Dr. Tarek Elbashiti Assoc. Prof. of Biotechnology

1. Bioseparation Techniques Centrifugation Chapter 6B Dr. Tarek Elbashiti Assoc. Prof. of Biotechnology

2. Centrifugal Filter • A centrifugal filter with a membrane filter or chromatography column can be inserted in a spin tube for carrying out a variety of separation and purification functions on biosolids. • The centrifugal filter can be a micro-spin tube 15-20 mL with an insertion of a membrane module for separation or a chromatography column (also referred to as membrane absorber) for purification. • The centrifugal filter is inserted in a 50 mL spintube. Typically, the combo configuration can be used where centrifuges can reach 12,000 g.

4. The following is an example of both separation and purification of RNA using a centrifugal filter. • The objective is to separate RNA from a suspension containing RNA, DNA, salts, suspended particles, and other impurities. • The centrifugal filter is first inserted with a microfiltration membrane, after which the mixture is added. • Under centrifugation, micron-sized suspended contaminants and particles are removed by the microfiltration membrane.

5. The microfilter is removed from the spintube, and the filtrate is decanted to a separate container. • After cleaning, another microfilter with a chromatography column is inserted in the spintube. • The liquid sample, now free from micron-sized suspended solids, is first chemically conditioned before pouring in the chromatography column. • The liquid mixture runs through the column under G-force that further enhances the rate of the drainage process.

6. Concurrent with liquid draining through the chromatography column, RNA is preferentially adhered to the column. • Next, the column is washed under centrifugation with desalting liquid to remove salts and contaminants. • Further, chemical/buffer liquid is added to the column subject to centrifugation to favor removal of DNA. • Upon completion of removing DNA, purified RNA is released under centrifugal field from the column by elution using a conditioning agent.

7. This example illustrates that the separation and purification process can be readily tailor-made for the specific process. • Each step benefits from the enhanced centrifugal body force despite the liquid possibly being viscous. • Normally takes a long time to flow through a column were this drainage process being carried out under the Earth's gravitational acceleration.

8. Major centrifugation methods for bioanalysis • There are two major forms of centrifugation commonly encountered; namely • Differential centrifugation and Density gradient centrifugation. • Furthermore, as indicated earlier, centrifugation can be used in both preparative and analytical modes, providing powerful tools for bioanalysis.

9. Differential centrifugation • This technique is sometimes referred to as differential sedimentation, and is essentially a process of successive centrifugation (single or repeated steps) with increasing centrifugal force (g). • Separation is predominantly dependent on particle mass and size, where heavier particles or cells settle first at lower g values (e.g. intact cells can sediment at around 800 g).

10. However, in many cases, differential centrifugation is used to separate out intracellular matter, and thus this method is important for so-called subcellular fractionation. • During sub-cellular fractionation, various markers can be used as a quality control measure, giving an assessment of the quality of separation of individual fractions; • for example, DNA can be used as a marker for the step sedimenting nuclei,

12. while the enzyme succinate dehydrogenase can be used as a marker for the step sedimenting mitochondria. • Obtaining a pure organelle fraction from differential centrifugation is nearly impossible.

14. Density gradient centrifugation • As the name implies, density gradient centrifugation utilizes a specific medium that gradually increases in density from top to bottom of a centrifuge tube. • This means that under centrifugal force, particles will move through the medium and density gradient and stop (suspended) at a point in which the density of the particle equals the density of the surrounding medium.

15. The medium used depends on the desired outcome, with four general categories: (i) alkali metal salts (e.g. caesium chloride); (ii) neutral water-soluble molecules (e.g. sucrose); (iii) hydrophilic macromolecules (e.g. dextran); and (iv) synthetic molecules (e.g. methyl glucamine salt of tri-iodobenzoic acid). • The density gradient can be established in the tube either (zonal) or (isopycnic). • Both zonal and isopycnic centrifugation are illustrated in the figure below.

17. For zonal density gradient centrifugation there are a number of media that can be used, however the most common of these is sucrose; • whereas the most common medium for isopycnic density gradient centrifugation is caesium chloride (CsCl). • Equilibrium sedimentation uses a gradient of a solution such as caesium chloride or sucrose to separate particles based on their individual densities (mass/volume). • It is used as a purifying process for differential centrifugation.

18. A solution is prepared with the densest portion of the gradient at the bottom. • Particles to be separated are then added to the gradient and centrifuged. • Each particle proceeds (either up or down) until it reaches an environment of comparable density. • Such a density gradient may be continuous or prepared in a stepped manner. • For instance, when using sucrose to prepare density gradients, one can carefully float a solution of 40% sucrose onto a layer of 45% sucrose and add further less dense layers above.

19. The homogenate, prepared in a dilute buffer and centrifuged briefly to remove tissue and unbroken cells, is then layered on top. • After centrifugation typically for an hour at about 100,000 x g, one can observe disks of cellular components residing at the change in density from one layer to the next. • By carefully adjusting the layer densities to match the cell type, one can enrich for specific cellular components.

21. Isopycnic centrifugation • is a technique used to separate molecules on the basis of density. • (The word "isopycnic" means "equal density".) • Typically, a "self generating“ density gradient is established via equilibrium sedimentation, and then analyte molecules concentrate as bands where the molecule density matches that of the surrounding solution.

22. To illustrate the process, consider the fractionation of nucleic acids such as DNA. • To begin the analysis, a mixture of caesium chloride and DNA is placed in a centrifuge for several hours at high speed to generate a force of about 105 x g (earth's gravity). • Caesium chloride is used because at a concentration of 1.6 to 1.8 g/mL it is similar to the density of DNA.

23. After some time a gradient of the caesium ions is formed, caused by two opposing forces: • diffusion and centrifugal force. • The sedimenting particles (caesium ions) will sediment away from the rotor, and become more concentrated near the bottom of the tube. • The diffusive force arises due to the concentration gradient of solvated caesium chloride and is always directed towards the center of the rotor.

24. The balance between these two forces generates a stable density gradient in the caesium chloride solution, which is more dense near the bottom of the tube, and less dense near the top. • The DNA molecules will then be separated based on the relative proportions of AT (adenine and thymine base pairs) to GC (guanine and cytosine base pairs). • An AT base pair has a lower molecular weight than a GC base pair and therefore, for two DNA molecules of equal length, the one with the greater proportion of AT base pairs will have a lower density, all other factors being equal.

25. Different types of nucleic acids will also be separated into bands, e.g. RNA is denser than supercoiled plasmid DNA, which is denser than linear chromosomal DNA. • Caesium chloride allows for greater precision in separating particles of similar density. • In fact, with a caesium chloride gradient, DNA particles that have incorporated heavy isotopes (13C or 15N for example) can be separated from DNA particles without heavy isotopes.

26. Protein Yield • For a soluble protein expressed from extracellular process, one important measure of the separation performance of the centrifuge is the protein yield Y. • Yield is defined as the ratio of the amount (e.g. kg/min or gm/min) of protein recovered in the liquid product to the amount (kg/min or g/min) of protein in the feed to the centrifuge. • A complete recovery of protein without loss is 100%.

27. Usually the yield should be very high before the separation process can be considered viable. • A 90% or higher yield is not untypical. • The specific yield depends on how difficult the separation is. • For continuous-feed centrifuge, the volumetric rate (L/m) and protein concentration of both feed and centrate need to be measured respectively for calculation of yield.

28. For batch-feed centrifuge, the volume and protein concentration of both feed and supernatant (i.e. centrate) should be measured respectively for the yield calculation. • It is evident that liquid loss in the concentrate or cake affects yield as the protein is dissolved in liquid, therefore the amount of liquid in the concentrate should be minimized.