Optimizing Analysis Conditions, Part 1: Mobile Phase

There are a lot of things to consider when analyzing a new sample with your OMNISEC system.  Analysis conditions, such as the column set, preparation solvent, mobile phase, sample concentration, injection volume, and even other parameters such as flow rate and system temperatures can all influence the results. 

In this post, I’m going to focus on the mobile phase and use some example data to guide you through my process of attempting to improve the observed chromatography.  It should be noted that not every sample or sample type has an optimized solution such as the one presented below.  Hopefully the strategy I employed below provides you with ideas, and is also a reminder that sometimes you can get lucky!

Initial mobile phase – 0.05 M Na2SO4

The sample was described to me as a neutral, water-soluble polymer.  Therefore, since the most important requirement of any mobile phase is that it solubilizes the sample, I set up the sample to run in an aqueous solution of 0.05M Na2SO4 (sodium sulfate) using a set of mixed-bed aqueous columns. 

Optimizing chromatography with OMNISEC - adjust mobile phase - bad data

As you can see in the data above, the chromatography left something to be desired.  Only two of the detectors displayed any sort of response that resembled a sample peak: the refractive index (RI) and right angle light scattering detector.  And the signals of those two detectors only somewhat corresponded to each other.

Additionally, the detector responses never quite returned to baseline, suggesting that the sample was interacting with the column and not eluting properly.  Additives such as salts and/or organic co-solvents are often employed to minimize these column interactions, so that’s where I headed with my next attempt.

Second mobile phase – 5% methanol in water

In my experience, methanol is an excellent mobile phase additive when dealing with samples that are potentially sticking to the column set.  Another common choice is acetonitrile.  They are miscible with water and both have refractive indices that are almost identical to that of water, which is very convenient because that means their addition doesn’t affect the dn/dc value of your sample!

Therefore, I prepared a mobile phase of 5% methanol in water, placed it on the system and flushed the columns and detectors, and then re-analyzed the sample. 

Optimizing chromatography with OMNISEC - adjust mobile phase - decent data

Not perfect, but progress!  I now had signs of quality data: decent to strong responses in all detectors, sample peaks that corresponded with each other (even if they weren’t perfect), and observed the signals return to baseline following the sample peaks.  There was still a bit of noise on the back side of the light scattering peak, and that matched with the lopsided RI peak and shoulder in the viscometer signal.  This suggested to me that there was still a bit of interaction between the sample and column set, but that the majority of the sample had eluted as hoped.  Since a little bit of methanol had helped, for my next attempt I decided to use even more!

Final mobile phase – 20% methanol in water

I went with 20% methanol in water, which is the highest concentration of a co-solvent I’ve used.  That’s not to say you can’t go higher, but in my experience, this is where I had maxed out.  Something to note, with methanol-water combinations, the viscosity of the solution is high, so you’ll need to monitor your pump pressure and potentially reduce your flow rate.   Once I had the system converted and equilibrated, I re-analyzed the sample.

Optimizing chromatography with OMNISEC - adjust mobile phase - great data

As you might expect, since I was able to select this example after seeing the results, the chromatography was beautiful!  But I didn’t know it would turn out that way before checking.  The interaction between the sample and column set was completely eliminated and the peaks in all detectors are strong and clean.    

Final thoughts

I hope the example described in this post helps you understand the thought process behind my progression from one set of conditions to the next, in the pursuit of optimizing the chromatography of this sample.  Stay tuned for at least one follow-up where I’ll discuss optimizing a column set for your samples. 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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