Earlier this year I highlighted the different types of GPC/SEC standards available and where to find them on our eStore. In this post, I’m going to share some tips and tricks about preparing your standards and making them last.
But first, what standard should you be using?
In my previous post, I discussed how the type of standards you use is dependent on what detectors you have available. If you have a light scattering detector, you should use Narrow + Broad standards. If you don’t have a light scattering detector, then you’ll need to use a series of Column Calibration standards.
After you’ve settled on a type, you’ll need to determine which specific standards are best suited for your application. It is important to remember that your column set can affect which standard you use, too. I’ve organized them below based on each standard chemistry.
- Polystyrene – THF, toluene, chloroform, DCM, ethyl acetate
- Polymethylmethacrylate – THF, chloroform, DCM, DMF, DMAc, HFIP
- Polyethylene oxide & dextran – aqueous mobile phases (e.g., 0.05 M Na2SO4)
- Pullulan & dextran – 5% acetic acid in water, DMSO
- BSA (not sold by Malvern Panalytical) – aqueous buffers for protein applications
How to prepare your standards
Once you’ve chosen which standards to use, the hard part is over! If you use Malvern Panalytical standards, preparation is easy. For most standards, simply add 10 mL of the mobile phase to the standard vial. The standards are pre-weighed and designed to produce an appropriately concentrated solution (about 1-2 mg/mL) when diluted with 10 mL. After that, let the standards sit (or agitate gently) at ambient temperature for at least 4h to ensure complete dissolution before injection. If possible, I prefer to let standards dissolve overnight.
There is one exception for the 10 mL rule, and that’s pullulan. When preparing a pullulan standard, you should add 2 mL of plain water to the vial, regardless of what mobile phase you’re using. This holds true even when running a mobile phase of DMSO on an organic column set.
How to make your standards last
A common question I receive is “How long will a prepared standard last?” Once a standard has been dissolved, I recommend using it within one week, with a maximum of two weeks. After that, degradation can occur and potentially affect the standard’s peak shape. The polystyrene and PMMA standards are stable for up to two weeks in solution if kept sealed and out of direct sunlight. The PEO, pullulan, and dextran standards are stable for up to one week in solution if kept refrigerated, or 48 hours at room temperature, and out of direct sunlight.
However, I know that 10 mL of a standard solution is a lot to go through in that short amount of time! So, here’s my suggestion for making them last:
- Dissolve standard in 10 mL of appropriate solvent
- Place aliquots of known volume in empty vials (e.g. 2 mL aliquots)
- Let solvent evaporate; dry under vacuum if possible
- Seal & label individual vials (with original concentration & volume required)
- When needed, reconstitute with original volume of aliquot
This procedure will allow you to stretch one standard vial into multiple preparations. You should carefully label your new individual vials with the original standard concentration and requisite solvent volume. Without that information, the narrow standards cannot be used to calibrate a method!
Lastly, I wanted to share a quick note on preparing standards to conserve solvent instead of standard. You might find this helpful if you’re working with an expensive solvent, such as HFIP. In that case, instead of using 10 mL to dissolve the PMMA Narrow + Broad standards, you can use 5 mL. Since the resulting standard solution is twice as concentrated, you can set the injection volume to 50 µL, half of the typical 100 µL, to compensate. Just make sure that you enter the correct concentration of the 5 mL solution in the sequence!
Storing your standards
The best way to store standards is to keep them dry (i.e. undissolved) and at room temperature. The polystyrene and PMMA standards are stable for 10 years under these conditions. For PEO, pullulan, and dextran, the undissolved standards are good for 4 years at ambient conditions.
In conclusion, I hope this helps you utilize your GPC/SEC standards more efficiently. Furthermore, if you have any questions please don’t hesitate to contact us or email me directly at firstname.lastname@example.org.