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micro or ultra filtration | Sonika | 2004-04-21 | Click here to register. |
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| I would like to know what kind of filtratio unit, will work best to seperate polymer ( Mw 70 Kda) from a vesciles of size say, 100-200 nm | | |
| lil stevie | 2004-04-21 | Click here to register. |
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 | The 100-200 nm vessicles are about the same size as a 1,000 kD (1 million dalton) globular protein. To separate this from the 70 kD polymer you'd want a filter rated at about 300 kD.
The best technique to use would depend upon the stability of the vessicles and the sample size and throughput required. (I tend to think of vessicles as fragile when frequently they are rather robust.)
If your vessicles are fragile or this is a one-off type of thing, then dialysis would be your best bet. It is must more gentle than filtration and needs no equipment other than the dialysis membrane. A good choice would be one of the 300 kD MWCO CE membranes listed at http://www.spectrapor.com/0/9/5a.html Pick a tubing flat width based on your sample size. Look at the Vol./Lgth. and pick one that would give an easy to work with length. After the dialysis your vessicles would be at about the same concentration as at the start, but the polymer would be very dilute in the surrounding buffer.
If your vessicles are more robust, then filtration is a faster technique that can be used. For very small (less than 20 mL) samples, you can use the MicroKros filters to evaluate the separation. The pressure for these filters is generated by pushing on plastic syringes. It is good for an initial evaluation but becomes very tedious if you have very many samples. The description of the MicroKros filters starts at http://www.spectrapor.com/0/1/6.html and you should probably pick a 400 kD module from page http://www.spectrapor.com/0/1/9.html
After a filtration separation, the 70 kD polymer will be slightly diluted and the vessicles will be more concentrated.
For repeated applications or larger samples, various pump-driven filtration units are available. |
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| Sonika | 2004-04-23 | Click here to register. |
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| Thanks for the detialed reply. Based on it, i have some further questions.
1. I agree that dialysis can be easier and gentle for separation of the polymer from the vesicles but since Lipsomes are prone to oxidation and since i would need to make this sepation more^number of times for given batch of Liposomes, it would be useful to know how much time do you think one should keep these liposomes for dialysis?
2. To me the other approach of filtration sounds better provided one can do that with minimum pressure say 1 bar or less without demaging the Liposomes and depeneding on the pore size of hollow membranes. Could you explain separation of LIposomes from polymer and their place of exit in these mini or midiKros modules? Is it possible to use some pump for this application and we would need this sepation of Polymer everytime liposomes is coated with polymer to have a multilayer of polymer around Liposome?
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| lil stevie | 2004-04-23 | Click here to register. |
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| Dialysis separations are usually 6 to 24 hour long steps.
The Mini and Midi Kros modules are used with a pump. The Microkros, which is usually just used to evaluate and get a feel for the technique, is powered with plastic syringes.
When using a pump system your sample is pumped from its reservoir, down the inside of a hollow fiber (0.5 or 1 mm diameter), out the other end and back to its reservoir. While it's passing through the hollow fiber a little bit of the water, polymer, and other small species move through the pores in the hollow fiber to the outside. The constant movement down the length of the fiber keeps the liposomes from collecting on the fibers and slowing down the filtration.
Generally, after some polymer and water have left the sample and it has become more concentrated, additional water (or buffer) is added to the sample to keep it from becoming too concentrated. This lets you run the filtration until all of the polymer has been removed without overly concentrating the liposomes.
Your liposomes would be recovered from the solution in the reservoir they started in. The free polymer would have moved through the hollow fiber and been collected from the tubing fittings on the sides of the cartridge.
In terms of run time, if you limit the pressure to only 1 psi then you should get liquid through the membrane at a rate of about 10 liters per square meter of membrane per hour. If you use a cartrdige with 35 square centimeters of membrane area the throughput will be about 3 milliliters per hour. Using a larger cartridge or higher pressure will increase this throughput.
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