by
lmh » Thu Jan 23, 2020 12:33 pm
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.