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some concept of Novel Separation Processes are Asymmetric Membrane, Centrifugal Separation Processes, Cloud Point Extraction, Colloidal Particles, Common Stationary Phase.Main points of this lecture are: Electrophoretic Separation, Migration, Separation, Biomolecules, Isoelectric, Negatively Charged, Aspartic Acid, Borine Serum Albumin, Electrophoretic Mobility, Spherical Ion
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Separation processes, membranes, electric field assisted separation, liquid membrane, cloud point extraction, electrophoretic separation, supercritical fluid extraction
Different rates of migration of charged particles/molecules in an electric field are useful for separation. This principle can be utilised quite effectively for the separation of biomolecules as most of them are having charges. Each such molecule has a characteristic pH, known as isoelectric pH, at which, the biomolecule is neutral. One can control the operating pH by addition of few drops of acid or alkali. Thus, if the operating pH is set above the isoelectric pH (pI), the molecule becomes negatively charged and if the operating pH is set below the isoelectric pH, it is positively charged.
Compound Mw pI(25^0 C) Aspartic acid 2. Lysine 9. Ovalbumi 43000-45000 4. Borine serum albumin 68000 4. Misoglobin ~17000 7. Cytochrome C ~12000 9.
As discussed in Chapter 4, the electrophoretic mobility of a charged spherical ion in an electrolytic solution becomes,
(^6) p u v^ ze = (^) E = (^) πη R (9.1)
For charged colloids, the system contains small amount of electrolytes to carry appreciable current. This causes redistribution of counter and co-ions around charged colloids i.e., within electrical double layer.
3 3
stronger. There exist several methods to avoid convection: i. Efficient cooling reduces Δ T and prevents thermal degradation. ii. Electrophoresis is conducted at 4^0 C, where water density is maximum. iii. Difference between plates ‘ h ’ is kept small or electrophoresis is done in small tube, pores of a gel, membrane or paper capillary. Introduction of gel/membrane to stop convection leads to sieving of molecules/particles which is undesirable as it involves hindered migration. Electroosmosis is another complicating factor in electrophoresis. Fixed charges on chamber wall or gel are subjected to a force due to presence of the electric field. In this case, charges are fixed and fluid moves. This fluid movement is defined as electroosmosis.
Effect of electroosmosis is reduction in separation. The extent of reduction is a function of geometry of the flow channel. The deleterious effects of electroosmosis can be prevented, if wall is coated by a material like methyl cellulose which haas a low zeta potential, so that the mobility due to electroosmosis is marginalized.
To prevent the natural convection, electrophoresis is conducted in a stabilizing porous media, e.g., gel or membrane. This method is known as zone electrophoresis. This system
behaves exactly like, chromatography. Some stabilizing media should be used. These should have the following characteristics: i. Inert, should not react with species. ii. No residual charges on the media so that no ion exchange or electroosmosis takes place. iii. Pore size should be greater than the molecular size so that no hindered transport occurs.
A typical schematic of laboratory scale gel electrophoresis set up is presented in Fig. 9.1.
Cooling Plate
Support Gel Sample Plate
Cover
Cathode
Anode
Fig. 9.1: Laboratory gel electrophoresis cell
A 1 A 2
O
C 2 C 1
Distance
Concentra
tion
Δσ
A 1 , A 2 : anions O: zero charge C 1 , C 2 : cations
it is remarkable that each protein-SDS complex has the same charge to mass ratio. Therefore, all complexes have similar electrophoretic mobility in free solution. But SDS- protein complexes have different sizes and can be separated based on protein size by using sieving property of polyacrylamide gel. This method is helpful in estimating relative masses.
Like chromatography, a pulse of feed results a Gaussian distribution response at the exit of the set up.
max, (^2 )^2 exp (^2) 1, i i^ i i c c z^ z
peak. The total electrophoretic velocity of the species becomes, v net = uE + uosmE (9.8),
where the electric field strength is,
E V = (^) L (9.9)
L is the maximum length of migration. If tR is the average retention time to migrate a distance zi, then the expression of retention time is,
( ) Ri^ i^ i i i osm t z^ z L = (^) u = (^) u + u V (9.10)
The expression of zi is thus obtained.
z i = v ti Ri = ⎛⎜⎝ ui VL^ + uosm VL ⎠^ ⎞⎟ tRi (9.11)
Since, the chromatographic run is for a fixed time, we have, tRi = t exp t. Thus, different particles will traverse different distance. The width of the concentration curve is given by Einstein’s equation.
Deff i (^) , is effective axial dispersion coefficient in electrophoresis system. Thus inserting the expression of experimental time, the following equation is obtained.
( ) 1,^2 i^2 eff i^ ,^ i i osm
D z L
The resolution (R) between two zones can be obtained as,
R (^) ( )
(^12) (^1) exp 1 2 (^4 )
t eff
V t u u L (^) D
( ) (
(^12) (^14 2) Deff u max v (^) uosm u 1 (^) u 2 = ⎛⎜^ ⎞⎟ ⎜⎝ (^) + ⎟⎠ −^ ) (9.14) Where, t exp (^) t = (^) v max L^ = (^) ( u max + L 2 uosm ) V and^ Deff^ =^ D^ eff^^1 + 2 Deff^2. This is generally set to the fastest migrating species travels a distance ‘ L ’. This is done by adding a dye as marker. It may be noted that R increases if v increases.
Solved Problems
1. A batch electrophoresis is done in PAGE where μosm =0; μA =1.0510-5^ cm^2 /v.s. ; DA=DB= B 1.510 -7^ cm /s.^2 E= 125 v/cm. for 2.5 hrs Find location of peaks (distance from feed well), peak widths and resolution of two proteins?
Deff = D^ A^ + DB = × − cm s σ (^) 1,^2 i = 2 Deff tR i , = 2 × 1.6 × 10 −^7 × (^) ( 3 × (^3600) ) = 3.46 × 10 −^3 cm^2
Resolution = R = 14 E (^) 2 t D exp eff ( μ 1 −μ 2 )
= 14 × 100 × (^2) ×^3 1.6 ×^3600 × 10 − 7 ( 1.02 − (^1) ) × 10 −^5 = 0. R can be increased by decreasing E or by increasing texp.
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