Color inside the lines: informative shading between the limits in the OMNISEC software
When analyzing your samples using the OMNISEC software you’ve probably noticed colorful shading between the limits. You may have even wondered what that shading means, or if it means anything at all. Well, it does! In this post I will discuss what the shading between the limits means and why it’s important.
What shading are you talking about?
When you place integration limits around a sample peak in the OMNISEC software the space between the limits is shaded a light blue color. If you have indicated a sample peak that has enough detector response to calculate any results, molecular weight, intrinsic viscosity, etc., then a subset of the shading will have a red-purple tint where results can be calculated.
In the image below, I have placed two sets of limits. Limit set 1 does not include a peak, while limit set 2 does. I did this intentionally to demonstrate the light blue color corresponding to the space between the limits (1) in contrast to the red-purple tint that covers most of the sample peak from about 17-24 mL in limit set 2.
But what does the shading mean?
The detector responses within limit set 2 are strong enough to provide calculated results. However, once the detector signals return to baseline, or rather all but one detector signal returns to baseline, the red-purple shading stops and only the light blue shading remains (at 24 mL). There is still a little bit of refractive index peak area that has not reached the baseline, but since the light scattering and viscometer signals have returned to baseline no molecular weight or intrinsic viscosity data can be calculated past 24 mL.
Likewise, on the front side of the peak, right around 17 mL, the light scattering and viscometer peaks begin their ascent in the area with the light blue shading. It is not until the refractive index signal rises from the baseline that the red-purple shading begins, indicating that now molecular weight and/or intrinsic viscosity can be calculated at this point.
It is important to note that the concentration data provided by the refractive index detector in this example can alternatively be obtained from the UV-Vis photodiode array (PDA) detector. This holds true for all subsequent examples discussed in this post.
What results are indicated by shading?
Excellent question! There are four different calculated results that can be represented by various shading. They are: molecular weight, intrinsic viscosity, hydrodynamic radius, and radius of gyration. The available options are listed in the Result Types section of the main toolbar, as shown below.
The two greyed out options, concentration and molecular weight distribution, are not available on the Raw Data chromatogram where the shading is present but are accessible in the Derived Data and Distribution Plot, respectively.
How many different color shadings are there?
Technically, you can have six different color shadings within your limit set. The light blue indicating the limits, the red-purple tint for at least one calculated result, and then four different shades for each result type. Light red for molecular weight, a lavender for intrinsic viscosity, a slightly darker blue for hydrodynamic radius, and a slightly darker red for radius of gyration. Hopefully those color descriptions make sense and you’re able to differentiate between them!
What do they all look like?
It’s probably easier to tell the difference if I show you, rather than describe subtle color differences. Additionally, since the point of these shadings is to highlight the detector responses, they include an element of transparency. This means that when shaded areas overlap, a different light red-blue-purple color can be created.
In the figures below I will show what each result shading looks like individually. I’ll include the Result Types section of the tool bar to make it easier to see which is activated.
Molecular weight
Intrinsic viscosity
Hydrodynamic radius
Radius of gyration
Can you activate multiple Result Types at once?
Yes! You can choose to turn on as many or as few Result Types as you prefer. When displaying multiple shaded areas, toggling the results on and off can make it easier to identify which is which.
And to get ahead of your next question, I’ll explain why you might want to have multiple Result Types represented on the Raw Data chromatogram. As you can see from the individual Result Types displayed in the above figures and the multiple Result Types displayed in the image below, not all data are calculated over the same retention volume range.
In multi-detector GPC/SEC each calculated result relies on a different combination of detector responses. For instance, as mentioned above, molecular weight can be calculated as long as there is enough refractive index (or UV-Vis) and light scattering data. Intrinsic viscosity relies on refractive index (or UV-Vis) and viscometer data. Hydrodynamic radius calculation depends on both molecular weight and intrinsic viscosity data, therefore it can only be calculated where there is sufficient refractive index (or UV-Vis), light scattering, and viscometer data. And radius of gyration can only be calculated for sample fractions that display angular dependence, i.e. the right angle and low angle light scattering responses differ.
It’s worth pointing out here that the low molecular weight / angular dependence cutoff for radius of gyration when using data from the SEC-MALS 20 detector is similar to that from the right angle and low angle light scattering detector.
Why does this matter?
To provide data that best represents your sample, OMNISEC determines results that are not limited to retention volumes at which they are directly calculated. This allows you to obtain data for the earliest and latest eluting fractions of your sample, outside of the shaded area for that particular parameter. At the extremes of the sample peak the results are likely extrapolated based on the directly calculated data.
This is noticeable in the Derived Data view with the molecular weight, intrinsic viscosity, hydrodynamic radius, and radius of gyration Result Types activated, as shown in the image below. The Result Types plots in the Derived Data view are solid where they are directly calculated, corresponding to the shaded area in the Raw Data view. The dotted portions of each plot indicate the result is extrapolated until the placement of the limit.
From the above image, it is clear that the molecular weight calculated for the sample (gold line) extends over most of the peak. The intrinsic viscosity (sky blue) and hydrodynamic radius (dark green) are directly calculated over the same range, which makes sense because the calculation of hydrodynamic radius depends on both molecular weight and intrinsic viscosity, so it will mirror the range of the more limited calculated result. The calculated radius of gyration (dark purple), relying on light scattering data, starts earlier than the others but stops by about 21 mL, indicating the point at which the right angle and low angle light scattering responses are similar enough that no angular dependence is observed.
This comparison visualizes an advantage of hydrodynamic radius versus radius of gyration as a molecular size calculation: it can be calculated down to smaller molecular sizes and lower molecular weights.
These calculated and extrapolated data portions are also observable in the Distribution Plot, as shown below. Since the data is plotted against LogMW on the x-axis, molecular weight is not an available option. This highlights the importance of quality intrinsic viscosity data when generating a Mark-Houwink plot. And again, the extended range towards low molecular weights of hydrodynamic radius versus radius of gyration is illustrated.
Final thoughts
In conclusion, I hope this explanation of the different colors and shadings between the limits helps you better understand the OMNISEC software’s calculations and the data it provides for your sample. Initially, the different colors might look like a visual gimmick, but they’re actually offering you quite a bit of information at a quick glance. If you have any questions, please don’t hesitate to contact us or email me directly at kyle.williams@malvernpanalytical.com.
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