Hi Imtiaj Uddin

In practice, I think it doesn’t make a huge difference whether you load the sample directly through the pump inlet or use a sample loop. Personally, for a first purification step, I usually load samples directly — often with much larger volumes than 20 mL — and it works perfectly fine.

Doing multiple 5 mL injections with a loop will certainly work,, but it’s mostly just time-consuming. A 20 mL loop (or a Superloop) can save time, but it’s an extra expense. I’d only go for that option if the sample cannot be concentrated (due to precipitation or instability) and if it really can’t wait through several injections before being loaded onto the column for some reason (buffer, etc...). Did you already try to concentrate?

Technically, using a loop maybe is the “cleaner” way as it is only dedicated to sample loading, whereas the pump tubing also carries buffers — so there’s a theoretical risk of cross-contamination. But in parctice, as long as you flush the lines thoroughly with distilled water before switching solutions, direct loading works without problems.

Hope this helps clarify things!

Alexandre Almeida Thank You very much for your answer. I am new to protein purification, and I would greatly appreciate advice for this experiment and for future steps in my work.