Gene Lauffer of Menardi for kind permission to use images of filtration units from their website (Figures 10.7 and 10.10 in this book). Shuichi Ogawa of Asahi Kasei for kind permission to use the image of membrane filtration unit from their website (Figure 11.16 in this book).
Overview of bioseparations engineering
- Introduction
- What is separated in bioseparation?
- Economic importance of bioseparation
- Nature of bioseparation
- Basis of separation in bioseparation processes
- Bioseparation techniques
- The RIPP scheme
- Example of bioseparation
- Current trends in the bioseparation
This type of separation can be achieved by zonal centrifugation which involves introducing the mixture to a location within a. An indirect way to perform solute-solvent separation is by precipitation, which involves the selective precipitation of the target solute.
Properties of biological material
Introduction
Nucleic acids such as DNA and RNA are linear molecules (see Fig. 2.3) and their sizes cannot be expressed in terms of the Stokes-Einstein diameter. The height of the suspension in the container was 30 cm and the density of kaolin is known to be 2.6 g/cm3.
Molecular weight
With macromolecules such as proteins and nucleic acids, size can be estimated using indirect methods. The diffusion coefficient can be measured using some of the experimental techniques discussed in the previous section.
Sedimentation coefficient
The diffusivity of a protein that has a Stokes-Einstein radius of 2 nanometers in a certain liquid is known to be 4.5 x 10"11 m2/s. Predict the diffusivity of another molecule that has twice the Stokes-Einstein radius in the same liquid at the same temperature.
Osmotic pressure
It should be noted that the osmotic pressure acts from the lower concentration side to the higher concentration side.
Electrostatic charge
This is due to the presence of a large number of phosphate groups on these molecules.
Solubility
However, at higher ionic strengths, protein solubility is found to decrease significantly with increasing salt concentration.
Partition coefficient
Light absorption
The emitted light in fluorimetry is measured at right angles to that of the incident light (Fig. 2.5) to avoid interference of the transmitted light. Which molecule has the largest diameter and by what percentage is this diameter greater than that of the others.
Introduction
Convective mass transfer occurs in flowing fluids especially when the flow is turbulent in nature i.e. In convective mass transfer, solute flux occurs due to a combination of molecular diffusion and vortex diffusion.
Molecular diffusion in liquid medium
In equation (3.2), the flux is denoted NA to distinguish it from JA. which is generally used for flux purely due to molecular diffusion. If we consider a simple case of molecular diffusion, e.g. Similarly, the flux of B from 2 to 1 is given by: .
Measurement of diffusivity
There are several other techniques for measuring diffusivity, some of which are variants of the technique discussed above. These correlations link diffusivity to solute and fluid properties such as molar volume, molecular weight and fluid viscosity.
Diffusion of solutes in dense solid
Diffusion of solutes in porous solid
The mass transfer coefficient of the protein in the radial direction was found to be 1 x 10"6 m/s. In this problem, the diffusion length is the radius of the tube, which is 0.5 mm.
Experimental determination of mass transfer coefficient
Estimation of mass transfer coefficient
Inter-phase mass transfer
Unsteady state mass transfer
To simplify the problem, we assume that the concentration of the solute in the solution next to the plate remains constant. We also assume that the solute concentration on either side of the solution-slice interface is identical, i.e. the partition coefficient is one.
Equilibrium and rate processes
A membrane with a porosity of 0.3, an average pore size of 0.01 microns, an average pore tortuosity of 1.1, an area of 2 cm2, and a thickness of 0.1 mm separates two water-filled, well-mixed chambers, one with a volume of 10 mL and the other with a volume of 20 ml. Human immunoglobulin G (HIgG) diffuses through a porous medium with a porosity of 0.5, a tortuosity of 1.8, and an average pore diameter of 0.45 microns.
Cell disruption
- Introduction
- Cells
- Cell disruption using bead mill
- Cell disruption using rotor-stator mill
- Cell disruption using French press
- Cell disruption using ultrasonic vibrations
- Cell disruption using detergents
- Cell disruption using enzymes
- Cell disruption using organic solvents
- Cell disruption by osmotic shock
The cell disruption induced within the rotor-stator mill can be described using the equations discussed for a bead mill. Cell disruption occurs mainly due to the high shear rate impact from the cells within the hole.
Precipitation
- Introduction
- Factors utilized for precipitation
- Precipitation using organic solvents
- Precipitation using anti-chaotropic salts
Solvents such as ethanol and acetone precipitate proteins by reducing the dielectric constant of the solution. S = protein solubility (kg-mol/m3) Sw = protein solubility in water (kg-mol/m3) A = constant.
Centrifugation
Introduction
This means that the particle will experience increasing values of G as it moves towards the bottom of the centrifuge tube. 34;max = radial distance from the axis to the bottom of the tube (cm) rmin = radial distance from the axis to the top of the tube (cm) rpmmax = maximum rotation speed (/ min).
Preparative centrifuge
The feed flow in a tubular bowl centrifuge is shown in Fig 6.5: the diameter of the tubular bowl is r, while the diameter of the annular space is ra. The second term in square brackets is called the sigma factor (S) of the centrifuge.
Ultracentrifugation
The liquid flows up the device along the central regions and is discharged from above. The top of the tubes was 5 cm away from the shaft and it took 15 minutes at a rotation speed of 10,000 rpm to completely sediment the cells.
Extraction
Introduction
Such phase diagrams rely on the fact that all possible combinations of the three components can be represented by the area within the triangle. The tie lines are straight lines connecting the compositions of the two phases, which are in equilibrium with each other.
Theory of extraction
When the equilibrium distribution is reached, the chemical potential of the solute in both phases must be the same. The transfer of a solute between two partially or completely immiscible liquids requires close contact between the two.
Aqueous two-phase extraction
This eventually leads to the separation of the two liquids into different layers due to density difference. In PEG/dextran aqueous two-phase extraction of proteins, the distribution behavior depends to a large extent on the relative polymer composition.
Batch extraction
The equilibrium ratio is given by CE = 100 CR1, where CR and CE are the concentrations of citric acid in the raffinate and extract, respectively, and are expressed in g/1. a) Concentration of citric acid in raffinate and extract. If the extract obtained in this way then comes into contact with a further 100 liters of an aqueous solution of citric acid (concentration = 1 g/l), calculate: . c) Citric acid concentration in the raffinate and extract phases of the second extraction.
Single-stage continuous extraction using immiscible solvents The single-stage continuous extraction process is merely an extension of
At pH 3.0 the equilibrium relationship is given by CE = 40CR, where CR and CE are the concentrations of antibiotics in the raffinate and extract respectively and are expressed in g/1. At pH 7.0 the equilibrium relationship is given by CE = 37Q, where CE is the concentration of the antibiotic in the aqueous extract phase while CR is the concentration in the organic refining phase.
Batch extraction using partially miscible solvents
Continuous cross-flow extraction using immiscible solvents Every extraction process is limited by an equilibrium constant.
Cross-current continuous extraction using immiscible solvents Any extraction process is limited by the equilibrium constant. In order to
Staged counter-current extraction
The same equilibrium relationship holds for all phases, i.e., the value of K is independent of the solute concentration. The aqueous feed enters the extractor at a rate of 100 kg/h while the organic solvent enters the extractor at a rate of 20 kg/h.. a) Extract composition and raffinate.
Differential extraction
The term (QR I {kR a)) is the film transfer unit height on the raffinate side and is denoted by hR. NTUE = number of transmission units on the outlet side (-) hE = height of the transmission unit on the outlet side (m).
Supercritical fluid extraction
Gaseous carbon dioxide is first converted into a supercritical fluid by adiabatic compression, which is then fed into the extraction vessel. The carbon dioxide is separated from the ethanol-water mixture and sent back to the compressor.
Adsorption
Introduction
These physical forms result in very high specific surface areas (i.e. surface area per unit amount of adsorbent). Particulate adsorbents can be used in the form of a suspension or in the form of a packed bed.
Separation mechanisms
The ionic strength of the feed solution also affects the amount of molecule bound on the adsorbent. Therefore, a positively charged protein can be eluted from a cation exchange adsorbent by increasing the pH of the solution above the isoelectric point of the protein.
Adsorption isotherms
Adsorption of proteins based on hydrophobic interactions takes place at very high concentrations of anti-chaotropic salts, typically 1 M or more. The Freundlich isotherm does not predict the saturation of the interface with the solute.
Diffusional limitations in adsorption processes
The Langmuir isotherm is useful when there is a strong specific interaction between the solute and the adsorbent. External mass transport (which is controlled by diffusion from the base solution to the surface of the adsorbent).
Batch adsorption
The adsorption follows a linear isotherm and the concentration of the hormone in the liquid is 0.01 g/1. 80% of the hormone could be adsorbed in the batch mode by 10 ml of affinity adsorbent.
Packed bed adsorption
The last step is the elution step which is performed by pumping through the packed bed, a fluid that facilitates desorption of the target molecule. The sequence of events in packed bed adsorption can be followed by monitoring the composition of the effluent stream (see Figure 8.17).
VMM 1
- Other types of adsorption devices
The depletion concentration (C") represents the concentration corresponding to the saturation of the filler layer with the target molecule. We must further assume that the shape of the concentration profile within the filler layer does not change with time.
Chromatography
Introduction
This results in the sample being fed into the column with the mobile phase. In a chromatographic separation process, the concentration of the individual components in the column effluent stream must be controlled.
Theory of chromatography
This interaction results in the dynamic distribution of the solute between the mobile phase and the stationary phase during its journey through the column. This depends on the difference in the interaction of the two solutes with the stationary phase and the flow rate of the mobile phase, but is independent of the solute concentrations.
Shape and yield of a chromatographic peak
Chromatograms (see figures below) were obtained for two different compounds A and B by injecting pure samples of these substances into a 30 cm column. We want to separate A and B at the same mobile phase flow rate from a mixture containing the same amounts of these substances as used to obtain the chromatograms.
1 HTT ^
IIIUMI II
Binary chromatography
The second mobile phase in binary chromatography is chosen such that in its presence bound solutes cannot continue to interact with the stationary phase and are therefore released. Solute A appeared in the effluent at about the same retention time as in the mobile phase.
Hydrodynamic chromatography
If the effluent were collected between 4 and 6 minutes, what fraction of the amino acid would be recovered. If the retention time of the antibiotic at the optimal mobile phase rate is 3 minutes, predict the characteristic peak width.
Filtration
Introduction
Surface filtration: Particles are captured by a scanning action and held on the outer surface of the filter. They are retained within the filter by three mechanisms: direct interception, inertial impaction, and diffusive interception.
Filter medium
Pressure filtration can be operated by pressurizing the inlet with the help of compressed air or by maintaining the hydrostatic height of the liquid on the inlet side. Vacuum filtration is preferable from a safety point of view, since an explosion is more dangerous than an implosion.
Constant pressure cake filtration
