Hi all,
I am a marine biologist who has been independently researching the properties of horseshoe crab blood from a local population of crabs for about a
year now, and for the next stage of my research I had some questions about drying a sample. For context, one of the best ways to test the sensitivity
of the immune cells of a horseshoe crab is to produce a reagent called Limulus Amebocyte Lysate, which is commonly used in the biomedical industry for
endotoxin test kits. LAL is a heat sensitive reagent that starts to denature at around 36° C. When LAL is produced it is in an aqueous solution of
distilled water and a very small amount of 3% NaCl saline water (used for rinsing the amebocyte cells), and while I have produced countless samples of
LAL over the last several months, going forward I would like to dry the samples completely and remove all of the moisture from them, leaving behind
only the LAL. This is what biomedical companies do to prepare the reagent for storage/shipment, and I want to mess around with drying/re-constituting
the LAL to see if it impacts its sensitivity at all.
The most common technique used for this is lyophilization, but as I'm not affiliated with an actual lab I don't really have the budget for a quality
lyophilizer. I also don't have much experience with this aspect of chemistry, so I'm unsure of what types of techniques might work to dry the sample.
Do you guys have any suggestions for alternatives, or would other drying processes like rotary evaporation require temperatures that are too high for
the LAL? Would it be better to try and set up a custom lyophilization system using pressure chambers, a vacuum pump, and a makeshift water trap to dry
the sample?sauveurdumonde - 10-3-2022 at 04:17
Yes! If you have access to a vacuum capable of about 2-3% atmosphere, I believe water should boil off at about room temperature. Even if you don't
have this ability, any level of vacuum will increase vapour pressure of the water, and therefore increase evaporation rate. I don't know how
concentrated LAL solutions are so this may take a while.
Now of course if you do manage to drive off the water there will be NaCl left over from the saline solution. I don't know how to remove this since LAL
is an extract rather than a compound.Jenks - 10-3-2022 at 09:51
What volume of solution do you need to evaporate?
If it is air stable, you could just put the solution into a casserole dish and blow air over it, covering it with paper towel if necessary to keep out
dust.
If you are only dealing with small amounts, you can lyophilize by freezing the solution (careful not to crack the container) and then applying hard
vacuum until the ice evaporates. The salt in it may complicate this - as it gets more concentrated, the ice may need some cooling or insulation to
keep from melting.
The nice thing about lyophilization is that it gives nice fluffy, homogeneous product. If that isn't important, you could evaporate the water on a
rotovap from a cool water bath.
All the water evaporated needs to go somewhere, so to protect the pump you will want a cold trap, ideally cooled with something like acetone/dry ice.
If you are evaporating a lot of water, the trap will have to be emptied when it clogs - unless, that is, you have a well sealed system, in which case
you will have made a cryogenic pump!Limulice - 12-3-2022 at 19:50
Thanks guys! The solution is only about 5-10mL of distilled water so hopefully evaporating it won't take too long. I'll play around with the vacuum
pump and see how just evaporating it at room temperature goes, and if I don't like how that turns out I'll try putting together a freeze-drying setup
and I'll make sure to include an ice trap. Since I'm going to lyse the cells in distilled water right away after rinsing anyways I might try rinsing
with distilled water instead of the 3% NaCl just so I don't have to worry about the salt in the drying process. Metacelsus - 13-3-2022 at 04:16
