Chromatography Forum

Hi,
I'm looking for ideas about how to load samples in a low pressure prep scale LC system. I primarily use SEC to separate protein mixtures. My sample volumes can be anywhere from 2 to 20 mL depending on the column I'm using. Currently we load the samples onto the column manually by submerging a short tube leading from the inlet in the sample, connecting a luer syringe to a second short tube on the bottom of the column and "sucking" the sample onto the column. This method works well, minimizes dilution of the sample before the column, and makes it easy to keep air from entering the column. What I don't like is that it is a tedious process to make all the necessary connections, and it's easy to make an error when reconnecting everything. We've tried samples loops, but either we lose sample due to laminar flow effects, or the sample gets diluted too much with a partial loop fill. I've also tried GE's "superloop". It seems to work, but I'm concerned about carryover from one sample to the next. Another solution we considered was using a switching valve before the pump to load the sample and then start pumping the mobile phase. This also leads to unacceptable dilution of the sample, incomplete loading, or bubbles. Does anyone know of a product or method for loading large samples like this? I'm sure there's an obvious solution, but it's apparently too obvious for me to see.

I would just use a 20 ml luer syringe with a plastic 3-way T valve. Load the sample onto the syringe, connect to luer fitting, and turn valve to load onto the column, and turn again fill syringe with solvent and turn back to rinse syringe contents onto column, and then turn again to run column and disconnect syringe.

I'd also review the loops you used previously on a valve, as losses should be low and a singe rinse should clean the loop - if not consider changing the loop diameter/length, or your sample solvent, to minimise viscosity effects.

Mentioning the GE "superloop" makes me wonder whether you're using an ÄKTA system? If so, there is this "Sample Application Kit" which in fact is a pump, that's loading any given volume directly onto the column. Works perfectly.

Best Regards

Thanks for the suggestions. I've actually gotten a T valve with the intention of doing exactly what you said. It sounds like that will be the easiest and most cost effective approach.

I'm not using the AKTA system in this situation. I've used one before, with the sample application kit (p-960 pump, valves etc..), but this system was built from various parts to avoid the cost of purchasing another AKTA and to make use of some parts we already had. I like the sample loading mechanism on the AKTA systems, but like everything from GE, it costs about twice what it's "worth".

The "superloop" from GE works great in most aspects. They are designed so that you can continue buffer flow through the loop once the sample is loaded and thereby flushing the superloop and avoiding carryover to the next sample.

You haven't explained how you connected the superloop, but if you haven't got any spare valves then you can use a couple of these 3-way T valves to connect the loop to the column. Mind you, this doesn't remove the

tedious process to make all the necessary connections, and it's easy to make an error when reconnecting everything.

The only thing that will remove this is to use an automatic system, such as GE Healthcare's ÄKTAfplc.

Btw, I totally disagree that the ÄKTA systems costs about twice what it's worth. The ÄKTA systems are so great that they are worth every penny you spend on them. If you talk nicely to the sales reps they can often give you a significant discount, too. Please note that I'm in no way connected to GE Healthcare and these are my own opinions.