Bioseparation Techniques

1. Bioseparation Techniques 2. Properties of biological material

2. Fundamental properties of biological substances, relevant in separation processes: 1. Size 2. Molecular weight 3. Diffusivity 4. Sedimentation coefficient 5. Osmotic pressure 6. Electrostatic charge 7. Solubility 8. Partition coefficient

3. Size • The size of biological material is important in separation processes such as: • Filtration, membrane separation, sedimentation, centrifugation, size exclusion chromatography, gel electrophoresis, • The size of particulate matter such as cells, cell debris and macromolecular aggregates can be measured by direct experimental techniques such optical and electron microscopy. • Indirect methods such as Coulter counter laser light scattering techniques are also used for determining particle size.

4. Nature of the material to be sized, e.g. estimated particle size and particle size range solubility ease of handling toxicity Cost capital running Specification requirements Time restrictions Choosing a method for particle sizing

5. For thick particles, the sedimentation rate, i.e. the rate of settling under gravity in a fluid having a lower density can be used to measure particle size. • Gravitational settling is possible only with particles larger than 5 microns in diameter. • The equivalent radius (re) of a particle settling under gravity can be estimated from its terminal velocity:

6. Microscopy • Sieving • Sedimentation techniques • Optical and electrical sensing zone method • Laser light scattering techniques • (Surface area measurement techniques)

7. Optical microscopy (1-150µm) Electron microscopy (0.001µ-) Being able to examine each particle individually has led to microscopy being considered as an absolute measurement of particle size. Can distinguish aggregates from single particles When coupled to image analysis computers each field can be examined, and a distribution obtained. Number distribution Most severe limitation of optical microscopy is the depth of focus being about 10µm at x100 and only 0.5µm at x1000. With small particles, diffraction effects increase causing blurring at the edges - determination of particles < 3µm is less and less certain. Microscopy

8. For submicron particles it is necessary to use either TEM (Transmission Electron Microscopy) or SEM (Scanning Electron Microscopy). TEM and SEM (0.001-5µm)

9. Manual Optical Microscopy Advantages Relatively inexpensive Each particle individually examined - detect aggregates, 2D shape, colour, melting point etc. Permanent record - photograph Small sample sizes required Disadvantages Time consuming - high operator fatigue - few particles examined Very low throughput No information on 3D shape Certain amount of subjectivity associated with sizing - operator bias

10. Transmission and Scanning Electron Microscopy Advantages Particles are individually examined Visual means to see sub-micron specimens Particle shape can be measured Disadvantages Very expensive Time consuming sample preparation Materials such as emulsions difficult/impossible to prepare Low throughput - Not for routine use

12. Sieve analysis is performed using a nest or stack of sieves where each lower sieve has a smaller aperture size than that of the sieve above it. Sieves can be referred to either by their aperture size or by their mesh size (or sieve number). The mesh size is the number of wires per linear inch. Approx. size range : 5µm - ~3mm Sieving

13. Sieving may be performed wet or dry; by machine or by hand, for a fixed time or until powder passes through the sieve at a constant low rate Wet sieving Air-jet sieving Weight distribution

14. Advantages Easy to perform Wide size range Inexpensive Disadvantages Wear/damage in use or cleaning Irregular/agglomerated particles Rod-like particles : overestimate of under-size Labour intensive

15. Instrument measures particle volume which can be expressed as dv : the diameter of a sphere that has the same volume as the particle. The number and size of particles suspended in an electrolyte is determined by causing them to pass through an orifice an either side of which is immersed an electrode. The changes in electric impedance (resistance) as particles pass through the orifice generate voltage pulses whose amplitude are proportional to the volumes of the particles. Volume distribution Electrical sensing zone method – Coulter Counter

16. Obscuration of light source relates to particle size (area) Advantage of not requiring medium to be an electrolyte Optical sensing zone method

17. Laser Diffraction Particle Size Analysis (Particle size range 0.02-2000µm) Photon Correlation Spectroscopy (Particle size range :1nm to 5µm) Laser light scattering techniques

18. Particles pass through a laser beam and the light scattered by them is collected over a range of angles in the forward direction. The angles of diffraction are, in the simplest case inversely related to the particle size. The particles pass through an expanded and collimated laser beam in front of a lens in whose focal plane is positioned a photosensitive detector consisting of a series of concentric rings. Distribution of scattered intensity is analysed by computer to yield the particle size distribution. Laser diffraction • Volume distribution

19. Advantages: Non-intrusive : uses a low power laser beam Fast : typically <3minutes to take a measurement and analyse. Precise and wide range - up to 64 size bands can be displayed covering a range of up to 1000,000:1 in size. Absolute measurement, no calibration is required. The instrument is based on fundamental physical properties. Simple to use Highly versatile Disadvantages: expense volume measurement all other outputs are numerical transformations of this basic output form, assuming spherical particles must be a difference in refractive indices between particles and suspending medium

20. Large particles move more slowly than small particles, so that the rate of fluctuation of the light scattered from them is also slower. PCS uses the rate of change of these light fluctuations to determine the size distribution of the particles scattering light. Comparison of a "snap-shot" of each speckle pattern with another taken at a very short time later (microseconds). The time dependent change in position of the speckles relates to the change of position of the particles and hence particle size. The dynamic light signal is sampled and correlated with itself at different time intervals using a digital correlator and associated computer software. The relationship of the auto-correlation function obtained to time intervals is processed to provide estimates of the particle size distribution. PCS

23. Advantages: Non-intrusive Fast Nanometre size range Disadvantages: Sample prep critical Vibration, temperature fluctuations can interfere with analysis Restricted to solid in liquid or liquid in liquid samples Expense Need to know R.I. values and viscosity

24. Molecular weight • For macromolecules and smaller molecules, the molecular weight is often used as a measure of size. • Molecular weight is typically expressed in Daltons (Da) or g/g-mole or kg/kg-mole (It should be remembered that the IUPAC (The International Union of Pure and Applied Chemistry) accepted unit for molecular weight is the atomic mass unit (amu) not the Dalton, although they are the same in value). 1.66 × 10-27 kg. • Table 2.2 lists the molecular weights of some biological substances.

25. With nucleic acids, such as plasmids and chromosomal DNA, the molecular weight is frequently expressed in term of the number of base pairs of nucleotides present (bp). • One base pair is roughly equivalent to 660 kg/kg-mole. • Molecular weight being linked to size is used as a basis for separation in techniques such as gel-filtration, hydrodynamic chromatography and membrane separations.

27. The molecular weight of a substance also influences other properties of the material such as sedimentation, diffusivity and mobility in an electric field and can hence be an indirect basis for separation in processes such as ultracentrifugation and electrophoresis. • As with particle size, the molecular weight of certain substances can be polydispersed • i.e. may demonstrate a molecular weight distribution.

28. Examples include the polysaccharides dextran and starch, both of which have very large molecular weight ranges. • Size-exclusion chromatography is a column based method where separation takes place based on size.

29. Western Blot • Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples.

30. …Western Blot • Proteins are separated by gel electrophoresis, usually SDS-PAGE. • The proteins are transferred to a sheet of special blotting paper called nitrocellulose. • The proteins retain the same pattern of separation they had on the gel.

31. ..Western Blot • The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. • An antibody is then added to the solution which is able to bind to its specific protein. • The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time.

32. ..Western Blot • The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.

33. ..Western Blot

34. SDS-PAGE (PolyAcrylamide Gel Electrophoresis) • SDS-PAGE,sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, forensics, genetics and molecular biology: • to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight). • to separate proteins according to their size, and no other physical feature.

35. …SDS-PAGE • SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfATE) attached to it.

36. Fig.1Before SDS: Protein (pink line) incubated with the denaturing detergent SDS showing negative and positive charges due to the charged R-groups in the protein. The large H's represent hydrophobic domains where nonpolar R-groups have collected in an attempt to get away from the polar water that surrounds the protein. After SDS: SDS disrupt hydrophobic areas (H's) and coat proteins with many negative charges which overwhelms any positive charges the protein had due to positively charged R-groups. The resulting protein has been denatured by SDS (reduced to its primary structure-aminoacid sequence) and as a result has been linearized.

37. ..SDS • SDS (the detergent soap) breaks up hydrophobic areas and coats proteins with negative charges thus overwhelming positive charges in the protein. • The detergent binds to hydrophobic regions in a constant ratio of about 1.4 g of SDS per gram of protein.

38. ..SDS • Therefore, if a cell is incubated with SDS, the membranes will be dissolved, all the proteins will be solubalized by the detergent and all the proteins will be covered with many negative charges.

39. PAGE • If the proteins are denatured and put into an electric field (only), they will all move towards the positive pole at the same rate, with no separation by size. • However, if the proteins are put into an environment that will allow different sized proteins to move at different rates. • The environment is polyacrylamide. • the entire process is called polyacrylamide gel electrophoresis (PAGE).

40. ..PAGE • Small molecules move through the polyacrylamide forest faster than big molecules. • Big molecules stays near the well.

41. …SDS-PAGE • The end result of SDS- PAGE has two important features: 1) all proteins contain only primary structure & 2) all proteins have a large negative charge which means they will all migrate towards the positive pole when placed in an electric field.

42. The actual bands are equal in size, but the proteins within each band are of different sizes.

43. Sample of SDS- PAGE