DIALYSIS, ULTRAFILTRATION

AND LYOPHILIZATION

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Done By:

Sumayyah Muhammad Qasim

dialysis

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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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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.

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Dialysis

Dialysis

membrane

Conentrated

solution

Buffer

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.

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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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

PROTOCOL

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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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Applications and limitation of Dialysis

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

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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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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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).

Slide-A-Lyzer Dialysis Flasks(preformated dialyzer)

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Diagram of Slide-A-Lyzer Dialysis Flasks

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.

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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Highlights:

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

ARTICLE

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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.

ULTRAFILTRATION

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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

Definition: Ultrafiltration

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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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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PRINCIPLE

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)

Operational Setup

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Batch, Semi-Batch Operation Series Operation

-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

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Membrane Options

Polyethersulfone (Polymeric) Regenerated Cellulose Ceramic

-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

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

m.

Cassette

-Produces turbulent flow for

better medium-membrane

communication.

-Small footprint

-Large membrane surface

area

Hollow Tube

- High packing density

-Susceptible to structural

deformation

Hollow fibre moduke Spiral wound module

Cassette module

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

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

Water treatment in Germany

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.%)

Study on the effect of polymer concentration on

hollow fiber ultrafiltration membrane performance and morphology

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 fiberultrafiltration 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.

LYOPHILIZATION

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Lyophilization, or freeze drying, is a process in whichthe solvent (usually water) is:

-first frozen and then

-removed by sublimation

in a vacuum environmental

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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

PRINCIPLES INVOLVED IN FREEZE DRYING

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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

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.

PROTOCOL

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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

Continued …

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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.)

Lyophilization cycle is divided in three phases:

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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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.

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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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

Applications of freeze-drying

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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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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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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.

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

ADVANTAGES

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.

DISADVANTAGES

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