biochemical separation techniques

1. DIALYSIS, ULTRAFILTRATION
AND LYOPHILIZATION
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Done By:
Sumayyah Muhammad Qasim

2. dialysis
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4. PRINCIPLE
Diffusion is the random, thermal movement
of molecules in solution (Brownian motion)
that leads to the net movement of
molecules from an area of higher
concentration to a lower concentration
until equilibrium is reached.
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5. Dialysis
Dialysis is an operation
to separate dissolved
molecules based on
molecular weight.
◦ In practice, a biological
sample is placed inside a
tube of semi permeable
membrane, and placed
inside a much bigger
container.
Dialysis
membrane
Conentrated
solution
Buffer
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6. 1. Only small molecules diffuse through the collodion
membrane.
2. At equilibrium, the concentration of small molecules is
the same inside and outside the membrane.
3. Macromolecules remain in the bag.

7. The only two variables in this method are:
1. The type of membrane (most common are
cellophane & cellulose)
2. The size of pores or the molecular weight cut
off.
Only molecules or ions smaller than MWCO will
move out of the dialysis bag.
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8. PROTOCOL
1. Load the sample into dialysis column
2. Place the column into dialysis buffer (1 X PBS)
3. Dialysis at 4˚c overnight (change dialysis buffer for
3-4 times)
4. Dialysis column with appropriate molecular weight
cut off
5. Permeate smaller than MWCO(salt,ion)
Retentate concentrated macromolecules (proteins)
6. Collect clarified sample in a new tube
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9. Applications and limitation of Dialysis
1. Removal of salts and low molecular weight compounds
2. Buffer exchange
3. Concentration of macromolecules
4. Purification of biotechnological products
5. Medical applications: kidney dialysis and Haemodialysis
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10. Daily Application
 kidney -blood's toxins and waste products-
 Kidney failure-release of nitrogenous containing
waste products (urea and creatine) – azotemia
-causes metabolic acidosis leading to illness
 Solutes -potassium and calcium
-Sodium Bicarbonate
added to neutralize

11. Advantage of dialysis
1. Dialysis is still in use today for it is very simple and is still the only
way to deal with large-volume samples.
2. characterization of a candidate drug in serum binding assays or
detailed study of antigen-antibody interactions
3. proves to be the most accurate method available.
4. inexpensive and easy to perform
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12. Disadvantage of dialysis
Slow process several hours for completion, and thus, has been
replaced by gel filtration for most applications.
Other forms of dialysis includes flow-dialysis and pressure-dialysis
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13. Slide-A-Lyzer Dialysis Flasks
(preformated dialyzer)
 Thermo Scientific Slide-A-Lyzer Dialysis Flasks facilitate simple
and effective removal of buffer salts and small contaminants
from proteins and other macromolecules
 Volume upto 250 ml in 8 to 24 hours
 Molecular -weight cut off 2K, 3.5K, 10K and 20K
 Maximum sample recovery and sample purity
 The dialysis flasks are available in distinct colors, corresponding
to the pore size (MWCO) of the dialysis membrane: Purple
(20,000 daltons), Orange (10,000 daltons), Pink (3500 daltons),
and Blue (2000 daltons).

14. Diagram of Slide-A-Lyzer Dialysis
Flasks
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15. Thermo Scientific Slide-A-Lyzer Dialysis Flasks make sample loading and
recovery easy. Attach supplied float-ring and hydrate membrane for 2 minutes. Pour
sample into device. Remove air and cap. Dialyze for 8 hours to overnight (replace
buffer after 2 and 5 hours). Pour out sample to recover.

16. Highlights:
 Easy to use – Simply pipette or pour sample into flask and
begin dialysis
 Fast dialysis – flat flask chamber with two membranes
provides high surface-area to volume ratio, enabling
dialysis of a 250mL sample in 8 hours to overnight
 High recovery – rectangular flask design maximizes
recovery of entire sample volume via opening at top of
flask
 Multiple molecular-weight cut offs – select the membrane
MWCO that best suits your sample’s molecular weight
 Color-coded frames – easily identify membrane pore size
(MWCO) based on the frame color
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17. ARTICLE
Separation characteristics of dialysis membranes
Molecular weight cut-off (MWCO) specifications and
rates of buffer exchange with Slide-A-Lyzer Dialysis
Devices and Snakeskin Dialysis Tubing
Ph.D Paul Haney ;B.S Katherine Herting ;M.S Suzanne
Smith, April 18, 2013
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18. Figure: How dialysis membranes work. A dialysis membrane is a semi-permeable
film (usually a sheet of regenerated cellulose) containing various
sized pores. Molecules larger than the pores cannot pass through the
membrane but small molecules can do so freely. In this manner, dialysis may
be used to perform purification or buffer exchange for samples containing
macromolecules.
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19. ULTRAFILTRATION
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20. Definition: Ultrafiltration
 Operates according to principle of diffusion
under pressure
 Solutes and water are extracted
 Retains macromolecules i.e. Insulin
 Serves two purposes:
 Purification
 Concenration
 Differs from dialysis: dialysis are generally used for
simply purification purposes

21.  Ultrafiltration concentrates a protein solution using
selective permeable membranes. The function of the
membrane is to let the water and small molecules
pass through while retaining the protein. The solution
is forced against the membrane by mechanical pump,
gas pressure, or centrifugation
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22. PRINCIPLE
 It uses a pressure induced separation of solutes from
a solvent through a semi permeable membrane. The
relationship between the applied pressure on the
solution to be separated and the flux through the
membrane is most commonly described by the Darcy
equation:

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23. Darcy equation
 J=TMP/μRt
 J is the flux (flow rate per membrane area),
 TMP is the transmembrane pressure (pressure
difference between feed and permeate stream),
 μ is solvent viscosity
 Rt is the total resistance (sum of membrane and
fouling resistance)

24. Operational Setup
Batch, Semi-Batch Operation Series Operation
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-Largest tank volume and membrane
area required.
-Conversion per pass is low, but with
multiple passes, virtually any
concentration can be achieved
-Fresh medium continually added
to feed tank
-Continual re-pressurization
required

25. Membrane Options
Polyethersulfone (Polymeric) Regenerated Cellulose Ceramic
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Excellent hydrolytic stability-
High flux, high retention-
Wide range of operating pH-
Durable and thus -
economically efficient
-Expensive
-Susceptible to fouling
and deformation
-Good flux and retention
-Comparatively fragile
-Narrow operating pH
range
-Lower flux than polymer
membranes

26. Membrane Modules
Spiral Wound
-Cannot produce turbulent flow
with the flow rates at which we
are operating
-Small footprint- 5 – 25 mm with
lengths from 0.6 - 6.4
Cassette
-Produces turbulent flow for
better medium-membrane
communication.
-Small footprint
-Large membrane surface
area
Hollow Tube
- High packing density
-Susceptible to structural
deformation

27. Cassette module
Hollow fibre moduke Spiral wound module

28. Applications
 Drinking water-used for the removal of particulates
and macromolecules from raw water to produce
potable water.
 Protein concentrate -dairy industry- processing of
cheese whey to obtain whey protein concentrate
(WPC) and lactose-rich permeate .
- more energy efficient
- consistent product quality, 35-80% protein
product depending on operating conditions
-Do not denature proteins as they use moderate
operating conditions

29.  Other Applications
 Filtration of effluent from paper pulp mill
 Cheese manufacture, see ultrafiltered milk
 Removal of pathogens from milk
 Process and waste water treatment
 Enzyme recovery
 Fruit juice concentration and clarification
 Dialysis and other blood treatments
 Desalting and solvent-exchange of proteins (via
diafiltration)
 Laboratory grade manufacturing

30. Water treatment in Germany

31. Study on the effect of polymer concentration on
hollow fiber ultrafiltration membrane
performance and morphology
M. I. Mustaffar, A. F. Ismail, R. M. Illias
Membrane Research Unit, Faculty of Chemical &
Natural Resources Engineering,
University Teknologi Malaysia, Locked Bag 791, 80990
Johor Bahru, Malaysia
 Fabricated
 External coagulant –Tap water
 Bore fluid- mixture of potassium acetate and water
(20/80 wt.%)

32.  Three newly developed polymer solution were
formulated by using turbidimetric titration method
with varying polymer concentration in the range of
18-22 wt.%
 Experimental results -the flux of the hollow fiber
ultrafiltration membranes decreases while the
rejection for particular solute increases with an
increase in polymer concentration.
-outer skin layer –thicker-denser-increasing polymer
concentration –too slow flux-rejection of cyclodextrin
-spinning asymmetric hollow fiber membranes -dilute
polymer solution- thin and porous skin layer- leading
high value of flux- but a relatively low percentage of
rejection for cyclodextrin separation.

33. LYOPHILIZATION
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34. Lyophilization, or freeze drying, is a process in which
the solvent (usually water) is:
-first frozen and then
-removed by sublimation
in a vacuum environmental
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35. PRINCIPLES INVOLVED IN FREEZE
DRYING
 Water is removed from frozen state by sublimation
 Drying is achieved by subjecting material to
temperature and pressures below triple point.
 The major factors that determine the phase which
substance takes place depends on
1. temperature
2. Pressure
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36. If temperature is b/w sea level freeze
point(320F/ 00 C) and the sea level B.P
(2120F/1000C) the water takes a liquid form.
If the temperature increases above 320F while
keeping the pressure below 1 atm, the water is
warm enough, but there is no enough pressure
for a liquid to form. It become a gas
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37. PROTOCOL
1. Prepare ice box (dry ice)
2. Add chilled ethanol to reduce mist.
3. Transfer samples into ice box.
4. Remove cap and seal with parafilm.
5. Puncture holes with syringe needles.
6. Turn on lyophizer and close ballast.
7. Wait for vaccum to reach <100 mT, and condenser
temperature should be atleast -40˚c
8. Load the sample.
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38. Continued …
9. Open the valve
10. Let it run for 3 hours at least or overnight
11. After the run is complete, switch the valve to
release vaccum
12. Take out the samples
13. Remove parafilm and replace with cap
14. The sample is freeze dried
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39. Lyophilization cycle is divided in three phases:
 An initial freezing process, carried out in such a way
that:
 The product exhibits the desired crystalline structure.
 The product is frozen below its eutectic temperature.
 A primary drying (sublimation) phase during which:
 The partial pressure of the vapour surrounding the
product must be lower than the pressure of the
vapour from the ice, at the same temperature.
 The energy supplied in the form of heat must remain
lower than the product's eutectic temperature (the
highest allowable product temperature during the
conditions of sublimation.)

40.  A secondary drying aimed at eliminating the final
traces of water which remain due to absorption, and
where:
 The partial pressure of the vapor rising from the
product will be at its lowest levels.
 At the completion of the process, the treated product
will have retained its form, volume and original
structure-as well as all its physical, chemical and
biological properties. It can then be stored (provided
packaging is effective to the reduction of moisture
migration) for an almost indefinite period of time. As
the product is porous, it can be re-dissolved by the
simple addition of a proper solvent.
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41. Freeze drying
 In planning for the long-duration Apollo missions,
NASA conducted extensive research into space food.
 Techniques developed in 1938 - Nestlé -freeze drying.
In the United States
 Action Products later commercialized this technique
for other foods, concentrating on snack food
resulting in products like Space ice cream.
 The foods are cooked, quickly frozen, and then slowly
heated in a vacuum chamber to remove the ice
crystals formed by the freezing process.

42.  The final product retains 98% of its nutrition and
weighs much less than before drying.
 The ratio of weight before and after drying depends
strongly on the particular food item but a typical
freeze-dried weight is 20% of the original weight.
 Today, one of the benefits of this advancement in
food preservation includes simple nutritious meals
available to handicapped and otherwise homebound
senior adults unable to take advantage of existing
meal programs.
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43. Applications of freeze-drying
 Pharmaceutical and biotechnology
-shelf life of the products
-easily stored, shipped
-to produce tablets or wafers
 Food and agriculturally-based industries
-freeze-dried ice cream
-remains in good condition, longer than wet food
-Instant coffee
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44.  Technological industry
-In chemical synthesis -more stable, or easier to
dissolve in water for subsequent use.
-late-stage purification procedure-remove solvents,
concentrating substances with low molecular weights
-proteins, enzymes, microorganisms, and blood
plasma
 In bacteriology freeze-drying is used to conserve
special strains.
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45. Other uses
 Organizations -Document Conservation Laboratory -
the United States National Archives and Records
Administration (NARA) have done studies on freeze-drying
as a recovery method of water-damaged books
and documents. While recovery is possible,
restoration quality depends on the material of the
documents. If a document is made of a variety of
materials, which have different absorption properties,
expansion will occur at a non-uniform rate, which
could lead to deformations. Water can also cause
mold to grow or make inks bleed. In these cases,
freeze-drying may not be an effective restoration
method.
 To restore water damaged materials, such as rare and
valuable manuscripts.
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46.  Advanced ceramics processes sometimes use freeze-drying
to create a formable powder from a sprayed
slurry mist. Freeze-drying creates softer particles with
a more homogeneous chemical composition than
traditional hot spray drying, but it is also more
expensive.
 Freeze drying is also used for floral preservation.
Wedding bouquet preservation has become very
popular with brides who want to preserve their
wedding day flowers
 A new form of burial which previously freeze-dries the
body with liquid nitrogen has been developed by the
Swedish company Promessa Organic AB, which puts it
forward as an environmentally friendly alternative to
traditional casket and cremation burials.

47. ADVANTAGES
 Thermolabile materials can be dried
 It is porous and uniform. The reconstitution - easy.
 Denaturation does not occur
 Migration of salts and other solutes does not take place.
 Loss of volatile material is less.
 Moisture level can be kept as low as possible.
 Sterility can be maintained

48. DISADVANTAGES
 The process is very slow and uses complicated plant, which is very
expensive.
 It is not a general method of drying, but is limited to certain types of
valuable products that cannot be dried by any other means.
 The period of drying is high. Time cannot be shortened.
 It is difficult to adopt the method for solutions containing non-aqueous
solvents.
 The product is prone to oxidation, due to high porosity and large
surface area, therefore product should be packed in vacuum or using
inert gas or in container.

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