3 types of calculated molecular weight data

If you’re in the business of analyzing macromolecules, there is a good chance that molecular weight is a parameter of interest.  This is because molecular weight corresponds to polyer chain length, which is related to the material’s physical properties.  A convenient analytical technique to characterize a sample’s molecular weight is GPC/SEC.  However, there are a variety of GPC/SEC systems with different available detector configurations (like OMNISEC!), and these different detector combinations provide a range of calculated data.  And even though they can all provide molecular weight values, those molecular weight values aren’t all created equally!

If you are reading a journal article, relying on data from a supplier, reproducing a polymerization from the literature, or anything else, it is important to understand how the molecular weight values are calculated.  This post will describe three types of calculated molecular weight data so that you’ll be ready next time you reference a molecular weight.

Are you talking about molecular weight averages?

Not exactly, although a brief description of molecular weight averages is warranted.  Macromolecules are produced by smaller building-block molecules connecting to one another.  While proteins exhibit a single molecular weight value, due to their specific amino acid sequence, many natural and all synthetic polymers exhibit a molecular weight distribution, meaning a sample contains chains of varying lengths.  Therefore, molecular weight averages, such as number average molecular weight (Mn), weight average molecular weight (Mw), and z-average molecular weight (Mz) are used to describe polymer samples.  This linked page of the Polymer Science Learning Center provides a fantastic explanation of molecular weight averages.

Ultimately, I’m using this post to describe the practical differences when those molecular weight averages are calculated by different analysis methods, and what that means about the resulting values.  For more further details on each of the analysis methods themselves, please see my previous post on the topic.

Relative molecular weight

When using a single concentration detector and a conventional calibration analysis, the resulting molecular weight data is described as ”relative.”  The molecular weight of each data slice of your sample is taken from a calibration curve created from known standards, as shown in the figure below.  For example, your sample could have a Mw of 50 kDa relative to polyethylene oxide (PEO) standards.  Unfortunately, that value only indicates that your sample is the same size as a 50 kDa PEO sample.  It doesn’t mean anything about the actual molecular weight of your sample!

GPC/SEC molecular weight calculation from conventional calibration curve

Remember, GPC/SEC separates based on molecular size – not molecular weight.  Think about a volleyball and a bowling ball as your samples: they’re both about the same size, so they would elute at the same retention volume.  Therefore, their relative molecular weights will calculate as the same.

Relative MW comparison of bowling ball & volleyball - they're the same!

However, anyone who has picked up both of these objects will know that a bowling ball has much more mass than a volleyball.  But since relative molecular weight values are based only on retention volume, which is based only on molecular size, the difference in mass is not detected.

Relative molecular weights are great if the sample being analyzed is the same as the standards used to generate the calibration curve.  But if the sample and standards are different, the accuracy of the calculated relative molecular weight values will reflect those differences. 

If you see molecular weight values reported as relative, make sure you keep in mind the standards (and system conditions) employed, and remember that the actual molecular weight of the samples might be nothing close to the reported value!

Universal calibration molecular weight

The addition of a viscometer to the detector array allows a universal calibration curve to be generated.  The inclusion of a viscometer allows for differences in molecular structure and density to be taken into account.  The key relationship is hydrodynamic volume (molecular size) is proportional to molecular weight times intrinsic viscosity (IV).

Hydrodynamic volume is proportional to MW x IV

While universal calibration molecular weight still relies on a calibration curve, and is thus dependent on system factors such as flow rate, temperature, etc., the calculated molecular weight values are accurate regardless of how similar your samples are to your standards. 

If we return to the bowling ball and volleyball, the molecular weight calculated by universal calibration will likely be accurate.  The two elute at the same retention volume by virtue of being the same size, which means the hydrodynamic volume is equivalent.  However, the viscometer detector’s response to each will be quite different.  The dense structure of the bowling ball means it has a lower IV than the volleyball.  Therefore, since we’re now using Mw x IV, the difference in molecular weight is identified.

Universal calibration comparison of bowling ball and volleyball - works, but could be better

When reading about molecular weight values determined by universal calibration, it is important to take note of the system conditions used for analysis.  But more likely than not, the universal calibration molecular weight values can be trusted!

Absolute molecular weight

A GPC/SEC system with a static light scattering is the ideal way to calculate molecular weight.  Since the molecular weight of the sample is related to the intensity of light it scatters, which is measured by the light scattering detector, the retention volume of the sample is now irrelevant.  That means no calibration curve is required!  When molecular weight is calculated using a system with a light scattering detector, those values are described as “absolute,” in contrast to the molecular weights calculated using calibration curves in the absence of a light scattering detector.

A system with a light scattering detector will correctly determine the molecular weights of the bowling ball and volleyball samples.  And in a more convenient fashion, since calibration curve standards are not necessary (to be clear, all light scattering detectors require calibration, regardless of manufacturer; this can easily be done with a single, narrow standard).  It is important to note that the sample’s dn/dc value is required, but fortunately that’s something the OMNISEC software can help you determine.

Comparison of bowling ball and volleyball - same size, different mass

Next time you are reviewing molecular weight values, hopefully they are described as “absolute” or determined with a light scattering detector.  Those are values you know you can trust!

Final thoughts

In conclusion, I hope this post helps you understand the process of assessing these three different types of calculated molecular weight values.  If you are interested in analyzing samples to determine all three yourself, I have good news – OMNISEC can offer all of options described above!  If you have any questions, please don’t hesitate to contact us or email me directly at kyle.williams@malvernpanalytical.com.

Related content