Separation of Ethylene Glycol, Formamide, and DMSO

[ebasarah]

Thanks mbicking,

What Polar Bonded phase column were you using?
Regular RP phase can't have 100% water. Did you use amino bonded normal phase column?
I really appreciate if you can give me more details
DMSO and Formamide have relatively strong absorption at 195-215nm but Ethylene glycol has poor absorption at 190nm.

Every experiment was done in triplicate, run blank, and co-injection to make sure I see what I see.

Thanks!

Ernest Basarah

[Uwe Neue]

In my opinion, a HILIC coating is defeating the purpose. Pure HILIC relies on the adsorption of water on the surface of the packing. You can do that very well with silica or a zwitterionic surface, or a partially charged surface like an amine. I think that a diol is much weaker than any of the other options. I have experience with the silica, which is why I am recommending it.

I still give HILIC more of a shot than RP. In RP, the packing with the strongest retention is the Atlantis T3. It gives roughly double the retention compared to a packing with an embedded polar group, but indeed both are compatible with water.

There is a possibility that you can get things done in water as well, by salting out effects. A long, long time ago, I did sugar separations on the unendcapped Resolve C18 0.5M NaCl. The enginneers did not like this though since the NaCl is no good for steel.

[ebasarah]

Thanks Uwe,

There is a possibility that you can get things done in water as well, by salting out effects

Does the salting effects bring the analytes from water to organic solvent?
Could you elaborate more about salting effects, thanks!

[ebasarah]

Hi Uwe,

let me redo my previous reply.

There is a possibility that you can get things done in water as well, by salting out effects

Does the salting effects bring the analytes from water to organic solvent?
Could you elaborate more about salting effects, thanks!

[Uwe Neue]

I still think that the HILIC approach with a HILIC silica column is your best option.

To do the game with the salting out effect, you use a C18 that is compatible with 100% water. I had used Resolve C18. You run a high salt concentration as the mobile phase. You can think about what kind of salt you want to use. As I said, I do not recommend NaCl, unless you have a special HPLC instrument or you like to see your service engineer more often.

A high salt concentration increases the retention of very polar analytes on a water compatible C18 in 100% water. It is similar to the salting out into an organic solvent in extraction procedures, which maybe something that you may be familiar with.

[ebasarah]

Thanks Uwe,

So Far, I had tried using Luna NH2, Spherisorb ODS (2), Luna Hilic under various solvent mixtures.

Luna NH2 and Luna Hilic gave same result, three analytes came together when I used 90% ACN/water.

Surprisingly, spherisorb (normal C-18, 10% Methano/water) gave separation between DMSO and formamide but EG came at the same time with Formamide.

So the salt in salting out effect is not acting only as a buffer but more like decreasing solubility of analytes by occupying water molecules.

I thought phosphate buffer at low pH or any salt with pH too acidic or basic are more corrosive than NaCl? What is the common milder salt is safe to use for this purpose?

Thanks again for your input!


Ernest Basarah

[mbicking]

For my separations, I used a Prontosil EPS phase (polar embedded phase). I believe it is an amide. The Supelco RP Amide seems to have similar selectivity.

I would try one of these phases, and I like the salting out effect the Uwe suggested. You could use just about any non-corrosive salt, including the phosphates (although a pH change will result) or sodium sulfate.

Good luck and let us know how it comes out.

[Uwe Neue]

Sodium sulfate is a reasonable choice. Not corrosive, and reasonably soluble.

With respect to packings, my first choice is the ancient Resolve C18. It is a highly coated, unendcapped C18 that is running without difficulty in 100% water. It is similar to the Spherisorb ODS (2) that you are using. The advantage of Resolve is that I can guarantee that it will not have any dewetting character in 100% water (been there, done that). My next choice is Atlantis T3, which has been designed to give maximal retention in standard aqueous mobile phases without any dewetting. However, it is endcapped, and if indeed you need to use a high salt concentration to get retention, it may run into difficulties. The same is true for the Spherisorb ODS (2).

Packings with embedded polar groups generally give roughly 2x less retention than the Atlantis T3 recommended above. Otherwise, I would either go for the Supelco RP amide mentioned above or for the Symmetry RP18 packing. Many of the other packings with embedded polar groups contain residual amine, which limits there stability. I do not know how the Prontosil EPS phase is made, but I advice caution when picking a random EPS phase.

Since you have the Spherisorb in your hands already, I suggest to try it first in water with sodium sulfate. If it does not give you enough retention, I would go for the Resolve C18 first, and then for Atlantis T3.

[mardexis]

This is a nice discussion on HILIC. Slighty off the HILIC though...do you have a GC available with an FID?

[ebasarah]

Thanks all for the input...

I will update you if anything interesting happens.

I had tried using pottassium phosphate salts 0.05M with 100% water.
What is the concentration range to achieve effective salting effect?
Usually I don't do more than 0.1M, please advise.


I also had looked into GC, and in fact, I found a literature of GC which separated DMSO, Formamide, and Ethyle Glycol. That is our last option since we don't have GC here.

RP- Amide bonded phase sounds promising and hopefully could get decent retention times for Formamide.


Thanks!

[mbicking]

The salting out effect is directly proportional to the ionic strength, which is directly proportional to the concentration of the salt. However, high salt concentrations can cause other problems with the LC, so I would stay well below 0.1 M unless absolutely necessary.

Try this approach:
Do the injection with water only.
Then prepare 100 mL of a solution of 1 M sodium sulfate (12.6 g/100 mL).
Dilute 10 mL of this solution to 1 L of water, to produce a 10 mM solution. Equilibrate and inject.
You can prepare 20 mM and 50 mM solutions using the same approach.

My personal preference would be to stop at 50 mM, although others may be comfortable with going to a more concentrated solution.

Let us know how it works out.

[Uwe Neue]

I a fully aqueous environment, I am routinely running salt concentrations up to 1 M, typically around 300 to 500 mM.

[mbicking]

Uwe:
I realize it is possible and common to use higher concentrations of dissolved solids, especially with some of the aqueous SEC columns, but I am always trying to maximize component lifetime, and every little thing helps. And I know you are familiar with all of the potential problems and will take all of the precautions necessary to minimize problems.

Unfortunately, many less experienced analysts are not aware of all of these things, so I am always extra cautious when I am making recommendations to people that I haven't worked with before. I visit many labs around the country, and there is a very wide range of understanding about how to care for an LC. I would hate to have someone finish working with a 500 mM buffered mobile phase and then try to immediately clean the system with acetonitrile, resulting in massive precipitation everywhere.

[Einar Ponten]

Hi,

Now this really start to be puzzling. However, there are different kind of HILIC columns working according to the same principle.

As you describe the mobile phase shall consist of >50-60% of a solvent like acetonitrile [propanol is however stronger eluting than acetonitrile] and the rest aqueous with or without (as Uwe suggest) a buffer. The advantage not to have a buffer salt in the mobile phase is obviously the reduced risk for UV absorbtion. However, it is "well known" that mobile phase buffer salts contribute to build up the water layer on the stationary phase, which in turn contribute significantly to the retention. It is consequently a compromise, but generally it is recommended that one have 5-20 mM of overall ion strength.

Regarding your question about column stationary phase chemistries the Waters Atlantis HILIC is in principal described as a (sophisticated) plain silica phase and the Luna Diol as a propriatory phase with non-chargeable diol groups, if silanolic interactions are assumed to be none.

In principal one would expect both these to give reasonable results, while final tuning of resolution might be difficult as you indicate by already using high ACN ratio for these polar compounds.

I still suspect that your sample is not adjusted before injection (see above). If you inject samples with higher water content than the mobile phase these columns will probably not show sufficient resolution due to the stronger elution power of the sample solution than the mobile phase.

Try to reduce the injection volume and buffer ion strength. Take great care about what kind of buffer salt you are using and make sure the acetonitrile is "low UV" grade.

The bottom line is that I really think this should work and we are more than happy to provide you with a free copy of "A Practical Guide to HILIC" that can be downloaded from our website. If further attempts still not are successful our Support Team are ready to support you.