Loading capacity

[luis_vn]

I am somewhat new to working with large columns, I have always worked with analytical columns. Now I have to work with a column of the following dimensions: RP C18 column, 300 mm x 50 mm and a particle size of 15 µm. It will be used to purify peptides. What load can the column have? Or any advice you can give me to work with this column, my phases are A, 0.1% TFA water and B ACN.

[TylerSmith123]

Hi Luis,
Just to preface: a majority of my work is preparative and is done on a 250 mm x 150 mm column with a particle size of 5 um. We use a PFP phase (historically) but run within acetonitrile and the separations are very similar to a C18.

Loading capacity can be hard to estimate and is really specific to your column, analyte matrix and analyte itself. Essentially, retention time and peak width are independent of how much you inject up to a point. Above this point is what I consider "overloading", which is typically where prep work operates (if you're looking for maximizing yield and throughput). Essentially, you will be loading so much on your column that the retention time would be affected if you went any higher or your peaks become to wide and may become indiscernible. According to my manual, the column becomes overloaded once you retention time decreases by 10% of it's normal value.
Luckily for you, you have a nice and wide column with a large pore size, so it seems looks like you could load a pretty good amount on this column. I'd begin testing with injections that range from 5 mg and up. When you start seeing issues (I'm not sure of your ultimate goal whether it be purity, amount or speed so I will assume purity and amount), such as coelution or retention decreases, I would call that your maximum load and then go from there.

I'm sorry about the complicated response, it is just very variable depending on the analysis being done. My analytes are plant extracts and are extremely dirty, but if my sample is not obsidian black and sludgy before I inject it on the column, I am not satisfied (I inject 300 mg/ml water-soluble polymer extracts at volumes between 200 ul - 1000 ul, typically 50- 100 mg each injection). If my sample was more pure though, I'd expect my total injectable volume to DROP because I would see decreases in retention and peak width that would interfere with the goal of my analysis.

Note: I am not familiar with preparative peptide purification and these analytes could have a significant impact on your analysis.

Shoot a few runs from 5 mg up to 50 mg, along with standards, and compare the retention times and peak width/plates. After a certain point it will be clear what is unusable for your application.

Tyler Smith

[Multidimensional]

Here is how we do it in professional laboratories where prep HPLC is used (Column up to 10" internal diameter).
Pack an analytical column, 4.6mm x length, with the same length and support (same particle size as prep). This allows for easy scaling. Perform a loading study using the small column and same mobile phase. The data obtained will provide you with a starting point for the 2" diameter Prep column. A full loading study should be done with the Prep column too, but having the analytical data will allow you to know where to start (concentration/volume). Note that "overloading" in PREP LC may be OK as we sometimes do this intentionally to run in "Displacement mode chromatography" which allows for larger sample loads while still being able to heart-cut and fractionate many samples (take slices, then re-analyze the fractions on your analytical HPLC).

Don't forget to optimize the flow path of the detector flow cell too on the PREP detector to keep it on-scale and get the best results (and not saturate the detector).