Hello, I've recently synthesized a short (17aa) peptide via manual SPPS. I was instructed to submit a small pipette tip's worth of material onto a capillary column to obtain a rough estimate of the purity of the crude material (Waters 2695 pump/ 996 UV detector; 1% MeCN/ min gradient) and then move onto a semi-preparative column for bulk purification. I believe this is a quick and non-specific method of workup but id like to know how to properly scale up from an analytical column (250 x 4.6 ID) to semi-preparative (250 x 22 I.D) while maintaining a similar chromatogram.
1. How much material should I be injecting onto an analytical column to obtain a chromatogram with strong signal yet avoid saturation? (I read sample capacity is ~12mg but injection vol should be ~1% column volume which is roughly 40 uL.)
2. Once I establish a serviceable analytical profile, how can I scale up to semi-preparative work?
Apologies if this question is very basic. I am reading through a couple introductory HPLC texts and trying to learn on the job so to speak. Any help is appreciated!
I simply would maintain the first HPLC conditions you worked with, you may inject from 10 to 20 uL no more, of course collecting on your retention time, you will obtain purity but a lot of volume (mobile phase). The other thing you can do is use an FPLC column (to purify proteins) but couple it to your HPLC detector, of course, this will change your retention times and maybe you should use another mobile phase, but yet you will get a pure protein (not so pure but acceptable).
and to scale shouldn't be a problem with the second option I provided...
Ofen use next rules:
m1 / m2 =d12/ d22 , m = mass mg , d- diameter mm
V1 / V2 =d12/ d22 , V = flow ml/min
if l1=l2 as in your case l=250 and both columns have same particle size
Once you have an analytical method, you can duplicate the method on a larger scale by working in column volumes (CV). It is best if the columns are the same packing- this means same manufacturer and media, including pore size as this affects peptide retention, too. Using different types of C18 may lead to differences in retention due to differences in selectivity.
CVs can be measured with the void volume- I have good luck using iodide (KI, NaI) salts or nitrate salts because the anions absorb UV light and don't interact with the reverse phase packing.
Once you know the CVs for your columns, find the length of your gradient steps in your analytical method as column volumes; your preparative method will use the same method in CVs, including the solvent composition at the start and end of each gradient segment. Knowing the CV of your preparative column and the flow rate you will use, you can convert back from CV to time and program your gradient. This method is complicated by the dwell volume of the instrument, which is the volume from wherever the gradient is formed in your system to the head of the column, including any sample loops the flow path after injection. Depending on the manufacturer of your HPLC system, "dwell volume" may have other names, such as "gradient delay". As the latter term suggests, it tends to delay the gradient, causing later elution. If you are using the same HPLC as for the analytical run, you compound may elute slightly earlier in a gradient because the dwell volume is smaller compared to the column volume as compared to your smaller analytical column. Antonina Kokisheva 's equations become column volumes as they are proportional scale-up, assuming the columns are the same length and particle size. If the columns are different lengths, working in CV is more general.
Another method is described in the references below, starting from a scouting run. For peptides, the calculated gradient is often very close and sometimes needs adjustment. Peptides are more sensitive to slight changes in pH and solvent composition than small molecules so the calculations described seem to get you very close to an preparative gradient for peptides.
Conference Paper Calibration of analytical HPLC to generate preparative LC gr...
Article An overview of analytical-to-preparative liquid chromatograp...
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