If my solvent includes DMSO, what do I need to know for DynaPro Plate Reader or NanoStar measurements?
Introduction
Dimethyl sulfoxide (DMSO) is an important solvent/additive that is commonly employed to study small-molecule drug compounds, and can be used in your DynaPro® Plate Reader or NanoStar®. However, there are several important factors that you need to consider when using DMSO with your DynaPro instrument in order to collect high-quality dynamic light scattering (DLS), static light scattering (SLS) and particle concentration data.
Considerations when working with DMSO
While relatively inert and non-toxic, DMSO is a hygroscopic solvent that dissolves a wide variety of different polar and non-polar molecules—this makes it very easy to contaminate your samples with unwanted particles. Please heed the following best practices for minimizing DMSO-related particle contamination, as well as specific guidelines regarding safe handling of DMSO.
1. Always work with fresh, filtered solvents in clean glassware, and transfer “working volumes” by pouring rather than directly pipetting.
- To maintain clean glassware, rinse out your solvent reservoir with ultra-pure water followed by alcohol to aid in drying prior to filling with your filtered solvent that contains DMSO.
2. Ensure chemical compatibility with DMSO.
- Filters and housings: While we encourage you to optimize your DMSO-compatible filtration system to suit your analyte(s)—we generally recommend Whatman™ polypropylene-based Anotop™ filters, which are adequate for filtering aqueous solvents containing 0-100% DMSO. Either discard the first few drops of filtrate or wash the filter with solvent in order to remove particulates from the dry filters.
- Microwell plates: For Plate Reader users, it is important to confirm with your plate manufacturer that your plates, lids, and covers are all DMSO-compatible. We recommend Aurora® Microplates’ cycloolefin plates, which have specifically been validated for use with the DynaPro Plate Reader. Please see the Plate Reader data on the right, which were collected using the above filter and microwell plate recommendations.
3. Flammable substances such as DMSO pose a general fire hazard risk.
- NanoStar: this instrument has been engineered to minimize fire hazard risk and electronic damage that can occur when working with volatile liquids. Additionally, quartz cuvettes are compatible with a broad range of organic solvents, as well as DMSO. For these reasons, we generally recommend using the NanoStar when working with volatile organics.
- DynaPro Plate Reader: since DMSO is relatively non-volatile at 25 °C, it can safely be used with your Plate Reader at ambient temperature; however, exercise caution when using high DMSO concentrations with a Plate Reader or when operating above 25 °C.
Filtered versus unfiltered DMSO: The slow-decaying autocorrelation function (ACF) for unfiltered 100% DMSO (red trace) indicates that this DMSO stock has been contaminated. This issue was remedied by filtering the same 100% DMSO stock through a 0.02µm, Anatop filter, prior to taking the DLS-measurement (yellow trace). Both measurements were taken using a DynaPro Plate Reader with Aurora 384-well plates.
Adjusting parameters in DYNAMICS to collect high-quality data with DMSO
It is important to adjust solvent and sample parameters in DYNAMICS® that are impacted by the presence of DMSO.
Solvent viscosity and refractive index: Viscosity and refractive index are both affected by solvent composition. Thus, depending on the amount of DMSO in your solvent, you may need to change these values to get accurate hydrodynamic radii and particle concentrations.
Sample dn/dc (for Mw-S measurements): The dn/dc value of a molecule depends on the solvent composition. The correct dn/dc value should be ascertained and entered when taking SLS measurements for molar mass determination, a feature available with the Plate Reader III, and all NanoStar models.
When using DMSO in your solvent, we recommend creating a new sample and solvent profile in DYNAMICS. Once you have designated a new profile, you can input the new sample and solvent parameter values. Please see the DYNAMICS User Guide for more information regarding how to assign sample and solvent profiles to your measurements using Experiment Designer or the Event Schedule.
Note: The dn/dc, viscosity and refractive index values can be determined in a number of ways: referencing the literature, using models in DYNAMICS or measuring them yourself. While the "aqueous" temperature model in DYNAMICS may be appropriate for low concentrations of DMSO, we recommend cross-checking the literature or experimentally determining the viscosity and refractive index of your solvent at the same temperature(s) in which you will conduct your experiment.
Include a DMSO-negative control
Aggregation screens are often collected on samples that are initially dissolved in 100% DMSO and serially diluted with buffer. When doing this, it is important to measure an equivalent negative-control solvent that contains the same final concentration of DMSO and buffer as your samples. To make a DMSO-negative control, simply perform the same serial dilutions with pure DMSO that you do with your samples. We recommend using the same initial DMSO stock for both the solvent and sample, as well as performing these serial dilutions simultaneously. This will not only keep your experimental system conditions as uniform as possible, but it may also help troubleshoot any potential downstream issues related to particle contamination. For more information about conducting aggregation screening with DMSO, please see TN7002 – Screening for Compound Aggregates.
Note: Given DMSO’s high refractive index (~1.48), it will scatter more light compared to aqueous solvents. Thus, to maintain adequate instrument sensitivity when working with DMSO, you should aim to have the total scattering intensity of your analyte (Normalized Intensity) at least 2-3x that of the pure DMSO. This can be quickly checked within the Datalog Grid.
Conclusion
DMSO is an important solvent/additive for studying drug compounds, often with DLS. Due to DMSO’s high flammability risk, it is important that you follow proper safety guidelines, especially when working with it in a DynaPro Plate Reader. By heeding these guidelines, as well as implementing appropriate sample-based preparation and good experimental design practices, you will be ready to obtain high-quality DLS data with DMSO in your DynaPro Plate Reader and NanoStar.
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