Loading sample in FPLC loops - (Aug/19/2009 )
Hi everyone,
The FPLC manual says:
"The sample volume loaded should be, at maximum, 50% of the loop volume"
On the other hand, some web pages say:
"The sample volume should ideally be larger than the sample loop so that no air bubles will be present in the loop after loading"
What is the correct way for you and why?
Many thanks
Sample loops. I think the other 50% is buffer.
Super loops- you can load the whole thing, but make sure that if you inject, say 5mL, you tell the machine to inject less (i.e 4.5mL) to prevent air bubbles getting in. (Air is seriously annoying)..
At any rate, ALWAYS have someone there to supervise you. The columns and machines are extremely expensive, and I know because i got air bubble in my column and that was bye bye $2500.00
There are 2 schools of thought about sample loading. The first says that you load ~ 1/2 the volume because in the process of filling the loop, a sample front develops at the boundary between the buffer and the sample, and sample mixes with the buffer already in the loop. 1/2 volume is an estimated volume to ensure none of the sample in this mixture gets expelled to waste (this is also why it's a good idea to keep the expelled buffer from each loading if the sample is precious, because you can always add it again later). It's typically used if you have very precious sample.
The second school of thought when loading sample loops is interested in the smallest number of separation runs. In it, you empty the loop by blowing air through it (while the loop is unplugged from the rest of the system!!!). You then load your sample into the loop until sample comes out the (open) end of the loop. You now have a loop that is 100% filled with your sample, with no dilution at the "leading edge".
Six of one, half a dozen of the other.
swanny on Aug 24 2009, 11:03 AM said:
The speed at which you inject the sample can also have an effect here via laminar flow. With the low turbulence inside chromatography tubing, the faster you inject the greater the convex nature of the solvent front and the greater the chance that your sample will pass through the loop in a thin stream not touching the sides.
DRT on Aug 24 2009, 01:49 PM said:
swanny on Aug 24 2009, 11:03 AM said:
The speed at which you inject the sample can also have an effect here via laminar flow. With the low turbulence inside chromatography tubing, the faster you inject the greater the convex nature of the solvent front and the greater the chance that your sample will pass through the loop in a thin stream not touching the sides.
Yeah, good point.
PS: you're not a kiwi, are you?
Agree.........rest is sample buffer. Air will surely kill yr column.
Best,
TC
Luria Bertani on Aug 21 2009, 06:48 PM said:
Super loops- you can load the whole thing, but make sure that if you inject, say 5mL, you tell the machine to inject less (i.e 4.5mL) to prevent air bubbles getting in. (Air is seriously annoying)..
At any rate, ALWAYS have someone there to supervise you. The columns and machines are extremely expensive, and I know because i got air bubble in my column and that was bye bye $2500.00
swanny on Aug 25 2009, 11:27 AM said:
Was it the inverted vowels that gave it away?
Doing a stint up state from you in the Northern Rivers region, (though if this winter weather, 37oC, is an indication of what the spring/summer has in store I think I’ll have to migrate to more temperate climes or reduce my work/beach ratio even more).
I usually load a bit more than sample loop. But just remember to flush the waste tubes with ACN after each sample.