w/w) sucrose.
54
Figure 13
During centrifugation (see text), the
populations of mouse and human erythrocytes were
separated as a function of their size differences.
The bands are at equivalent positions in both
rotor chambers.
56
Cushion (65% (w/w) sucrose), gradient, and sample were
loaded as described previously,
In this test, the rotor
was accelerated to 1,000 rpm using a Sorvall table-top
centrifuge.
erated.
After 2 minutes the rotor was slowly decel-
The resulting separation of the three bead popu-
lations is seen in Fig. 14.
The bands were at equivalent
gradient positions within each rotor chamber.
c.
gradient reorientation.
The lucite ver-
tical rotor provided an opportunity to directly examine
the phenomenon of gradient reorientation.
The rotor cham-
bers were partially filled with water and a small colored
zone formed by layering a solution of new methylene blue
(disolved in butanol) onto the surface of the water.
The
rotor was mounted on the table-top drive and illuminated
from behind.
Fig. 1 is a series of photographs illus-
trating the sequence of _events in gradient reorientation.
Reorientation was not completed until a speed of 2200 rpm
had been attained; this is equivalent to 206g at rave
In
a tali, narrow rotor or chamber, greater centrifugal force
is required to achieve complete reorientation than in a
rotor or chamber that is wider than it is tall (Hsu, 1969).
As is evident, the greatest change in the areas of the
paraboloids occurs at low speed.
Therefore, the lower
the rpm, the more critical the control of the acceleration
or deceleration in order to minimize any deleterious effects due to gradient reorientation and Coriolis forces.
57
Figure 14
Three populations of beads separated by
isopycnic centrifugation in a linear 10-100%
Percell gradient.
The bands appear at equivalent
positions in the rotor chambers.
59
Additional observations were made when the three
populations of beads were isopycnically banded in the
Percoll gradient (see above).
The gradient and the three
separated zones were subjected to rapid acceleration and
deceleration.
The disk-like particle zones were trans-
formed into inverted vortexes as the particles in each
zone swirled about the central axis of the chamber; particles at the circumferential edge of the zone traveled
faster than particles at the center.
Also, particles in
the zones at the top of the gradient swirled faster than
those in the zones below due to the viscosity relationship.
The extent of swirling induced by the Coriolis force
depended on the rate of change in rotor speed.
It is
clear that the Coriolis force should be minimized during
rotor acceleration to preserve the gradient profile and
to minimize broadening of the sample zone.
Controlled
deceleration is vital to maintaining the separation between
particles achieved during centrifugation.
The importance
of controlled rotor deceleration increases as the rotor
gradually approaches rest.
B.
Modification I of the SV-288 Vertical Tube Rotor
1.
Description.
A vertical channel was drilled
through the center of the hemispherical floor of four of
the eight chambers in a Dupont/Sorvall SV-288 superspeed
vertical tube rotor.
The chamber plungers constructed
60
are hemispherical to complement the floor of the chambers
(Fig. 19) and are axially threaded to accommodate the
plunger rods.
Four a-rings serve to effect a seal with
the chamber walls and floor.
With inserts in place,
each chamber has a capacity of 25 ml.
Cap assemblies were designed for the modified
chambers consisting of two components: a loading cap and
a sealing cap (Figs. 15, 16, 17, and 18).
The undersides
of the loading caps were conical, leading to an axial port
and the top of each cap accommodated Luer-lock fittings.
0-rings in the undersides of the caps produced a seal
with the surface of the rotor body.
0-rings in the top
of the loading caps contacted with the sealing cap.
A
recess in the sealing cap fitted with a rubber disk enclosed the Luer-lock fitting.
2.
Performance.
The four unmodified rotor cham-
bers were filled with water and sealed with the new cap
assemblies.
The rotor was accelerated to 10,000 rpm in a
Dupont/Sorvall RC-2B superspeed centrifuge.
At this
speed, one cap assembly failed, and the centrifuge was
quickly shut down.
The centrifugal force had stripped
the threads anchoring the cap assembly to the rotor body
and flung the cap assembly to the wall of the centrifuge
chamber.
After striking the wall the cap spiralled down
to the chamber floor, where it eventually came to rest.
61
Figure 15
The loading and sealing caps designed for
the modified SV-288 vertical tube rotor.
(A) under-
side of loading cap revealing conical surface and
axial port,
The o-ring at the outer edge effects
a seal with the top of the rotor body.
(B) top of
the loading cap with the Luer-lock fitting in place.
The o-ring effects a seal with the sealing cap.
(C) the underside of the knurled sealing cap showing the axial recess designed to accommodate the
Luer-lock fitting.
A flat rubber disk placed at
the bottom of this recess seals against the
Luer-lock fitting.
(D) sealing cap seen from above.
A
B
c
D
63
Figure 16
Cross section of the modified SV-288 rotor
and the associated plunger system.
(A) sealing cap
with the rubber disk; (B) loading cap with entrance
port formed by Luer-lock fitting; (C) hemispherical
plunger ·with four o-rings; (D) body of the SV-288
vertical rotor; (E) plunger rod; (F) top anchoring
nut; (G) floating plate; (H) bottom anchoring
nut; (I) the vertical support shaft.
Q-c
l--E
G---j
-F
l--H
65
Figure 17
Modified SV-288 vertical tube rotor with
loading caps positioned in each chamber,
Lines
attached to the Luer-lock fittings connect to a
distributor linking the chambers to the gradient
source.
67
Figure 18
Modified SV-288 rotor with cap assemblies
prepared for centrifugation,
69
The liquid in the exposed chamber was thrown out into the
centrifuge chamber, leaving the rotor severely out of
balance.
The violent perturbations of the rotor sheared
the bolts anchoring the drive but little permanent damage
was sustained by the centrifuge or the rotor itself.
C.
Modification II of the SV-288 Vertical Tube Rotor
1.
Description.
The cap assemblies were re-
designed to minimize the mass above the surface of the rotor as well as to eliminate plunging noted when loading the
lucite prototype.
In this design, the loading caps
(Jigs. 19 and 20) extended further into the rotor chambers to provide more support and lower the center of
mass of the caps,
The concave walls approaching the exit
port we~e replaced by a convex cone at an angle of 10°
from the horizontal.
Twelve small ports equally
distri-
buted about the circumference of the cone converged to
form a single channel leading to the protruding surface of
the loading cap (Jig. 20).
The distribution of the gra-
dient among the twelve channels substantially reduces the
flow rate of the incoming liquid, thereby reducing the
plunging effect.
The walls of the centrifuge tube adja-
cent to the collection ports acted as a baffle to deflect
and slow the moving liquid, further reducing the plunging.
The decision to place twelve collection ports at the circumference of the chamber was the result of the observa-
70
Figure 19
The re-designed cap assemblies and other
components of the modified SV-288 vertical rotor.
(A) plunger rods; (B) inserts or plungers; (C) rubber caps; (D) loading caps; (E) sealing caps.
UJ
"'0
I
••
••
()
72
Figure 20
Cross section of modified SV-288 rotor
illustrating (A) the re-designed sealing caps;
(B) loading caps; (G) inserts or plungers;
(D) rotor body; (E) plunger rods; (F) anchoring
nut; (G) floating plate (later replaced by a
motor-driven plate); (H) lower anchoring nut;
(I) the vertical support shaft.
~A
~---E
~-H
74
tion that following centrifugation, particles in a zone
are more concentrated at the periphery of the zone than at
the center.
This design provided a more efficient means
of collecting particle zones.
The two o-rings incorpor-
ated into each cap form seals with the rotor chamber and
surface of the rotor.
The top of the loading cap is re-
cessed and contains a protruding axial loading channel.
The sealing caps and the rubber caps serve to seal the
rotor chambers (Figs. 19 and 20).
The same chamber plungers previously described
(see above) were also used as a part of this second modification.
The floating plate was replaced by a motor-
driven plate in order to eliminate the uneven movement of
the plate during loading and unloading (Fig. 21).
A
reversible motor was used to rotate a vertical threaded
shaft and the plate (with eight arms extending from the
center) threaded to travel along the vertical shaft
(.mU;ch like a worm gear).
The slotted housing (Fig. 22)
prevents the plate from revolving along with the vertical
shaft,
2.
Operation without tubes.
The loading and
unloading of this device is accomplished in much the same
manner.. as the lucite vertical rotor apparatus.
The gra-
dient is drawn into the rotor chambers by the negative
pressure created by the descending plungers.
The loading
75
1•
Figure 21
Loading assembly for the modified SV-288
vertical tube rotor.
(A) spindle; (B) slotted
housing; (C) eight arm plate; (D) controls.
'
77
Figure 22
Fully assembled apparatus.
(A) loading
caps; (B) SV-288 rotor; (C) plunger rods; (D) slotted housing; (E) anchoring nuts.
79
rate is regulated with the speed control.
Once the
cushion, gradient, sample and overlay are loaded, the
rotor is sealed by placing the rubber caps in position
and tightening the sealing caps.
About 15 minutes was
required to complete the operation.
At the conclusion of
centrifugation, the gradients were collected individually
by re-engaging the plunger rods (one chamber at a time)
and slowly raising the horizontal plate.
3.
Operation with modified centrifuge tubes.
Modified centrifuge tubes have been previously described
(~heeler
et al., 1978) and can be mounted in the modified
chambers of the rotor.
The underside of the hemispherical
insert is axially threaded to accommodate the plunger rods.
The brass conical cap with a Luer-lock fitting is employed
only during loading and unloading.
The brass cap is re-
placed by the conventional rotor caps to seal the rotor
chambers for centrifugation.
These modified centrifuge
tubes also provide an accurate and efficient means of
collection(Fig. 23).
4.
Performance.
a.
static tests.
The ability to produce iden-
tical gradients using the appartus·was examined. Four gradients were made in the fashion previously described and
then collected individually as 1.0 ml fractions.
The
sucrose concentration in each fraction was determined
80
.
Figure 23
Special centrifuge tubes for use in the
modified SV-288 vertical tube rotor.
(A) modi-
fied centrifuge tubes; (B) brass loading cap;
(_C) plastic insert or plunger;
(D} T-bar.
The
syringe barrels are for use in other tube-type
rotors,
82
by refractometry and the results are shown in Fig. 24.
The gradients vary considerably less than gradients produced by any of the other methods examined.
The average
variance between values was 13%.
In a second test, a cushion of 55% (w/w) sucrose
was loaded into the chambers followed by a 17 ml exponential 21.4 to 50%
(~/w)
sucrose gradient.
A 1.25 ml· sam-
ple (prepared by diluting blood 50-fold with 8% (w/w)
sucrose was then drawn in, followed by sufficient overlay
to completely fill the chambers.
The gradients were im-
mediately collected and the oxyhemoglobin concentration
in each fraction determined colorimetrically (Sheeler
and Barber, 1964).
The red cells were lysed by a concen-
tration of 0.04% (v/v) ammonium hydroxide.
The colori-
metric measurements were made with a Beckman-DB Spectrophotometer at 575 nm.
Fig. 25 summarizes the results.
Variation among the four tests is very low, especially
when unavoidable variations introduced by the collection
of gradients as numerous fractions are considered.
It
is clear that the apparatus provides an effective means
for producing several gradients with a minimum of variation, and also provides an efficient and accurate method
of collecting gradients.
b.
dynamic tests.
Rotor chambers were loaded
with 4 ml of 55% (w/w) sucrose cushion, followed by a
17 ml 21.4 to 50% (w/w) sucrose exponential gradient.
83
Figure 24
Gradients produced in the chambers of the
modified SV-288 rotor.
The curve represents
median values of the sucrose concentrations in
corresponding fractions of the four gradients.
1.4500
1.4400
1.4300
1.4200
1.4100
X
w 1.4000
0
~
w
>
i=
1.3900
0
ct
...w
0::
0::
1.3800
1.3700
1.3600
1.3!100
1.3400
1.3300 ~-.r--r--r--r--~-.--,-~r--r--r--r--.--.--.---r--r--.--.--.---r--,--.--~
2
3
4
5
6
7
8
9
I 0 II 12 13 14 15 16 17 18 19 20 21 22 23 24
FRACTION NUMBER
85
Figure 25
Gradients and sample zones formed in· the
chambers of the SV-288 rotor.
The gradient profile
represents the median values of the sucrose concentration in corresponding fractions of the four
gradients.
The histogram represents the median
values for oxyhemoglobin.
1.4500
1.2
(
1.4400
1.1
1.4300
1.0
1.4200
0.9
I. 4100
o.8 '2
z
I()
.....
X
UJ
0
0.7
1.4000
UJ
~
(.)
z
ct
CD
0.6 a:
UJ
>
i=
(.)
e
1.3900
0
<J)
ct
CD
a:
ct
IL.
UJ
a: 1.3800
0.5
1.3700
0.4
I. 3800
0.3
1.3500
0.2
1.3400
0.1
UJ
>
~
0
1.3300
2
3
4
5
6
7
8
9
10
II
12 13
14
15
FRACTION NUMBER
16
17
18
19
20 21
22
23
24
..J
UJ
a:
87
1.25 ml of sample (human blood diluted 30 fold) was then
layered onto the surface of the gradient and an overlay
used to fill the chambers.
The rotor was accelerated to
1,100 rpm for 90 minutes in a Sorvall superspeed centrifuge.
No leakage occurred during centrifugation.
The
gradients were individually collected and assayed (Fig. 26).
Oxyhemoglobin absorbances and sucrose concentrations
in corresponding fractions verify the uniformity among
the gradients produced.
What variation was introduced
during the collection of the gradients could be compensated by expressing oxyhemoglobin concentration as a function of sucrose concentration (Fig. 27).
88
Figure 26
Results of the isopycnic banding of
erythrocytes in four gradients produced in the
modified SV-288 rotor.
Gradient profile is shown
as mean values and the range indicated by vertical
bars.
The oxyhemoglobin contents of corresponding
fractions are depicted using histograms with vertical bars representing the range of absorbance
values obtained.
1.4450
2.2
1.4350
2.0
1.4250
1.8
1.4150
1.6
1.4000
1.4 ~
2z
I()
X
Ul
Ul
0
~ 1.3950
1.2
Ul
0
z
<t
al
0::
>
i=
0
~ 1.3850
1.0
0::
u.
<Jl
al
<t
Ul
Ul
0::
>
0.8
1.3750
~
..J
Ul
0::
1.3650
0.6
1.3550
0.4
1.3450
0.2
2
3
4
5
6
7
8
9
10
II
12 13 14 15 16 17 18 19 20 21
FRACTION NUMBER
22 23
90
Figure 27
Results of isopycnic banding of erythrocytes.
Distribution of hemoglobin (erythro-
cytes) in each fraction as a function of sucrose
concentration.
.22
0
.20
.18
i'
z
"',._
.16
0
•
•
!!?.
UJ
u
z<(
m
. 14
cr
0
<f)
.12
Cll
<(
UJ
> .10
i=
<(
..J
UJ
cr
0
.08
.06
.04
.02
0
•
1.:3500
1.:3700
1.:3900
REFRACTIVE
0
1.4100
INDEX
1.4:300
IV
CONCLUSIONS
A persistent problem in density gradient centrifugation has been the production of the many identical
gradients required when using multi-tube rotors.
Anum-
ber of possible solutions to this problem were examined.
The simplest (and still the most common approach) is
to produce the gradients individually.
Using two differ-
ent kinds of gradient makers, this method was shown to
fail to produce identical gradients.
A number of other
approaches were also examined:
1.
A radially symmetrical static manifold was
used to divert a stream of gradient from the gradient'
maker into several lines leading to centrifuge tubes.
The
inability to insure that the flow rates in each of the
distributor lines is equal resulted in a failure to produce identical gradients.
2.
Attempts were made to produce multiple iden-
tical gradients using a rotating manifold that made transient contact with a number of lines designed to apportion
the gradient equally among the centrifuge tubes.
92
This
93
device was also fraught with problems and performed unsatisfactorialy.
Other rotating devices have been described in the
literature.
In most of these a distributor is mounted on
the centrifuge rotor and apportions the gradient among
the tubes or buckets while the rotor is spinning (Albright
and Anderson, 1957; Siakotos and Wirth, 1967; Candler
et al,, 1967).
The construction of such distributors re-
quires extremely accurate machining, making them impractical as well as costly.
Recently, our laboratory intro-
duced an inexpensive, and simple device with which as
many as eight identical gradients could be produced
(Sheeler et al., 1978).
This device.employs
a system
of interconnected plungers ·to simultaneously draw the
gradient liquid into several modified centrifuge tubes.
Since the plungers descend at the same rate, all of the
tubes fill equally.
Further evolution of this approach,
,culminating in its incorporation directly into the design
of a vertical rotor, is presented in this thesis.
Vertical tube rotors were first introduced in
1977.
Since the chamber in a vertical rotor remains per-
pendicular to the direction of the centrifugal force, the
gradient must reorient from vertical to horizontal.
The
path length is much reduced in the vertically oriented
gradient and the centrifugal force is greater and more
uniform throughout, making vertical tube rotors more
94
efficient than conventional tube rotors.
Gradient reori-
entation also optimizes the resolution by reducing the
cross-sectional area of the starting zone.
However,
from
a practical standpoint resolution is equally dependent
upon the smoothness and gradualness of the reorientation
and the control of the Coriolis effect.
Hsu (1968) and
Hsu and Anderson (_1968) examined the shear forces within
a reorienting gradient, concluding that the most significant disturbance would occur at the top and bottom of the
gradient, recommending the use of a cushion and overlay.
The swirling created within a gradient by any rapid
changes in the rotor speed (either acceleration or deceleratj_on) ;i:.s much more likely to substantially decrease the
resolutton than the shear forces,
Various levels within a gradient swirl at different rates due to the different viscosities of the liquid
at different levels.
Increases in viscosity therefore
tend to oppose the Coriolis force.
In order to maximize
resolution during reorienting density gradient centrifugation, it is vital that both the acceleration and deceleration be carefully controlled.
As rpm decreases, the
sur£ace area of the paraboloids within a gradient increases
and the stabilizing effect of centrifugal force decreases.
The last few revolutions during deceleration (and the
first few during acceleration) are the most critical.
Other factors affecting resolution, including wall effects
95
and convection, are less important in vertical tube rotors
than conventional rotors,
A lucite vertical rotor was constructed incorporating a system of interconnected plungers to allow
the simultaneous production of identical gradients.
The
effectiveness of this approach was tested by separating
populations of erythrocytes on the basis of their sedimentation rates and three populations of inert colored
particles isopycnically.
Results indicated that the gra-
dients were produced with a minimum of variation.
An SV-288 vertical tube rotor was modified to incorporate a similar system of plungers and new rotor components were constructed to allow gradient production in
a manner similar to that for the lucite rotor.
Gradients
can be produced in the rotor chambers themselves utilizing the caps and plungers shown in Fig. 19.
Gradients
may also be produced in modified centrifuge tubes seen in
Fig, 23.
In either mode, any number of identical gra-
dients can be produced in the time required to produce
a single gradient.
The uniformity and reproducibility
of the gradients produced has been shown by both analysis
of the individual gradient profiles and by the recovery
of zones in corresponding fractions of the gradients.
The modified SV-288 vertical rotor may be employed in
three different modes (Fig. 28).
Gradients may be pro-
duced (1) directly in the rotor chambers themselves,
96
Figure 28
The several ways in which the modified
SV-288 vertical tube rotor may be used.
(A) an
empty rotor chamber; (B) conventional caps;
(C) brass, conical tube-loading cap; (D) the chamber-loading cap; (E) complete assembly sealing
rotor chamber.
---A
- -·B
c
~-o
98
(2) in modified centrifuge tubes, and (3) in the conventional (intended) manner.
Of course, the apparatus may
also be employed to produce gradients in tubes for use in
other types of centrifuge rotors.
The apparatus described here offers a practical
solution to the problem of producing multiple, identical
density gradients - a problem
that has plagued centri-
fugal technology for many years.
The new approach pro-
vides an accurate and efficient means of producing several
virtually identical gradients and equally important, provides an efficient means of recovering separated particles
entrained within the density gradient.
99
REFERENeES
Albright, J.F. and Anderson, N.G. (1958). A Method for
rapid fractionation of particulate systems by gradient differential centrifugation. Expl. Cell Res.
15:271.
Anderson, N.G. (1955). Studies on isolated cell components VIII. High Resolution gradient differential
centrifugation. Expl. Cell Res. 9:446.
Anderson, N.G. (1962). The zonal centrifuge. A new
instrument for fractionating mixtures of particles.
J. Phys. Chem. 66:1984.
Anderson, N.G. (1966). An introduction to particle separations in zonal centrifuges. Natl. Cancer Inst ..
21:9
Anderson, N.G. (1968). Preparative particle separation
in density gradients. Quart. Revs. Biophys. 1:217
Anderson, N.G., Price, C.A., Fisher, W.D. and Burger, C.L.
(1962a) . Reorienting gradient (reograd) systems.
The Joint NIH-AEC Zonal Centrifuge Development
Program Semi-Annual Report, ORNL-3415 Special:l6
Anderson, N.G., Price, C.A. and Canning, R.E. (1962b).
Rotor A-VII. The Joint NIH-AEC Zonal Centrifuge
Program Semi-Annual Report, ORNL-3415 Special: 16
Anderson, N.G., Barringer, H.P., Babelay, E.F. and Fisher,
W.D. (1964a). The B-IV zonal ultracentrifuge. Life
Sci. 3:667.
Anderson, N.G., Price, C.A., Fisher, W.D., Canning, R.E.,
and:Burger, C.L. (1964b). Analytical techniques for
cell fractions. IV. R~.orienting gradient rotor for
zonal centrifugation. Analyt. Biochem. l:l
Anderson, N.G., Barringer, H.P., Babelay, E.F., Nunley, C.
E., Barthus, M.J., Fisher, W.D. and Rankin, C.J.
(1966). The design and operation of the B-IV zonal
centrifuge systems. Natl. Cancer Inst. Monogr. 21:137
100
REFERENCES(Continued)
Anderson, N.G., Waters, D.A., Fisher, W.D., Cline, G.B.,
Nunley, C.E.,and Rankin, C.T. (1967). Analytical
techniques for cell fractions. V. Characteristics of
the B-XIV and B-XV zonal centrifuge rotors. Analyt.
Biochem. 21:235
Bauer, J.H. and Pickels, E.G. (1936}. A high-speed
vacuum centrifuge suitable for the study of filterable viruses. J. Exp. Med, 64:503
Beams, J.W. (1938).
Phys. 10:245
High speed centrifugation.
Rev. Mod,
Beams, J.W. and Pickels, E.G. (1935). The production of
high rotational speeds. Rev._ Sci. Instr. 6:299
Beams, J.W., Linke, F.W. and Sommer, P. (1938). A vacuum
type air driven centrifuge for biophysical research.
Rev. Sci. Instr. 9:248
Behrens, M. (1939). Zell-und Gewebetrennung. Handbuch
der Biologischen Arbeitsmethoden, Vol. 5, Pt. 10,
No. 2:1363, Edited by E. Abderhalden. Berlin,
Urban & Schwarzehberg.
Bernard, C. (1865). in Introduction a l'Etude de la
Medecine Experimentale, Pt, 2. Chapter 1.
Bock, R.M. and Ling, N.S. (1954). Devices for gradient
elution in chromatography. Analyt. Chern. 26:1543
Brakke, M.K. (1951). Density gradient centrifugation: A
new separation technique. J. Am. Chern. Soc. 73:1847
Brakke, M.K. (1955). Zone electrophoresis of dyes, proteins, and viruses in density gradient columns of
sucrose solution. Arch. Biochem. Biophys. 55:175
Britten, R.J. and Roberts, R.B. (1960). High resolution
density gradient sedimentation analysis. Science
131:32
Candler, E.L., Nunley, C.E. and Anderson, N.G. (1967).
Analytical techniques for cell fractions. VI. Multiple gradient-distributing rotor (B-XXI). Analyt.
Biochem. 21:253
101
REFERENCES(Continued)
Chanutin, A., Ludwig, S. and Masket, A.V. (1942).
Studies on the calcium-protein relationship with
the aid of the ultracentrifuge. I. Observations
on calcium caseinate. J. Biol. Chern. 143:737
Choules ,· G. L. (1962) . A linear-gradient mixing devices
for viscous solutions. Analyt. Biochem. 1:236
Claude, A. (1964a). Fractionation of mammalian liver
cells by differential centrifugation. I. Problems,
methods and preparation of extract. J. Exp. Med. 84:
51
Coombs, D.H. (1975). Density gradient fractionation by
displacement. Analyt. Biochem. 68:95
Davis, G.A., Santen, R.J. and Agranoff, B.W. (1965). The
production of a linear density gradient by means of
a proportioning pump. Analyt. Biochem. 11:4
DeDuve, C. (1964). Principles of tissue fractionation.
J. Theor. Biol. 6:33
Dounce, A.L., Witter, R.J., Monty, K.J., Pate, S. and
Cottone, M.A. (1955). A method for isolating intact
mitochondria and nuclei from the same homogenate and
the influence of mitochondrial destruction on the
properties of cell nuclei. J. Biophys. Biochem. Cyto.
1:139
Elrod, L.H., Patrick, L.C. and Anderson, N.G. (1969).
Analytical techniques for cell fractions. 13. Rotor
A-XVI, a plastic gradient-reorienting rotor for isolating nuclei. Analyt. Biochem. 30:230
Gordon, J. and Ramjoue, H.P. (1977). A simple design of
an apparatus for the generation of sucrose gradients
for large scale separation of ribosomal sububits.
Analyt. Biochem. 83:763
Greenbaum, A.L., Slater, T.F. and Wang, D. (1960).
somal-like particles in the rat mammary gland.
188:318
LysoNature
Gregor, H.D. (1977). A new method for the rapid separation
of cell organelles. Analyt. Biochem. 82:255
"'
102
REFERENCES(Continued)
Henriot, E. and Huguenard, E.
8:443.
(1927).
Hinton, R.H. and Dobrota, H. (1969).
maker for use with zonal rotors.
- 30:99.
J. de Phys. et Rad.
A simple gradient
Analyt. Biochem.
Hofman, L.F., Moline, C., McGrath, G. and Barron, E.J.
(1978). Use of vertical rotors to facilitate estrogen and progesterone receptor analysis. Clin. Chern.
24:1609
Hsu, H.W.
(1971). Transport phenomena in zonal centrifuge rotors. III. Particle sedimentation in gradient
solutions. Sep. Sci. Q:699
Hsu, H.W. and Anderson, N.G. (1969). Transport phenomena
in zonal centrifuge rotors. II. A mathematical analysis of the areas of isodensity surfaces in reorienting gradient systems. Biophys. J. 9:173
Jordan, V.C. and Prestwich, G. (1977). , Binding of (3 H)tamoxifen in rat uterine cytosols: a comparison of
swinging bucket and vertical tube rotor sucrose density gradient analysis. Mol. Cell Endocr. 8:179
Leif, R.C. (1968a). Density gradient system. I. Formation
and fractionation of density gradients. Analyt.
Biochem. 26:271
Lei£, R.C. (1968b). Density gradient system. II. A 50
channel programmable undulating diaphragm peristaltic
pump. Analyt. Biochem. 26:283.
Lindgren, F.T., Elliott, H.A. and Gofman, J.W. (1951).
The ultracentrifugal characterization and isolation
of human blood lipids and lipoproteins, with applications to the study of atherosclerosis. J. Phys.
& Coll. Chern. 55:80.
Ludewig, S., Chanutin, A. and Masket, A.V. (1942). Studies on the ca++ prbtein relationship with the aid of
the ultracentrifuge. II. Observations on serum. J.
Biol. Chern. 143:753.
103
REFERENCES (Continued)
McEwen, B.S., Allfrey, W.G. and Mirsky, A.E. (1963).
Studies of energy yielding reactions in thymus nuclei
I. Comparison of nuclear and mitochondrial phosphorylation. J. Biol. Ghem. 238:758.
Margolis, J.
device.
(1969). A versatile gradient-generating
Analyt. Biochem. 27:319.
Masket, A.V. (1941). A quantity type rotor for the ultracentrifuge. Rev. Sci. Instr. 12:277.
Masket, A.V., Chanutin, A. and Ludewig, S. (1942). Studies on the calcium-protein relationship with the aid
of the ultracentrifuge. III. Influence of augmentation
of serum with calcium and phosphate salts. J. Biol.
Chern. 143:763.
Michov, B.M. (1978). A concentration gradient system.
Analyt. Biochem. 86:432.
Sartory, W.K. and Halsall, H.B. (1978). Design of a generalized n-solute mixing chamber gradient generator.
Analyt. B·iochem. 88:539.
Schumaker, V.N. (1967). Zone centrifugation. Adv. Biol.
and Med. Physics II. (edited by Lawrence, J.H. and
Gofman, J.W,). Academic Press, New York.
Sheeler, P. (in preparation). Centrifugation in Biology
and Medical Sciences.
Wiley-Interscience, New York.
Sheeler, P. and Barber, A.A. (1964). Comparative Hematology of the turtle, rabbit and rat. Comp. Biochem.
Physiol. 11: 139.
Sheeler, P. and Wells, J.R. (1969). A reorienting gradient zonal rotor for low-speed separation of cell
components. Analyt. Biochem. 32:38.
Sheeler, P., Gross, D.M. and Wells, J.R. (1970). Zonal
centrifugation in reorienting density gradients.
Biochem. Biophys. Acta 237:28.
Sheeler, P., Doolittle, M.H. and White, H.R. (1978). Method and apparatus for producing and collecting a
multiplicity of density gradients. Analyt. Biochem .
. 87:612.
104
REFERENCES(Continued)
Siakotos, A.N. and Wirth, M.E. (1967). A method for the
mass production of density gradients. Analyt.
Biochem. 19:201.
Spragg, S.P. and Rankin, C.T. (1967). The capacity of
zones in density gradient centrifugation. Biochem.
Biophys. Acta __ 141: 164.
Turner, R.H., Snavely, J.R., Goldwater, W.H., Randolph, M.
C., Sprague, C.C. and Unglaub, W.G. (1951). The
study of s·erum proteins and lipids with the aid of
the quantity ultracentrifuge. I. Procedure and principal features of the centrifugate of untreated normal serum as determined by quantitative analysis of
samples from ten levels. J. Clin. Invest. 30:1071.
Wells, J.R., Gross, D.M. and Sheeler, P.
(1970). A large
capacity re-orienting density gradient zonal centrifuge rotor. Lab. Prac. 19:497.
R.W.G. and Lagsdin, J .B. (1937), Quantity heads
for the air-driven ultracentrifuge. Rev. Sci. Instr.
8:427.
~vyckoff,