Various loops in the autosampler, why?

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Hello,

I have such questions:

1. why we use different loops (e.g. 50, 100, 250ul etc) in the autosamplers instead of only the largest one, e.g. 500ul? This was understandable for manual valves - loop overfilling required, but in autosamplers there is partial filling? I think it's about the shape of the peaks (loop tube diameter), but what is the physical explanation for this? What are the benefits when the loop capacity is close to the injection volume?

2. is there any approximate relationship between the capacity of the syringe in the autosampler pump and the volume of the aspirated sample to obtain good repeatability?

Additionally I will ask if after changing the syringe should I do any checking PQ or it is a standard simple operation? Sometimes we make injections up to 200ul.

We currently have a 500ul syringe with a 250ul loop and for small injections (ca 10ul) there is very poor repeatability, sometimes tragic. HPLC Acquity Waters.

Regards
Greg
The syringe usually matches the expected injection size so as to get decent precision. Imagine a syringe pump operating with a full stroke of about 5cm on a 50uL syringe. If you want to make a 5uL injection, it's going to have to move 5mm. If it has an error of 0.1mm this will be 0.1uL on 5uL, 2%.
If you now fit a 500uL syringe, and do a 5uL injection, it will move only 0.5mm, and an error of 0.1mm would correspond to 20%, which is terrible.

So it makes sense to have a syringe size that is well matched to your injection size.

If your syringe isn't big enough to draw 500uL into the loop, there's no point in having a 500uL loop. In fact an unnecessarily large loop would add complexity because although you could, in theory, draw the sample into the very end of the loop, and wash it back out from the end of the loop into the column, you'd need the system to remove the loop from the flow as soon as it's judged that the sample-plug is washed out. The reason is that the loop is otherwise an excessive volume between the pump and the column.

In a loop-injection system, if you don't take the loop out of the system as soon as the plug has gone through, and the loop is enormous, then you're starting your run with a huge splodge of solvent in the loop, so you'd need to know where it came from, and what it is, and you wouldn't be able to start a gradient. In a 300uL/min method, if you have a 500uL loop, you have a delay of 1.67min before anything the pump does gets to the column!

In a high-pressure injection system where the loop is actually the tubing upstream of the needle, into which the sample is drawn, before the needle is placed in its seat and the run begins, a huge loop is simply a vast delay, though at least it's certain to contain solvent from the pump.

These delays can cause weird problems. For example, if you're running a short column and judged you only needed a 1.5min re-equilibration between gradient runs, then with your 500uL loop and a 300uL/min flow rate, the equilibration hasn't even started when you make the next injection! The inevitable consequence will be that everything elutes as one huge injection peak.
Hello,

@ IMH thank you for your comprehensive explanation.
Maybe someone else will suggest something about changing the syringe?

Regards
Greg
I think there's still something to be said for filling the loop -- that's not just a manual injection thing. We get great reproducibility with an old Varian Prostar 410 in "full loop" mode.
Filled-loop injection is *very* reproducible so long as you "overfill" the loop: pushing a sufficient volume of sample through to be sure of completely flushing the loop. A good rule of thumb that I have always used is at least twice the loop volume (three times is better).
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Yes, and it avoids all complications relating to what else is in the loop! Loop injection systems in partial loop mode often end up putting some sort of needle wash solvent in the part of the loop that's not occupied by sample. There's no point in being super careful to match your sample injection solvent to the mobile phase if you add an extra dollop of completely inappropriate solvent from the other half of the loop.
But if you're working with biologists who have struggled to produce 50uL of ludicrously dilute sample, there is a certain appeal in a 5uL partial loop injection compared to using 40uL to flush a 10uL fixed loop. It's a matter of matching tools to jobs!
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