Hello.

The concentration and dilution will depend on your method, the detector to be used, the HPLC column, the polydispersity of the standards (the CoA should have that), and the Mw (from CoA as well; smaller Mw requires lower conc.).

To keep it simple, for SEC, simply dissolve the powder in your mobile phase. A good starting point is 0.5 mg/mL - 2.0 mg/mL if you don't know how you want your sample yet; otherwise, prepare it at the sample concentration. The material dissolves in both organic and water mixtures.

Prepare all your different molecular weights the same way since your Mw between the 4 bottles doesn't seem too different; preferably that would be at least 5 points, but in your case you only have 4. You can do one injection with only one standard to check your peak shape and response as different detectors will produce different signal (RID, UV, PAD, MALLS, etc), but as I said, 0.5 - 2.0 mg/mL should be fine. The injection volume will also depend on the HPLC column, detector, and sample conc., but 50-100 uL is a good range.

Edit: I forgot the filtering part. The standards should come out clear. For HPLC, you always need clear solutions, so if they are not, then they will need centrifugation, settling, or filtering. Myself, I don't filter samples unless I have done filter study to know which filter is suitable. Instead, I would let the solution settle for a few minutes and decant from the top and vial into HPLC vials.

Hi Dokyeoung,

Maybe you can read Iron sucrose injection monograph from USP/BP for example. Hope it helps

Styrene will absorb like crazy in the UV, so you will need very little if you are doing UV detection. Other detection systems will likely require more material. You have to dilute to the point that there is no flat top to your peak. A flat top can either result from overloading the column or saturating the detector. Lower concentration is the solution to both. You have picked a very small MW range for your standards, so your SEC resin must have very precise pore sizes to resolve these.

Note polystryrene sulfonate is a polydisperse polymer (dispersity index = 4), so unless your standards are fractionated, the peaks will be very broad and may appear to overlap significantly. There is always an issue of whether the vender is reporting the number average MW or the weight average. You can probably confirm the number average MW distribution by MS (negative ion mode), at least in this MW range, but you are going to have to deconvolve the charge multiple charge states, so will need a high resolution MS.

You haven't specified the 'organic substances' in your question. sulfonated polystyrene is typically used as a aqueous MW standard. It will behave differently in an organic solvent. Precision in SEC work requires that the polymer be unraveled (random walk hydrodynamic radius), so you need to pick a standard that has the same affinity for the mobile phase as the standard. Aqueous Peptide work is best done using purified peptides and proteins as standards. Organic mobile phases can use synthesized polymers of similar solubility parameters as the target materials. Check the Polysciences catalog, not sure who owns them now.

Always remember in SEC that log(MW) is inversely proportional to retention time on the column. I would also do multiple runs on the standards you are using, because of the narrow MW range to see what kind of precision your column and HPLC will produce. I'm not sure that any SEC column will be able to accurately detect this small a range of MW.