Dialysis

Dialysis

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  Dialysis
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   Dialysis -A process, by which small molecules are selectively removed from a sample containing mixture of both small and large molecules (Based on size).  Semi-permeable membrane is used that allows small molecules to pass freely through, holding the large molecules inside.  Membranes- made of cellulose derivatives.  Cellulose – naturally occurring homopolymer of glucose. Glucose units connected through β (1→4) linkages.
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  Molecular weight cut-off Dialysis membranes – characterized by their Nominal Molecular weight cut-off (NMWCO).  NMWCO depends primarily on the pore size of the membrane.  Nominal – The separation is based not only on the size but also on the shape and charge of the molecule.  Molecules small enough to pass through the tubing (often water, salts and other small molecules) tend to move into or out of the dialysis bag, in the direction of decreasing concentration.  Larger molecules (often proteins, DNA, or polysaccharides) that have dimensions significantly greater than the pore diameter are retained inside the dialysis bag.
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  Protein purification  Toremove the salt from a protein solution.  Dialysis bag will be filled with the concentrated solution containing proteins.  Molecules that are small enough to pass through the pores of the membrane diffuse out of the bag into the buffer solution or dialysate.  Molecules go from an area of high concentration to low concentration.  When the level of concentration is equal between the bag and the buffer, there is no more net movement of molecules.
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   The bagis taken out and inserted into another buffer, causing the concentration to be higher in the bag relative to the buffer. This causes more diffusion of molecules.  This process is repeated several times to ensure that all or most of the unwanted small molecules are removed.  In general, dialysis is not a means of separating proteins, but is a method used to remove small molecules such as salts.  At equilibrium, larger molecules that are unable to pass through the membrane remain inside the dialysis bag while much of the small molecules have diffused out.
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  Pretreatment of dialysismembrane  To remove undesirable impurities such as glycerol, heavy metals, sulphides  Membranes generally supplied with 10% glycerol (added as humectants to prevent the brittleness of the membrane) and can be removed by washing in running water – distilled water  Always store the membrane at 4C in immersed condition.  Since the membrane is cellulosic nature- high affinity for heavy metals, and these heavy metals bound to tubing during manufacture.  Heavy metals can be removed by boiling the tube in 10 mM EDTA solution.  To remove sulphide, the tube is boiled for 10 min in 2% sodium bicarbonate.
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  Procedure  Tubings ofrequired length is taken and both inside and outside of the tubing is rinsed with water.  One end of the tubing is tied securely with the thread, through the open end, fill the tubing with distilled water and check for leakage.  Water is removed, the sample solution is filled 2/3 using a pipette or funnel  The open end also closed securely by tying with a thread.  The bag is placed in the buffer solution (50-100-fold buffer volume with respect to sample volume can be used) and dialyzed for 3-4 hours at required temperature.  Small molecules will be removed from the bag and the buffer can be changed after 3-4 hours.
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  Factors affecting therate of dialysis Solvents  The rate of dialysis is the highest in distilled water.  Most biomolecules like enzymes, proteins and nucleic acids is not stable in distilled water.  Dialysis is done in buffer of desired pH and ionic strength in which the molecules are stable. Temperature  Rate of dialysis increases by increasing the temperature of the solvent.  Temperature  rate of diffusion of small molecules.  But most of the macromolecules are sensitive to high temperatures and hence done at 4C.
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  Pressure  Pressure Rate of dialysis. Concentration gradient  The driving force in dialysis is concentration gradient.  Large concentration gradient between inside and outside of the bag increases the rate of dialysis. Diffusion constant  Small, spherical molecules are dialyzed faster than large, fibrous molecules.  Thickness, area and porosity of the membrane  Rate of dialysis is faster in thin membranes than in thick membranes.  Large membrane area effects dialysis than small membrane area.  High porosity increases the dialysis than low porosity.
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  Reverse dialysis  Duringdialysis osmosis causes water molecules to enter dialysis bag.  If high molecular weight compound like Sephadex G-25, G-50 or sucrose is used than that of solvent, the water molecules move out of the dialysis bag.  This process is known as reverse dialysis or reverse osmosis.  In this process, the amount of sample can be reduced to few ml. Electrodialysis  Application of electric field enhances the rate of dialysis and known as electrodialysis.  Cation and anion exchange membranes are available for electrodialysis.
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  Applications  To removesmall molecules such as salts from protein and Nucleic acid samples.  To introduce new buffer system to samples.  To electroelute DNA or RNA fragments after electrophoresis separation.