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Multiple sedimentation
Copyright All rights
(D 1972 by Academic Press, Inc. reproduction in my form reserced
of
Cell Research 73 (1972) 16 l- 169
Experimental
MULTIPLE A Method for Analysis
SEDIMENTATION and Separation
of Particle
Mixtures
P.-b;. ALBERTSSON Department
of Biochemistry,
University
of Umed,Urned,Sweden
SUMMARY 1. A new method for separation of particles, such as cells, is described. It is based on sedimentation at 1 g. The separation obtained by a single sedimentation is increased by a multistage procedure analogous to counter-current distribution between two immiscible phases. 2. The principle of the method is described. 3. Applications of the method on starch grains, Sephadex-gel particles, polystyrene latex particles and chlorella cells are described. In all cases separation according to size is obtained. The apparatus used is a modification of a thin-layer counter-current distribution apparatus described earlier (Anal biochem 11 (1965) no. 1; Science tools 17 (1970) no. 3). 4. The method shows a comparatively high selectivity and it is a mild method towards cells since no foreign material has to be included in the suspension medium.
Sedimentation is a powerful method for separating particles and molecules differing in size, density or shape. For large particles, earth’s gravitational force (1 g) is enough to achieve a fast sedimentation rate while for smaller particles and molecules centrifuges have to be used to speed up sedimentation. In both cases, after a certain time, a sediment enriched in faster sedimenting particles is obtained and may be separated from the supernatant. By such a one-step procedure, however, only a partial separation is obtained. To achieve a more efficient separation a multistage procedure can be used. This paper describes a procedure in which a series of sedimentation steps interspaced by mixings are carried out in order to multiply the effects obtained in a single sedimentation. A preliminary report of this procedure has been published earlier [l]. Its application to blood cells [l I] and the ll-
721807
unicellular algae chlorella [13] is described in separate reports. The theoretical treatment of the procedure is very similar to that of counter-current distribution using two immiscible phases. A similar procedure was used by Behrens & Marti for separating blood cells by repeated centrifugations [4]. Theory Sedimentation in a single chamber. Suppose
that we allow a suspension of uniform particles to sediment in a chamber as shown in fig. 1. After a certain interval of time, an upper layer, height h,, and lower layer, height hb, are separated from each other. We are interested in the ratio between the number of particles present in the top layer and the bottom layer. Let the sedimentation velocity of the particles be U, and suppose their concentration at start is c. If one neglects Exptl
Cell Res 73 (1972)
162 P.-A. Albertsson
n
Fig. 1. Sedimentation chamber. The entire chamber is filled with a suspension of particles. After a time, t, the particles have sedimented a distance of where u is the sedimentation velocity. After this time the two layers h, and h, are separated. The lower layer is enriched in faster-sedimenting particles.
or R J-vt ’ h,+vt
(2)
This ratio is analogous to the partition ratio in liquid-liquid extraction, i.e. the amount of substance in the upper phase divided by the amount of substance in the lower phase. We may therefore, by replacing the partition ratio by R,, use the same theoretical treatment as is used for countercurrent distribution. For calculation of a theoretical distribution curve for a single substance it is convenient to introduce p, the fractional amount of material in the upper layer and q, the fractional amount of material in the lower layer after the sedimentation time t such that Exptl Cell Res 73 (1972)
I
2
.;.:;:
transfer
.I... ... . ;:;2$ n
Brownian motion, then after a time t, the particles have moved a distance U. t. If A is the area of cross section, a number of particles equal to A *v * t * c therefore leave the upper layer, and enter the lower chamber. At the start (t =0) the number of particles in the upper and lower layers was A. h,* c and A*h,*c respectively. Therefore the ratio, R,, between the number of particles in the upper and the lower chamber at time t becomes
(1)
0
0
r 1
ri2
tram n
0
1
2
n
0
I
2
fer
completed
Fig. 2. Principle of multiple sedimentation. A series of chambers (see fig. l), numbered O-II are arranged in a circle. The figure shows a few chambers viz., those numbered 0, 1, 2 and n. All chambers, except the loading chamber 0, are charged with the same volume of the medium of suspension. Chamber 0 is charged with the same volume of a suspension of particles. (A) Start of the experiment. The particles which are uniformly distributed in chamber 0 start to sediment. (B) After a certain settling time the particles have sedimented a certain distance depending on their sedimentation velocity. (C) The upper layer of liquid in each chamber is transferred to the adjacent chamber with the next higher number. (0) Transfer is completed. (E) After the content of each chamber has been mixed, the sedimentation starts again. The particles are now distributed over the two chambers 0 and 1. Those with the higher sedimentation velocity are enriched in chamber 0. One step is now completed and the next can begin. For a drawing of the apparatus used in the experiments, see [2, 31.
pfq=l
(3)
and R, =f
(4)
Multiple sedimentation The multiple sedimentation process involving
a number of steps is illustrated in fig. 2 in a manner analogous to counter-current distribution. The values obtained for the fractional amount present in successive sedimentation cells are the terms of the binomial series (P + q)“, where IZ is the number of transfers. The total amount is constant throughout the entire process so that (p + q)” = 1. The amount in the rth chamber (r can be from 0 to n) expressed as a fraction, T,. r, of the total, after n transfers is given by n!
Tn.,= r!(n-r)!
p’. qcn-r)
(5)
Procedures for calculating a theoretical curve and comparing it with the experimental one are given in standard texts on countercurrent distribution [6, 71. When the amount in each chamber is plotted against r a distribution curve is obtained. The number, rmax of the chamber with the maximal concentration, i.e. the peak of the curve may be calculated from R, by the approximate relation r
- n.Rs mx
l+R,
which in combination n (hi - vt) r max= H
with (2) gives
163
tation time the distance travelled will decrease linearly with the sedimentation rate. Two different particle species will be more separated from each other the longer the time, t, which is allowed for sedimentation in a chamber. However, vt must be smaller than h, for the slower sedimenting particle; otherwise both particle species stay in the lower chamber. Therefore, h, should be chosen as large as possible for maximal separation, i.e. the lower sediment layer h, should be small compared to h,. For practical reasons h,, should not be made too small, however, because the volume capacity of the lower layer will then be too small. In the experiments below, the sediment layer is about one-tenth of the total height of the liquid. Apparatus
A thin-layer counter-current distribution apparatus used for aqueous polymer twophase system has been used for the multiple sedimentation experiments. The apparatus is described elsewhere [2, 31. The only modification introduced for the present experiments was that the bottom plate contained cavities which were 0.2 mm deep instead of 2 mm as is normally used for counter-current distribution. Procedure
(7)
where H is the total height, i.e. h, + h,. Thus, rmax increases with the number of transfers n. For a given number of transfers and for given h, and H values rmrtx decreases linearly with vt. Thus, the distance travelled by a particle species along the multiple sedimentation train will decrease linearly with sedimentation time or for a given sedimen-
The experiments were carried out in a plate with 60 cavities. The cavities are numbered 0, 1, 2 . . . 59. 0.7 ml of the suspension of particles to be analysed was introduced in cavity No. 0, and 0.7 ml of suspension medium was added to each of the other cavities. The cavities were shaken by rotary motion for lo-20 set, and the particles were allowed to settle during a given interval of time. After that the top plate was rotated 6 degrees, so that each top chamber was transferred to the adjacent bottom chamber. Then the plates Exptl
Cell Res 73 (1972)
164 P.-d. Albertsson
zfiO/23456 n Tube40~~ 01234567
40 20
::I 0123456
Shorf
r
,
01234567
axis
Long
axis
b
Fig. 3. (a) Abscissa: tube no.; ordinate: absorbance. Multiple sedimentation diagram of potato starch grains. Settling time 30 set, 60 transfers, 24°C. (b) Abscissa: micrometer readings of long and short axes; ordinate: number of grains of various size. Size distribution of grains in the different tubes of (u) by long and short axis. Since the grains were not spherical, the smallest and largest diameters were determined, Expd CelI Res 73 (1972)
Multiple sedimentation
165
tube10 160 120 80 40 200 0 ._lb 02468
02468
240
360 fubel8
200
320 280
260 120
240
eo
200
40
160
0 J;, o.?
4
6
120
8
80 40 0 tube
0
,
38
b
Fig. 4. (a) Abscissa: tube number; ordinate: absorbance. Multiple sedimentation diagram of Sephadex gel (b) Abscissa: micrometer readings of particles (Sephadex G75). Settling time 30 set, 58 transfers, +2-4°C. diameters (arbitrary units); ordinate: number of particles of different sizes. Size distribution of the particles in different tubes of (a).
were shaken again and the particles allowed to settle etc.; the procedure repeated to a total of 58 transfers. Then, the contents of each cavity were collected and analysed.
Absorbance due to the turbidity (400 nm) was measured in a spectrophotometer, and the sizes of the particles were determined microscopically. Exptl Cell Res 73 (1972)
166 P.-A. Albertsson
05 Fig. 5. Abscissa: tube no.; ordinate: AkOO. Multiple sedimentation diagrams of 1 polystyrene latex particles (7.6 pm in I
The amount of material which can be applied is limited by the volume of the lower, stationary layer of the cavities. Approx. 0.15 ml packed cells can be loaded in a single cavity of this apparatus. Since, for an experiment with 60 transfers, 6 cavities can be loaded without significant loss of resolution, approx. 1 ml packed cells can be fractionated in one experiment. In order to select a suitable sedimentation time the approximate sedimentation rate of particles should be known. This can be determined by visually following the sedimentation of particles in a vertical tube, for example a measuring cylinder. If the distances which the boundary has moved is plotted against time the approximate sedimentation rate is obtained from the slope of the curve. Particles
Starch particles were obtained from a commercial preparation of potato starch (Amend). The ,largest particles were removed by first allowing a suspension to stand for a while in a beaker. Sephadex particles were obtained by allowing Sephadex G75 superfine, lot 4995 (Pharmacia, Uppsala) to swell in 0.01 M NaCl for 48 h. Synchronized chlorella pyrenoidosa cells were kindly supplied by Mr ohm Taube, Department of PhysioloExptl Ceil Res 73 (1972)
_. _ . . . _.-mean diameter) obtained using different settling times. The scale to the right refers to the 1 min run, 58 transfers, + 24C.
gical Botany, Umea University. They had been cultivated according to reference [12]. Polystyrene latex particles were obtained from Serva, Heidelberg, as Dow latex. The sizes 7.6, 25.7 and 45.4 pm were used. For polystyrene and Sephadex particles it was necessary to add a small amount of detergent in order to get good wettability. Size determination
Size determination was, except for chlorella cells, carried out with a Zeiss light microscope equipped with an ocular micrometer. The sizes are given in arbitrary units. Chlorella cells were measured by Celloscope 302 (Lars Ljungberg Co., Sweden). It utilizes the same principles as the Coulter counter, i.e. it measures the excluded volume of electrolyte in a small capillary. The diameters of the cells (in microns) were calculated assuming all cells were spheres.
RESULTS The distribution diagrams from experiments with starch grains are shown in fig. 3 together with the size distribution of grains from some of the tubes. As may be seen there is a clear separation according to size. The
Multiple
sedimentation
167
Fig. 6. Abscissa: tube no.; ordinate: absorbance. Multiple sedimentation diagrams of three different polystyrene latex particles (7.6, 25.7 and 45.4 pm in mean diameter). Settling time 1 min, 58 transfers, +24”C. The scale to the right refers to the run with 7.6 pm particles.
larger particles are found, as expected, in the tubes with low number, and the smaller ones in the tubes with higher numbers. For an increase in tube number by 20 there is about a two-fold decrease in the diameter of the particles. A similar experiment with Sephadex particles is shown in fig. 4. A clear separation according to size is evident. Here, too, for an increase in tube number by 20, there is about a two-fold decrease in the diameter of the particles. Most of the Sephadex particles were spherical. A small number of them, however, had small buds sticking out from the spherical surface. These particles were almost all located on the right hand part of the diagram, tubes 40-60. The reduced sedimentation rate of these particles could be due to both a smaller size as well as a larger frictional coefficient resulting from the surface of the particles. Results from the multiple sedimentation of polystyrene latex particles are shown in figs 5 and 6. Since these particles are rather uniform in size they are suitable as test-particles. In fig. 5 are shown the diagrams for “7.6 pm” polystyrene particles obtained using different sedimentation times. As seen, the shorter the sedimentation time the further the particles travel along the row of sedimenta-
tion chambers. Different size polystyrene particles are compared in fig. 6. Fig. 7 shows the distribution diagrams obtained with two synchronous cultures of 2
0 0I 1
14 hrs.
LL 5. I-L--o-3. 25
/-+-La
3.8-48
w
2:::
20
40
60
20
40
60
m-w 20
40
60
Fig. 7. Abscissa: tube no.; ordinate: (a, b) absorbance (690 nm); (c, d) % cells. Multiple sedimentation diagram of the populations of synchronized chlorella cells. Settling time 8 min, 60 transfers, +2-4”C. (a) “0 h cells”, i.e. mainly young cells. (b) “14 h cells”, i.e. mainly older cells. (c) Size distribution of cells from (a). (d) Size distribution of cells from (b). Exptl Cell Res 73 (1972)
168 P.-A. Albertsson chlorella. One culture consists mainly of small cells, the other mainly of large cells prior to division. As seen the diagrams for the two cultures are quite different. The smaller cells have travelled further to the right than the larger cells. This is to be expected since the smaller cells have a smaller sedimentation rate. The size distributions of the different populations of cells are also given in fig. 7. DISCUSSION The multiple sedimentation technique presented here offers several advantages. It shows high selectivity because it is a multistage process. If time is not a limiting factor the resolving power can be increased simply by increasing the number of sedimentation steps. Each particle travels along the cavity train according to its sedimentation rate. The position of a peak therefore gives information on the sedimentation rate of a particle. The breadth or the shape of a multiple sedimentation peak gives information on the heterogeneity in sedimentation rate of a particle suspension. Since the sedimentation rate depends on size, shape and density of the particles all these parameters determine the shape of the diagram. Sedimentation may also be dependent on the concentration of particles. By analysing different fractions under a peak in the microscope for the size and shape of the particles, one can compare this with the multiple sedimentation peak and draw conclusions on difference in densities. Thus if a suspension of particles, uniform in size and shape gives a broader peak than expected from a uniform particle suspension it can be concluded that the suspension is not uniform in the density of the particles. The method can thus be used for analytical purposes in studying size and/or density distribution in a particle population. More Exptl Ceil Res 73 (1972)
work on defined particle suspension is needed, however, to determine the accuracy of the method in this respect. Since different particles are separated from each other into different fractions these can be collected and used for further chemical and morphological characterizations. This should be useful for particles in general, i.e. both of inorganic and biological origin. It should also be possible to change the sedimentation time during the process, thereby allowing analysis and separation of particles with a wide range of sedimentation rate. Other procedures for separation of cells and particles at unit gravity using density gradients or flowing liquids [5, 8, 9, lo] have demonstrated that many cells sediment fast enough to make separation at unit gravity feasible. A particular advantage of the method is that the composition of the sedimentation medium can be chosen almost at will and one can therefore use that which is the most suitable for the dispersion and the preservation of the cells. This is because the medium is constant in all chambers. This is a great advantage over gradient methods where a foreign substance has to be included for establishment of a density gradient. Multiple sedimentation should therefore be useful for separation of fragile cells such as tissue cells. This work has been supported by STU, the Swedish Board for Technical Development. I wish to thank G&e Johansson for valuable criticism.
REFERENCES 1. Albertsson, P-A, Abstracts from symposium on virus and cancer. Swedish Cancer Society, Saltsjobaden, p. 3 (1964). 2. - Analyt biochem 11 (1965) 121. 3. - Science tools 17 (1970) 53. 4. Behrens, M & Marti, H R, Naturwissenschaften 42 (1955) 610.
Multiple sedimentation 169 5. Berg, H C, Purcell, E M & Stewart, W W, Proc natl acad sci US 58 (1967) 1286. 6. Craig, L C & Craig, D, Technique of organic chemistry (ed A Weissberger) vol. III, part 1, 2nd edn. Interscience, New York (1956). 7. Hecker. E. Verteilungsverfahren im Laboratorium. Monbgraphien & Angewandte Chemie und Chemie-Inaenieur-Techniaue. No. 67. Verlan Chemie GmbH, WeinheimlBergstr., German; (1955). 8. Mel, H C, J theor biol 6 (1964) 159.
9. Miller, R G & Phillips, R A, J cell camp physiol 73 (1969) 191. 10. Peterson, E A & Evans, W H, Nature 214 (1967) 824. 11. Walter. H & Albertsson. , P-A. , Exntl - cell res 67 (1971) 218. 12. Walter, H, Eriksson, G, Taube, 0 & Albertsson, P-A, Exptl cell res 64 (1971) 486. 13. Walter, H, Eriksson, G, Taube, i5 & Albertsson, P-A, J cell biol 47 (1970) 220a. -Keceived October 20, 1971
Exptl Cell Res 73 (1972)
(D 1972 by Academic Press, Inc. reproduction in my form reserced
of
Cell Research 73 (1972) 16 l- 169
Experimental
MULTIPLE A Method for Analysis
SEDIMENTATION and Separation
of Particle
Mixtures
P.-b;. ALBERTSSON Department
of Biochemistry,
University
of Umed,Urned,Sweden
SUMMARY 1. A new method for separation of particles, such as cells, is described. It is based on sedimentation at 1 g. The separation obtained by a single sedimentation is increased by a multistage procedure analogous to counter-current distribution between two immiscible phases. 2. The principle of the method is described. 3. Applications of the method on starch grains, Sephadex-gel particles, polystyrene latex particles and chlorella cells are described. In all cases separation according to size is obtained. The apparatus used is a modification of a thin-layer counter-current distribution apparatus described earlier (Anal biochem 11 (1965) no. 1; Science tools 17 (1970) no. 3). 4. The method shows a comparatively high selectivity and it is a mild method towards cells since no foreign material has to be included in the suspension medium.
Sedimentation is a powerful method for separating particles and molecules differing in size, density or shape. For large particles, earth’s gravitational force (1 g) is enough to achieve a fast sedimentation rate while for smaller particles and molecules centrifuges have to be used to speed up sedimentation. In both cases, after a certain time, a sediment enriched in faster sedimenting particles is obtained and may be separated from the supernatant. By such a one-step procedure, however, only a partial separation is obtained. To achieve a more efficient separation a multistage procedure can be used. This paper describes a procedure in which a series of sedimentation steps interspaced by mixings are carried out in order to multiply the effects obtained in a single sedimentation. A preliminary report of this procedure has been published earlier [l]. Its application to blood cells [l I] and the ll-
721807
unicellular algae chlorella [13] is described in separate reports. The theoretical treatment of the procedure is very similar to that of counter-current distribution using two immiscible phases. A similar procedure was used by Behrens & Marti for separating blood cells by repeated centrifugations [4]. Theory Sedimentation in a single chamber. Suppose
that we allow a suspension of uniform particles to sediment in a chamber as shown in fig. 1. After a certain interval of time, an upper layer, height h,, and lower layer, height hb, are separated from each other. We are interested in the ratio between the number of particles present in the top layer and the bottom layer. Let the sedimentation velocity of the particles be U, and suppose their concentration at start is c. If one neglects Exptl
Cell Res 73 (1972)
162 P.-A. Albertsson
n
Fig. 1. Sedimentation chamber. The entire chamber is filled with a suspension of particles. After a time, t, the particles have sedimented a distance of where u is the sedimentation velocity. After this time the two layers h, and h, are separated. The lower layer is enriched in faster-sedimenting particles.
or R J-vt ’ h,+vt
(2)
This ratio is analogous to the partition ratio in liquid-liquid extraction, i.e. the amount of substance in the upper phase divided by the amount of substance in the lower phase. We may therefore, by replacing the partition ratio by R,, use the same theoretical treatment as is used for countercurrent distribution. For calculation of a theoretical distribution curve for a single substance it is convenient to introduce p, the fractional amount of material in the upper layer and q, the fractional amount of material in the lower layer after the sedimentation time t such that Exptl Cell Res 73 (1972)
I
2
.;.:;:
transfer
.I... ... . ;:;2$ n
Brownian motion, then after a time t, the particles have moved a distance U. t. If A is the area of cross section, a number of particles equal to A *v * t * c therefore leave the upper layer, and enter the lower chamber. At the start (t =0) the number of particles in the upper and lower layers was A. h,* c and A*h,*c respectively. Therefore the ratio, R,, between the number of particles in the upper and the lower chamber at time t becomes
(1)
0
0
r 1
ri2
tram n
0
1
2
n
0
I
2
fer
completed
Fig. 2. Principle of multiple sedimentation. A series of chambers (see fig. l), numbered O-II are arranged in a circle. The figure shows a few chambers viz., those numbered 0, 1, 2 and n. All chambers, except the loading chamber 0, are charged with the same volume of the medium of suspension. Chamber 0 is charged with the same volume of a suspension of particles. (A) Start of the experiment. The particles which are uniformly distributed in chamber 0 start to sediment. (B) After a certain settling time the particles have sedimented a certain distance depending on their sedimentation velocity. (C) The upper layer of liquid in each chamber is transferred to the adjacent chamber with the next higher number. (0) Transfer is completed. (E) After the content of each chamber has been mixed, the sedimentation starts again. The particles are now distributed over the two chambers 0 and 1. Those with the higher sedimentation velocity are enriched in chamber 0. One step is now completed and the next can begin. For a drawing of the apparatus used in the experiments, see [2, 31.
pfq=l
(3)
and R, =f
(4)
Multiple sedimentation The multiple sedimentation process involving
a number of steps is illustrated in fig. 2 in a manner analogous to counter-current distribution. The values obtained for the fractional amount present in successive sedimentation cells are the terms of the binomial series (P + q)“, where IZ is the number of transfers. The total amount is constant throughout the entire process so that (p + q)” = 1. The amount in the rth chamber (r can be from 0 to n) expressed as a fraction, T,. r, of the total, after n transfers is given by n!
Tn.,= r!(n-r)!
p’. qcn-r)
(5)
Procedures for calculating a theoretical curve and comparing it with the experimental one are given in standard texts on countercurrent distribution [6, 71. When the amount in each chamber is plotted against r a distribution curve is obtained. The number, rmax of the chamber with the maximal concentration, i.e. the peak of the curve may be calculated from R, by the approximate relation r
- n.Rs mx
l+R,
which in combination n (hi - vt) r max= H
with (2) gives
163
tation time the distance travelled will decrease linearly with the sedimentation rate. Two different particle species will be more separated from each other the longer the time, t, which is allowed for sedimentation in a chamber. However, vt must be smaller than h, for the slower sedimenting particle; otherwise both particle species stay in the lower chamber. Therefore, h, should be chosen as large as possible for maximal separation, i.e. the lower sediment layer h, should be small compared to h,. For practical reasons h,, should not be made too small, however, because the volume capacity of the lower layer will then be too small. In the experiments below, the sediment layer is about one-tenth of the total height of the liquid. Apparatus
A thin-layer counter-current distribution apparatus used for aqueous polymer twophase system has been used for the multiple sedimentation experiments. The apparatus is described elsewhere [2, 31. The only modification introduced for the present experiments was that the bottom plate contained cavities which were 0.2 mm deep instead of 2 mm as is normally used for counter-current distribution. Procedure
(7)
where H is the total height, i.e. h, + h,. Thus, rmax increases with the number of transfers n. For a given number of transfers and for given h, and H values rmrtx decreases linearly with vt. Thus, the distance travelled by a particle species along the multiple sedimentation train will decrease linearly with sedimentation time or for a given sedimen-
The experiments were carried out in a plate with 60 cavities. The cavities are numbered 0, 1, 2 . . . 59. 0.7 ml of the suspension of particles to be analysed was introduced in cavity No. 0, and 0.7 ml of suspension medium was added to each of the other cavities. The cavities were shaken by rotary motion for lo-20 set, and the particles were allowed to settle during a given interval of time. After that the top plate was rotated 6 degrees, so that each top chamber was transferred to the adjacent bottom chamber. Then the plates Exptl
Cell Res 73 (1972)
164 P.-d. Albertsson
zfiO/23456 n Tube40~~ 01234567
40 20
::I 0123456
Shorf
r
,
01234567
axis
Long
axis
b
Fig. 3. (a) Abscissa: tube no.; ordinate: absorbance. Multiple sedimentation diagram of potato starch grains. Settling time 30 set, 60 transfers, 24°C. (b) Abscissa: micrometer readings of long and short axes; ordinate: number of grains of various size. Size distribution of grains in the different tubes of (u) by long and short axis. Since the grains were not spherical, the smallest and largest diameters were determined, Expd CelI Res 73 (1972)
Multiple sedimentation
165
tube10 160 120 80 40 200 0 ._lb 02468
02468
240
360 fubel8
200
320 280
260 120
240
eo
200
40
160
0 J;, o.?
4
6
120
8
80 40 0 tube
0
,
38
b
Fig. 4. (a) Abscissa: tube number; ordinate: absorbance. Multiple sedimentation diagram of Sephadex gel (b) Abscissa: micrometer readings of particles (Sephadex G75). Settling time 30 set, 58 transfers, +2-4°C. diameters (arbitrary units); ordinate: number of particles of different sizes. Size distribution of the particles in different tubes of (a).
were shaken again and the particles allowed to settle etc.; the procedure repeated to a total of 58 transfers. Then, the contents of each cavity were collected and analysed.
Absorbance due to the turbidity (400 nm) was measured in a spectrophotometer, and the sizes of the particles were determined microscopically. Exptl Cell Res 73 (1972)
166 P.-A. Albertsson
05 Fig. 5. Abscissa: tube no.; ordinate: AkOO. Multiple sedimentation diagrams of 1 polystyrene latex particles (7.6 pm in I
The amount of material which can be applied is limited by the volume of the lower, stationary layer of the cavities. Approx. 0.15 ml packed cells can be loaded in a single cavity of this apparatus. Since, for an experiment with 60 transfers, 6 cavities can be loaded without significant loss of resolution, approx. 1 ml packed cells can be fractionated in one experiment. In order to select a suitable sedimentation time the approximate sedimentation rate of particles should be known. This can be determined by visually following the sedimentation of particles in a vertical tube, for example a measuring cylinder. If the distances which the boundary has moved is plotted against time the approximate sedimentation rate is obtained from the slope of the curve. Particles
Starch particles were obtained from a commercial preparation of potato starch (Amend). The ,largest particles were removed by first allowing a suspension to stand for a while in a beaker. Sephadex particles were obtained by allowing Sephadex G75 superfine, lot 4995 (Pharmacia, Uppsala) to swell in 0.01 M NaCl for 48 h. Synchronized chlorella pyrenoidosa cells were kindly supplied by Mr ohm Taube, Department of PhysioloExptl Ceil Res 73 (1972)
_. _ . . . _.-mean diameter) obtained using different settling times. The scale to the right refers to the 1 min run, 58 transfers, + 24C.
gical Botany, Umea University. They had been cultivated according to reference [12]. Polystyrene latex particles were obtained from Serva, Heidelberg, as Dow latex. The sizes 7.6, 25.7 and 45.4 pm were used. For polystyrene and Sephadex particles it was necessary to add a small amount of detergent in order to get good wettability. Size determination
Size determination was, except for chlorella cells, carried out with a Zeiss light microscope equipped with an ocular micrometer. The sizes are given in arbitrary units. Chlorella cells were measured by Celloscope 302 (Lars Ljungberg Co., Sweden). It utilizes the same principles as the Coulter counter, i.e. it measures the excluded volume of electrolyte in a small capillary. The diameters of the cells (in microns) were calculated assuming all cells were spheres.
RESULTS The distribution diagrams from experiments with starch grains are shown in fig. 3 together with the size distribution of grains from some of the tubes. As may be seen there is a clear separation according to size. The
Multiple
sedimentation
167
Fig. 6. Abscissa: tube no.; ordinate: absorbance. Multiple sedimentation diagrams of three different polystyrene latex particles (7.6, 25.7 and 45.4 pm in mean diameter). Settling time 1 min, 58 transfers, +24”C. The scale to the right refers to the run with 7.6 pm particles.
larger particles are found, as expected, in the tubes with low number, and the smaller ones in the tubes with higher numbers. For an increase in tube number by 20 there is about a two-fold decrease in the diameter of the particles. A similar experiment with Sephadex particles is shown in fig. 4. A clear separation according to size is evident. Here, too, for an increase in tube number by 20, there is about a two-fold decrease in the diameter of the particles. Most of the Sephadex particles were spherical. A small number of them, however, had small buds sticking out from the spherical surface. These particles were almost all located on the right hand part of the diagram, tubes 40-60. The reduced sedimentation rate of these particles could be due to both a smaller size as well as a larger frictional coefficient resulting from the surface of the particles. Results from the multiple sedimentation of polystyrene latex particles are shown in figs 5 and 6. Since these particles are rather uniform in size they are suitable as test-particles. In fig. 5 are shown the diagrams for “7.6 pm” polystyrene particles obtained using different sedimentation times. As seen, the shorter the sedimentation time the further the particles travel along the row of sedimenta-
tion chambers. Different size polystyrene particles are compared in fig. 6. Fig. 7 shows the distribution diagrams obtained with two synchronous cultures of 2
0 0I 1
14 hrs.
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Fig. 7. Abscissa: tube no.; ordinate: (a, b) absorbance (690 nm); (c, d) % cells. Multiple sedimentation diagram of the populations of synchronized chlorella cells. Settling time 8 min, 60 transfers, +2-4”C. (a) “0 h cells”, i.e. mainly young cells. (b) “14 h cells”, i.e. mainly older cells. (c) Size distribution of cells from (a). (d) Size distribution of cells from (b). Exptl Cell Res 73 (1972)
168 P.-A. Albertsson chlorella. One culture consists mainly of small cells, the other mainly of large cells prior to division. As seen the diagrams for the two cultures are quite different. The smaller cells have travelled further to the right than the larger cells. This is to be expected since the smaller cells have a smaller sedimentation rate. The size distributions of the different populations of cells are also given in fig. 7. DISCUSSION The multiple sedimentation technique presented here offers several advantages. It shows high selectivity because it is a multistage process. If time is not a limiting factor the resolving power can be increased simply by increasing the number of sedimentation steps. Each particle travels along the cavity train according to its sedimentation rate. The position of a peak therefore gives information on the sedimentation rate of a particle. The breadth or the shape of a multiple sedimentation peak gives information on the heterogeneity in sedimentation rate of a particle suspension. Since the sedimentation rate depends on size, shape and density of the particles all these parameters determine the shape of the diagram. Sedimentation may also be dependent on the concentration of particles. By analysing different fractions under a peak in the microscope for the size and shape of the particles, one can compare this with the multiple sedimentation peak and draw conclusions on difference in densities. Thus if a suspension of particles, uniform in size and shape gives a broader peak than expected from a uniform particle suspension it can be concluded that the suspension is not uniform in the density of the particles. The method can thus be used for analytical purposes in studying size and/or density distribution in a particle population. More Exptl Ceil Res 73 (1972)
work on defined particle suspension is needed, however, to determine the accuracy of the method in this respect. Since different particles are separated from each other into different fractions these can be collected and used for further chemical and morphological characterizations. This should be useful for particles in general, i.e. both of inorganic and biological origin. It should also be possible to change the sedimentation time during the process, thereby allowing analysis and separation of particles with a wide range of sedimentation rate. Other procedures for separation of cells and particles at unit gravity using density gradients or flowing liquids [5, 8, 9, lo] have demonstrated that many cells sediment fast enough to make separation at unit gravity feasible. A particular advantage of the method is that the composition of the sedimentation medium can be chosen almost at will and one can therefore use that which is the most suitable for the dispersion and the preservation of the cells. This is because the medium is constant in all chambers. This is a great advantage over gradient methods where a foreign substance has to be included for establishment of a density gradient. Multiple sedimentation should therefore be useful for separation of fragile cells such as tissue cells. This work has been supported by STU, the Swedish Board for Technical Development. I wish to thank G&e Johansson for valuable criticism.
REFERENCES 1. Albertsson, P-A, Abstracts from symposium on virus and cancer. Swedish Cancer Society, Saltsjobaden, p. 3 (1964). 2. - Analyt biochem 11 (1965) 121. 3. - Science tools 17 (1970) 53. 4. Behrens, M & Marti, H R, Naturwissenschaften 42 (1955) 610.
Multiple sedimentation 169 5. Berg, H C, Purcell, E M & Stewart, W W, Proc natl acad sci US 58 (1967) 1286. 6. Craig, L C & Craig, D, Technique of organic chemistry (ed A Weissberger) vol. III, part 1, 2nd edn. Interscience, New York (1956). 7. Hecker. E. Verteilungsverfahren im Laboratorium. Monbgraphien & Angewandte Chemie und Chemie-Inaenieur-Techniaue. No. 67. Verlan Chemie GmbH, WeinheimlBergstr., German; (1955). 8. Mel, H C, J theor biol 6 (1964) 159.
9. Miller, R G & Phillips, R A, J cell camp physiol 73 (1969) 191. 10. Peterson, E A & Evans, W H, Nature 214 (1967) 824. 11. Walter. H & Albertsson. , P-A. , Exntl - cell res 67 (1971) 218. 12. Walter, H, Eriksson, G, Taube, 0 & Albertsson, P-A, Exptl cell res 64 (1971) 486. 13. Walter, H, Eriksson, G, Taube, i5 & Albertsson, P-A, J cell biol 47 (1970) 220a. -Keceived October 20, 1971
Exptl Cell Res 73 (1972)