Back in June we released the OMNISEC v11.20 software that included column calibration methods. In a previous post, I summarized how the methods with calibration curves work and what information they offer. This post will offer some tips for setting up calibration curves and include helpful video guides for conventional and universal calibrations.
To generate conventional calibration curves, you’ll need to analyze a series of standards with known peak molecular weight (Mp) values. The more standards you use the more confident you can be that your calibration curve reflects your column set. But all those standards add up! If you have individual standards, you can combine three or four in the same vial to reduce the number of injections, and thus solvent and time. But don’t go mixing solutions just yet, there are a few things to keep in mind to ensure optimal results.
In order to maintain resolution between each peak, you’ll want to combine non-sequential standards. For example, if you have a series of twelve standards, 1-12 from lowest to highest molecular weight, I’d recommend combining standards 1, 5, & 9 in one vial. Next, I’d combine standards 2, 6, & 10 in one vial and 3, 7, & 11 in another. And last, standards 4, 8, & 12 to finish off the series. This example is illustrated in the figure below.
Additionally, remember that each time you combine two standards you’re diluting both. Therefore, if you’re going to combine three standards per vial, as in the example above, prepare each of the standards at three times the normal concentration (i.e. use one third of the typical amount of dissolution solvent). That way, after you’ve mixed three of them together each one will possess its appropriate concentration.
For further guidance please watch the conventional calibration video below.
Like conventional calibration, universal calibration curves require you to run a series of standards. In addition, a single narrow standard is also required to align and calibrate the detectors. Here’s a helpful tip: instead of obtaining a separate standard for this purpose, simply use one of the standards included in your series. This will work as long as the standard:
- possesses narrow dispersity of 1.10 or less
- is completely resolved (the peak begins and ends at baseline for the concentration and viscometer detectors)
- the molecular weight, dn/dc, and concentration of the standard are known
For a summary of the universal calibration process, please see the video below.
If you’re using a Mark-Houwink calibration, it is imperative that you know the Mark-Houwink parameters for both your standards and your sample. As you set up your method and step through each page in the Calculation method window, you might expect to see a place to enter these values. However, you won’t find it there – but don’t worry!
The place to enter the Mark-Houwink parameters for a particular sample type can be found, appropriately enough, in the Sample type database. Go to Tools > Sample types and you’ll access the database containing dn/dc, dA/dc, and Mark-Houwink parameters. Simply update an existing entry (non-default only; default entries can’t be altered) or create a new one by clicking the Add button. Since all these parameters are unique for each sample type in a specific solvent, I recommend indicating both the sample and solvent in the Description. Be sure to specify if the Mark-Houwink K value you’re using is linear or logarithmic!
Once you’ve done this, all you have to do is assign the appropriate Sample type to your standards and sample when setting up your sequence, and the OMNISEC software will take care of the rest!
The column calibration package significantly expands the capabilities of the OMNISEC software. If you like what you’ve seen, you can download the v11.20 software from our website and purchase the column calibration package from our online store. If you have any questions, please contact us or email me directly at firstname.lastname@example.org. Thanks, and enjoy!