Density Gradient Centrifugation DGC

DGC, developed in the 1950s, also relies on differential sedimentation behaviour to separate sample components, but compensates for some of the disadvantages of homogeneous media and also allows for the simultaneous separation of multicomponent mixtures. This is accomplished by the use of a density gradient, i.e. a liquid medium that increases in density from the layers nearest the axis of rotation to those farthest away. As will be discussed, this is achieved through variation in the concentration of an aqueous solute, or other gradient material, across the rotor. With minimal precautions, density gradients are surprisingly stable for extended periods, even with the rotor stopped. DGC separations are more extensively used for smaller-scale research applications in contrast to large-scale pelleting separations that are more common to industrial applications. DGC may be conducted as either rate or isopycnic separations.

Rate-zonal separations This technique, also called classification, is used to separate particles of similar density according to size. For batch separations, the sample mixture is layered on top of a preloaded medium, as shown in Figure 4. During a rate-zonal

Separation Mixtures Based Density
Figure 4 Rate-zonal separation in a swinging-bucket rotor. (Courtesy of Beckman Instruments, Inc.)

separation, larger particles sediment more rapidly, just as in a pelleting run. Also similar to a pelleting run, the maximum medium density is lower than the density of the particles being processed. However, unlike pelleting runs, the run must be stopped before particles reach the bottom of the tube or rotor wall, otherwise all sample components will simply sediment to the pellet.

Rate or setting velocity separations may be conducted with a homogeneous medium in batch or semi-batch mode. However, the use of density-gradient media offers several advantages. The steep gradient beneath the layer of sample suppresses premature sedimentation as well as convection currents in the liquid column, both of which lower the separation efficiency. In addition, the continuous increase in density, often accompanied by an increase in viscosity across the rotor, serves to slow the faster-moving particles and provide better resolution in the sample component bands. Increasing-viscosity gradients also lessen diffusional effects, though this advantage may be offset by an increase in the required run time. Rate-zonal separations are well suited for mixtures of particles of similar density that exhibit two or more well-defined modes of size distribution. However, owing to the additional steps and equipment required for DGC as opposed to pelleting, DGC separations are more commonly used to separate particle mixtures based on a parameter other than size, e.g. density.

Isopycnic separations These separations, which are based on differences in particle densities, are conducted in a density gradient. The density range of the gradient often spans the full range of particle densities so that particles never reach the rotor wall, regardless of run time. Instead, particles move through the gradient until they reach a position in which the medium density is the same as their own (Figure 5). As governed by the settling velocity equations (eqns [13] and [14]), particles introduced to the top of a performed gradient sediment relatively quickly at first, with movement slowing as the difference in density between particles and gradient lessens and essentially stopping once the particles reach a position in the rotor where the density of the medium is equal to their own. Particles remain in this terminal position even after the rotor is stopped; this allows them to be recovered as density fractions. Differences in particle size only affect their rate of movement, though this may ultimately dictate the required run time. When the range of particle densities exceeds the range of the density gradient, then a mixture of pelleting and isopycnic separations will occur as some particles fully traverse the rotor and pellet while others attain their isopycnic position and remain suspended. While most density gradients are formed by the loading of solutions of successively higher density to the rotor, it is possible to form such gradients in situ from a homogeneous solution at high

Percoll Sucrose Density Gradient
Figure 5 Isopycnic separation with a self-generating gradient. (Courtesy of Beckman Instruments, Inc.)

centrifugal speeds. This is achieved by routing the solutions to the rotor wall through veins in the central core. When such self-generating gradients are used, it is not necessary that the sample be layered on top of the solution but instead it may be mixed with the medium prior to loading (Figure 5). While self-generating gradients offer greater simplicity, they often require a significant increase in run time. For instance, though the advent of vertical tubes, faster centrifugal speeds, and overspeeding techniques have reduced run times to about one-third of those required only a few years ago, runs of 3 to 12 h are still typical for DNA banding experiments.

Isopycnic separation is a more powerful separation tool than rate-zonal separation in the sense that a generally greater number of particle types can be resolved. However, rate runs may still be preferred for separating large and/or fragile particles, since shorter run times and lower centrifugal forces are used. Run duration is crucial for a rate separation, whereas isopycnic runs simply require a minimum time for the particles to reach a stationary state. It is sometimes useful to conduct a two-dimensional separation in which, for instance, a rate-zonal run generates fractions of particles with similar S values that are further fractionated according to density in an isopycnic separation. The reverse process can also be performed to yield particles of similar density but different particle size distributions.

Gradient materials The selection of an appropriate gradient material is an important consideration as the gradient properties must be compatible with the separation objectives. The desired properties of an ideal gradient material, as set forth by Griffith and by Ridge, are summarized below.

The ideal gradient material should:

• span a density range sufficient to permit separation of the particles of interest without overstress-ing the rotor;

• be inert towards the fractionated materials, including biological activity;

• exert the minimum osmotic effect, ionic strength and pH;

• be removable from the product;

• be readily available and either inexpensive or easily recyclable;

• be sterilizable.

It should not:

• generate a prohibitively high viscosity;

• interfere with the assay technique (e.g. absorb UV or visible light);

• generate flammable or toxic aerosols.

From this list of properties, it is apparent that no single ideal gradient material exists, as each separation problem imposes its own set of requirements. Rather, selection can only be made after a careful evaluation of the gradient properties with respect to the requirements imposed by the separation to be conducted. The list of materials that have been used for gradient formation is extensive with examples of the more commonly used materials along with selected properties listed in Table 1.

With respect to biological inertness and low viscosity, the ideal aqueous gradient material is deuterium oxide (D2O). However, D2O is expensive and has a relatively low maximum density (1.11 g cm-3).

Sucrose was used in the pioneering density-gradient work of Brakke and, due to its low cost, transparency, ready availability and nontoxic nature, is still the most widely used. Densities to 1.33 g cm~3 can be achieved, which is sufficient for separating most cells and intracellular organelles. However, sucrose solutions are not completely physiologically inactive and often contain UV-absorbing components. Mannitol and sorbitol can be used as substitutes to

Table 1 Physical properties of gradient materials in aqueous solutions at 20°C (from Sheeler, 1981)

Gradient material

Tradename

Maximum solution concentration

20% w/w solution

Concentration

Density

Viscocity (cP)

Density

Viscosity (cP)

(% w/w)

(g cm~3)

(g cm~3)

Sucrose

65

1.33

182

1.08

2

Sucrose polymer

Ficoll

43

1.17

600

1.07

27

Colloidal silica

Ludox-SM

-

1.40

-

1.13

2

Colloidal silica

Percoll

23

1.13

10

1.11

8

Metrizamide

56

1.44

58

1.12

2

CsCl

65

1.91

1.3

1.17

0.9

Polytungstate salt

LST

85

2.89

14

1.20

-

Polytungstate salt

SPT

85

2.89

26

1.20

2

compensate for these deficiencies, but use of these sugars has disadvantages including higher viscosity and lower maximum densities. Polysaccharides also have a low osmotic pressure, but again are more viscous than sucrose solutions of equal density and may induce aggregation of the suspended sample via charge interactions.

Silica sols (e.g. Ludox™ and Percoll™), also called colloidal silica, are prepared from small silica particles in mildly alkaline solution. They provide low viscosities and osmotic pressures, even at high densities, and are transparent and inexpensive. Silica sols provide densities to 1.40 g cm~3. Their disadvantages include a tendency to gel at pH < 7 and problems in complete removal from the sample. Percoll™, prepared by coating the silica particles with a polymer, eliminates the gelling problem and provides low viscosity, low osmotic pressure solutions, greater stability at low pH, and densities to 1.21 g cm~3. However, this material is relatively expensive and removal from the sample can be a problem.

Salts are used to generate very high density aqueous solutions. Cesium chloride is by far the most widely used of this class. CsCl solutions can reach densities of & 1.9 g cm~3 at saturation while providing a very low viscosity at lower concentrations. Although expensive, CsCl can be readily recovered and purified. CsCl solutions also have a high osmotic pressure and are corrosive, though the titanium rotors generally used with this solute are relatively resistant. CsCl gradients are commonly used in applications ranging from the separation of viruses and dense cellular macromolecules such as DNA, to geological polymers found in coal or oil shale. Other salts that have been used to produce high density gradients include sodium bromide, sodium iodide, cesium bromide, cesium sulfate, cesium formate, cesium trifluoro-acetate, rubidium bromide and rubidium chloride. Though expensive, tungstate polymers such as sodium polytungstate (SPT) and lithium heteropolytun-gstate (LST) have recently been used to generate aqueous gradients well over 2.5 g cm~3. Applications for these materials include the separation of graphitic carbon and mineral components from fly ash. When using such high density salt solutions, the user should be aware that at high concentration, salts may precipitate on the rotor wall, thereby generating high point densities and the potential for catastrophic rotor failure.

For nonaqueous gradients, organic liquids such as toluene, methanol or kerosene may be blended to attain gradient densities lower than that of water (1.0 gcm~3). Of these, methanol presents an additional advantage of being water-soluble, thereby allowing gradients to be formed from a combination of the two. On the other end of the density scale, halogenated liquids such as diodomethane, bromo-form and tetrabromoethane can be used to prepare very dense solutions over 2.8 g cm~3. Problems associated with flammability, toxicity and attack of transfer lines and seals must be considered when using these materials.

Gradient formation and shape Gradient shape refers to the density profile across the tube or rotor as a function of gradient volume (Figure 6). Its choice is important as it governs the sedimentation rate in both rate and isopycnic experiments as well as the terminal position in isopycnic runs.

Gradients may be classified as step or continuous, as defined by the method of preparation. Step (discontinuous) gradients are prepared by the stepwise addition of solutions of successively higher density to the outer wall or bottom of the rotor. Steps gradients have the advantages that they may be formed without the need for a gradient generator. These gradients may also be readily tailored to provide larger volumes of separation media in the ranges that correspond to the density profile of the particles to be separated, thereby, permitting higher sample loadings. For continuous gradients, including the self-generating variety, the medium density varies in a continuous manner across the rotor or tube. Continuous gradients are classified as linear, exponential or isokinetic.

Figure 6 Gradient shapes: (A) linear; (B) exponential; and (C) isokinetic.

In a linear gradient, density increases linearly with distance from the axis of rotation (Figure 6A), and for cylindrical swing-out rotors, with increasing gradient volume as well. In an exponential gradient, the density increases or decreases exponentially across the rotor, producing convex or concave shapes, respectively, when plotted as a function of radial distance (Figure 6B). Isokinetic gradients are designed to produce a uniform sedimentation velocity throughout the gradient by counterbalancing the increase in centrifugal force particles experience as they traverse the gradient with an increase in the density and viscosity of the medium. Such gradients are often used in analytical rotors to study sedimentation behaviour. Simple linear sucrose gradients loaded in a swinging rotor provide a near isokinetic gradient.

Various methods are used to form gradients. The simplest approach is to form the gradient in situ, i.e. self-generating, by mixing the sample with a single-density medium prior to loading, then forming the gradient at high centrifugal speeds. While this is the simplest approach, higher speeds and longer run times are often required. Step gradients are also easily formed by simply pumping targeted volumes of successively denser solutions to the rotor wall. Inexpensive peristaltic pumps provide the simplest means of loading step gradients. The simplest liner-gradient generators consist of two equivalent cross-section cylinders that contain an initial and a limiting solution, respectively. The chambers are interconnected at the base with liquid from the limiting solution being drawn into and mixed with the initial solution as material from the initial-solution chamber is loaded. Exponential gradient generators are similar except that the cross-sectional area of one of the chambers changes in a predetermined manner as the chambers are depleted, thereby changing the relative volume contributed from the two chambers with time. More sophisticated gradient pumps are available including mechanical pumps that use cams to mix variable amounts of low and a high density solution prior to loading or programmable pumps, e.g. a liquid chromatograph pump, to generate the targeted gradient curve shape.

Several approaches are used to analyse and/or fractionate the rotor effluent. The simplest is to split the gradient into fractions according to volume, then subsequently analyse each fraction by chemical (density, absorbance, refractive index, fluorescence) or scintillation methods. However, this approach may be somewhat limited in resolution if the collected fractions are large, and thus represent a wider range in density. An alternative approach is to route the effluent through one or more in-line, low volume flow cells to monitor the gradient properties. Auto mated fractionators that select cut points and automatically switch collection vessels rely on such in-line detectors.

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