Introduction:
The dialysis process of a solute occurs at the membrane surface, in context to the concentration differences of two solutions on either side of the membrane. This process is affected by the variables of temperature, viscosity, and pressure gradient across the membrane.
Also it is affected by the solvent, pore size, and the nature of the membrane. The rate of dialysis is greatest in distilled water.
Dialysis (Which is not considered to be a form of chromatography) is a process that separates molecules according to size through the use of semi-permeable membranes.
“It is a simple process in which small solutes diffuse from a high concentration solution to a low concentration solution across semi-permeable membranes until equilibrium is reached. Since the porous membrane selectively allows smaller solutes to pass while retaining larger species, dialysis can effectively be used as a separation process based on size rejection”.
Advantages of lab dialysis:
• Very gentle conditions
• Easy operation
• Wide range of samples volume
• Many membrane types & MWCO’s
• Inexpensive materials
• Disposable membranes & devices
Dialysis application:
• Macromolecular purification
• Protein concentration
• Solute fractionation
• Contaminant removal
• pH change
• Desalting
• Buffer exchange
• Binding studies
• Electro-elution
Dialysis membrane materials:
“Synthetic and natural membranes are commonly used for diffusion, or more accurately osmosis. It allows the passage of small molecules but not larger ones (Filtration applications).
Membrane materials most often used include regenerated cellulose, cellulose acetate, polysulfone, polycarbonate, polyethylene, polyolefin, polypropylene, and polyvinylidene fluoride. Dialysis membrane is available in a wide range of size dimensions and pore size (Molecular weight cut-off). The main feature of this membrane is that it is porous”.
“The regenerated cellulose in Cellu·Sep® is derived from cotton: Cotton linters are dissolved in a solution and spread into flat sheets or extruded into tubes. The material is treated with glycerin to prevent the pores from collapsing and air dried at a certain temperature and pressure to form a rigid membrane. Cellu·Sep regenerated cellulose membrane has a symmetric pore structure which allows small molecules to migrate in either direction, making it ideal for experimental purposes”.
Cellophane is the most commonly use dialysis material. It is a thin, transparent sheet made of regenerated cellulose. The membrane contains traces of sulpher compounds, metal ions, and some enzymes.
Dialysis is best carried out with freshly prepared tubing since, once it is wet, it becomes very susceptible to attack by micro-organisms. If it has to be stored, then the tubing is best kept with a trace of benzoic acid in the solution.
Principle:
Starch consists of two large molecules: amylase of molecular weight 50,000 and amylopectin of molecular weight 1,000,000, neither of which will pass through a dialysis membrane. Salivary amylase converts the starch to maltose, molecular weight 360, which diffuses out into the dialyzate. A solution of our sample (Starch and salivary amylase) is placed into a dialysis bag such as cellophane and the bag is sealed (Usually made by knotting dialysis membrane at both ends). The sealed bag is then immersed in a relatively large volume of the solvent (distilled water). Maltose that is smaller than the pores of membrane allow moving freely across out of the membrane into the solvent. Starch (Larger molecule) have dimensions significant by greater than the pore diameter is retained inside the dialysis bag. Eventually, equilibrium is an achieved where the concentration of maltose equal inside and outside of the bag. However the volume of the solvent outside the bag is much greater than inside, over time, most of maltose will leave the bag. The passage of maltose into or out of the bag is in the direction of decreasing concentration, therefore displaying diffusion.
Apparatus & glassware:
For team of two students
• Test tube rack to hold 16 small test tubes
• Two beaker
• Magnetic stirrer and bar
• Cellophane tubing
• Dropper, Pipette (2 ml)
• Hot plate
Chemicals & other material:
• Salivary amylase (1 ml saliva diluted to 5 ml with distilled water)
• Iodine solution (5 mmol/L in 30 g/L Potassium iodide)
• Soluble starch (20 g/L)
• Sodium chloride solution (1g/L) buffered with 0.02 mol/L Sodium phosphate pH 6.8).
• Fehling’s solution.
Preparing of the dialysis tubing:
1. Cut a piece of dialysis tubing (i.e. the dialysis “bag”) you will need to hold your sample that has an extra couple of inches A suitable size and length is selected. Always handle the bag with gloves.
2. Boil the bag for 30 min in alkaline EDTA (Na2Co3, 10 g/L: EDTA, 1mmol/L) to avoid the loss of activity of molecules dialyzed and to make the dialysis sac soft. After boiling, the tubing is washed with distilled water, one end is knotted (Sealed).
3. Soak the bag in water. Never let the dialysis bag dry out once it has been wetted.
Procedure:
1. Label fourteen small test tubes 1-16. Number test tubes 1 S through 7 S - and 1 M through 7 M (“S” is for starch an M is for maltose). Arrange them in order on a rack.
2. In test tubes 1 M through 7 M place one ml or 20 drops of Fehling’s solution (Test for maltose).
3. Place 10 drops of iodine solution into each test tube numbered 1 S through 7 S (Test for starch).
4. Seal one end of the dialysis bag (The bottom) with a string. Carefully fill the sac with the reaction mixture by pipetting 2.5 ml starch, 0.5 ml phosphate buffer and then 0.5 ml salivary amylase. Quickly (At 0 min) pipette 1.5 ml of the reaction mixture (From inside the bag). Add 0.5 ml to tube # 1 S and 1 ml to tube # 1 M. Heat test tube # 1 M in a boiling water bath for 5 min. observes result for starch identification (Blue color). Observe result for maltose identification (Red ppt).
Note: being carefully not to puncture the bag. Also, don’t spill your sample! Seal the top of the bag.
5. Immerse the dialysis bag in a beaker or flask containing 100 ml of the solvent, distilled water.
Place the beaker or flask on a stir plate and stir in the cold for at least 90 min (It takes about 2 hours for dialysis to be complete). Stirring help in the entry of the water inside the sac and help in getting out of small molecules out of the sac.
6. Pipette 1.5 ml from the solvent (outside) every 15 min interval during the 90 min. Examine each fraction (of the 6 fractions) for the presence of starch or maltose as in step 4.
7. At the end of the experiment, cut the dialysis sac, carefully remove the dialyzed from the sac with a pipette and squeeze out as much as possible of whatever is left into the test tubes # 7 S (0.5 ml) and # 7 M (1ml). Examine the dialyzed (Inside the sac) for the presence of starch or maltose as in step 4.
Test for starch: Change of the color of iodine during separation
Test for maltose: Formation of red precipitate during the separation
0 min (inside) blue color
1 S 15 to 75 min (outside)
yellow color 2 S to 6 S 75 min (inside)
yellow color 7 S
0 min (inside) blue color
1 S 15 to 75 min (outside)
yellow color 2 S to 6 S 75 min (inside)
red color 7 S
1-a 1-b
0 min (inside) red ppt
1M 15 to 75 min (outside)
red ppt 2 M to 6 M 75 min (inside)
red ppt 7 M
2
Result sheet
Make a table of time intervals (min), and observe result for each fraction with iodine and Fehling’s tests. Note observation in table for Fehling’s test as follows: (+) for a few ppt, (++) for more ppt, (+++) for much more ppt and so on.
(1)
(2)
(3)
Time (min) Inside tubing
Comment
I2 test Fehling’s test 0
Time (min) Outside tubing
Comment I2 test Fehling’s test
15
30
45
60 75 90
Time (min)
Inside tubing
Comment
Outside tubing
Comment I2 test Fehling’s test I2 test Fehling’s test
90
Answer the following questions.
1. Explain your results
2. Why the separation done with stirring?
3. What role does the EDTA solution play in this experiment?
References:
1. http://www.spectrapor.com/dialysis/Fund.html 2. www.membrane-mfpi.com/labdialy.html 3. http://www.membrane-mfpi.com/t_instrs.html
4. http://web.siumed.edu/~bbartholomew/course_material/protein_methods.htm
