6 important things to consider when preparing your OMNISEC samples
A favorite saying among my colleagues is “the three most important things to acquire good data are sample preparation, sample preparation, and sample preparation.” And I couldn’t agree more! While the multi-detector configuration of OMNISEC offers an impressive amount of data from a single injection, the detectors are only responding to the sample fractions passing through. Therefore, it’s imperative that you offer a well-prepared sample to obtain the best results possible. Fortunately for you, in this post I’ll go through six things to consider when preparing your OMNISEC samples!
Solvent
This is the most important one. GPC/SEC is a solution-based technique, where the sample is carried through the column set and detectors by a liquid mobile phase. Therefore, your sample must be completely soluble (see below) and remain in solution through the entirety of the analysis. While you may apply heat to your sample to facilitate dissolution, I recommend that you confirm your sample maintains solubility once cooled to ambient temperature. The autosampler, column and detector compartments in OMNISEC are temperature controlled and can be heated but the injection valve and some of the connecting tubing is not heated.
In many cases the solvent used to prepare your sample is the same as the mobile phase. In fact, in those situations I recommend drawing the solvent to prepare your sample directly from the mobile phase reservoir. However, for some samples the preparation solvent (sometimes called dissolution solvent) doesn’t always match the mobile phase.
One reason this could be the case, which occurs frequently with protein and bio-related samples, is that the sample will be dissolved in water while the mobile phase will be an aqueous buffer. There are no solubility issues in this scenario, but the mismatched solvents result in a large peak in the RI, viscometer, and sometimes light scattering detectors when the preparation solvent elutes at the end of the chromatogram.
Another reason the preparation solvent could be different than the mobile phase is if the sample dissolves better in one solvent but has a larger dn/dc value in another. A common example is when working with PLGA and related materials: the PLGA samples are often dissolved in DCM or chloroform and injected into a mobile phase of THF. An important aspect to this strategy is that the mobile phase must sustain the solubility of your sample. If your sample precipitates once it is injected into the mobile phase this will not work and you will likely plug your system.
When your sample is properly dissolved it should be clear and transparent (not necessarily colorless) and not cloudy, as in the image above (left: soluble; right: not completely soluble). While preparing your OMNISEC samples, a good way to ensure your sample is completely dissolved is by passing it through a syringe filter with 0.2 µm pores during transfer to the autosampler vial. If the sample passes through the filter with little resistance, then you can be confident that it is soluble and ready for analysis. I’ll discuss filtering samples in more depth in a subsequent post, so stay tuned!
Concentration
An ideal sample concentration is just enough to provide strong, reliable responses in all detectors. As for specific values, typical sample concentrations range from 1-5 mg/mL. Samples that are low molecular weight or have low dn/dc values (< 0.050) will probably require a concentration on the high end of that range, or possibly higher. If a sample is high molecular weight and/or produces a particularly viscous solution, such as hyaluronic acid, optimal concentrations tend to be relatively low.
The column set in use can also influence your concentration. If you are only using one column, then you don’t need as high of a concentration since the sample fractions will not be separated as much as if you were using a set with three or more columns. Additionally, you don’t want to use a concentration that is too high so that the amount of sample will overload your column set (too concentrated to allow all sample molecules the opportunity to diffuse into the pores of the column gel) and affect the chromatography.
When preparing your OMNISEC samples it is critical to accurately determine their concentration if you do not already know their dn/dc value. This will allow the software to calculate a dn/dc value for your sample using the input concentration and injection volume and corresponding refractive index (RI) detector response.
Injection volume
Injection volume is related to sample concentration, as these are the two parameters that dictate how much sample is introduced to the system upon an injection event. As such, it serves as a convenient tool when preparing your OMNISEC samples. With the ability of OMNISEC’s autosampler to inject at 1 µL increments from 150 µL down to 1 µL, you can control how much sample is introduced at each injection…without having to prepare new sample solutions!
Typical injection volume for most samples is 100 µL. For proteins and other bio-related samples which may exist in limited quantities, injection volumes can range down to 20 µL. These lower injection volumes often work due to the relatively high dn/dc values (~0.185) of these materials in aqueous solutions.
If you are operating a REVEAL ULTRA as part of an APC-REVEAL system, then I recommend using lower injection volumes for all samples. Due to the smaller tubing and column volumes, injection volumes for these systems often range from 5-50 µL.
If you are trying to optimize your analysis conditions, running a series of varying injection volumes can help you determine the ideal amount of sample needed per injection. You only need to prepare a single sample solution (at a known concentration!) and then you can make injections of varying volume. The image below shows the RI response of 80, 60, 40, and 20 µL injections to determine the lowest volume that provides reliable detector responses. The autosampler and injection volume field in the sequence make this an easy experiment!
Temperature
I’ve already touched on applying heat to facilitate sample dissolution. Another aspect of temperature to consider when preparing your OMNISEC samples is the autosampler temperature. This is the temperature at which the samples will sit prior to injection. The OMNISEC autosampler has a temperature range of 4-60 °C. This means that samples can be heated to help maintain solubility until injection (but remember that the injection valve isn’t heated so the sample should still maintain solubility at ambient temperature). It also means that the samples can be cooled to ensure sample stability until injection. This is common when working with protein and other samples that are often refrigerated to prevent decomposition.
To maximize sample stability over the course of multiple injections of your samples, I recommend keeping your preparation and autosampler temperatures as low as possible. If you’re trying to decide between applying heat or extending dissolution time, I suggest using longer dissolution times. Speaking of which…
Stability over time
When preparing your OMNISEC samples it is important to maintain sample stability over the entire sample solution lifetime. I generally think about it as two different timeframes: the preparation/dissolution time and the idle time of the sample solution in the autosampler prior to injection.
Regarding the first, a common question is “how long should I let my samples dissolve?” This is, of course, extremely sample dependent so there is not a singular answer. Protein samples often dissolve within a few minutes. Whereas polymer samples can take many hours, depending on the sample type, molecular weight, and solvent. If possible, I recommend allowing polymer samples to dissolve overnight with gentle agitation. Whenever I can, I like to use a rocker instead of a stir bar, but if I am using a stir bar I keep the stir rate on low.
I do not recommend sonicating samples; in my experience this only causes them to degrade. And similarly, I do not recommend using a vortex when preparing protein samples, as this tends to result in aggregation.
As for sample stability in the autosampler, I’ve witnessed degradation of heated samples, and increasing aggregation of samples that continue to change in solution. To minimize thermal degradation, you can keep the autosampler temperature as low as possible. OMNISEC allows you to cool the autosampler vials down to 4 °C; just make sure you don’t freeze your solvent!
For the instances in which I’ve observed aggregates forming over time, it usually has to do with a small amount of water present in the sample producing a hydrophobic effect in the polymer. An example is when aqueous emulsions are diluted with an organic solvent, such as THF. The polymer dissolves in the THF and the water is miscible with THF, however even a small amount of water prevents the polymer from being completely stable over the course of multiple injections. As you can see in the four successive right angle light scattering chromatograms (red, purple, green, then black, respectively), the aggregate peak increases in magnitude over time.
Autosampler vial
The last thing to consider when preparing your OMNISEC samples is which autosampler vial to use. There are a variety of vials with different volumes to help make sure you are maximizing your sample solution. A standard vial is 2 mL, but if you don’t have that much sample solution, then a vial with a 350 µL inset will allow you to make a couple of injections, at least. I detailed the different styles of vials and included links to their respective pages on the Malvern Panalytical eStore in my previous post on OMNISEC consumables.
Sample preparation video
And last, here is an example of a typical sample preparation procedure.
Final thoughts
In conclusion, I hope this list helps you when preparing your OMNISEC samples. Furthermore, 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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