How to prepare a new HPLC column?

[hplc.chem]

Dear all,
One of my colleagues, who is an organic synthesis chemist, has synthesized an organic material with the potential to separate enantiomers. He asked me, as a HPLC analyst, if we can work together to develop a HPLC column based on this material as the stationery phase. However, as a HPLC USER, I was not sure how to develop such a column. I read some articles in which the workers developed new stationary phases by a series of chemical reactions, but I am not sure that the method described there can fit other materials.
Can you help me to know the basic principles behind preparing new stationery phases?
What experience and equipment are required for a chemist to prepare a new stationary phase?

Many thanks in advance!

[Uwe Neue]

See if you can get copy of my book on "HPLC Columns". It has a lot of good information on this subject. I can also send you other material, such as book chapters, if you contact me.

In principle, there are multiple ways to go about immobilizing a ligand to the surface of a chromatographic packing. How you do it depends on the need. You can just coat the surface, or you can immobilize the ligand to the surface via a chemical bond. An example of the first type are the older chiral phases from Daicel. Almost everything else is immobilized via a chemical bond to the surface. The most common technique is a silanization of the surface, but that may not be necessary, depending on how you want to use the packing.

[hplc.chem]

Thank you Uwe for your quick response and the valuable summary.

I already have your valuable book and it is really rich of information about HPLC columns. It was my start for this search. I went through some of the sections in the book, but to be honest, some of the information is above my background level.

What I am trying to find is a kind of detailed recipe for preparing stationary phases and packing them to HPLC columns. I am not sure if such recipes exist!

[Uwe Neue]

There is no generic recipe for preparing stationary phases. If you want, I can advise you and your colleague, once you give me more details.

[hplc.chem]

Many thanks Uwe for your help and for the advice offer.

What kind of details we have to gather before starting the process? I will ask my colleague about them.

[Uwe Neue]

A lot...

What is the chemical nature of the chiral material. If it is a polymer, with limited solubility, it can be attached to the surface simply via adsorption. Together with this comes the question, in which solvents you expect a chiral interaction.

If it is a rather small molecule with a wide range of solubility, the question becomes what functional groups are available for attaching it to a chromatographic surface without disturbing the chiral center. Once we know that we can think about the most convenient way to attach it to the surface. Often a long ligand is advisable that does not disturb the chiral interaction etc.

The common carrier is a silica-based packing with the right pore size for the ligand and the interaction.

Once the stuff has been made, we worry about packing it into a column, but this is a long, long way away.

[hplc.chem]

Thanks a lot Uwe.

I will discuss all this conversation with my colleague and I hope to have a good start with the help of you and all the friends in the forum.

Many Thanks Again...

[hplc.chem]

Hi all again,

After the discussion with my colleague, a question was raised. The compound that is proposed to be used in preparing the stationary phase has the ability to REACT CHEMICALLY with each enantiomer in a different way. Two products will be generated, one for each enantiomer.

I wonder if this might be a positive or negative point for using the compound as a stationary phase!

i.e. can we rely on chemical reactions as a mechanism for the separation of enantiomers?

Many thanks for any help.

[tom jupille]

can we rely on chemical reactions as a mechanism for the separation of enantiomers?

Possible, but I think it would be difficult. The easiest to imagine is if:
A. the reagent (and therefore the resulting diastereomers) is immobilized on the stationary phase,
B. the reaction is reversible, and
C. the equilibrium constants for the two diastereomers is different.
all three of the above conditions would have to be met.

That's the way many chiral columns work, with the difference that a diastereomeric complex (transient) is formed rather than true (covalent) diastereomers.

A more plausible use would be for pre-column derivatization.

[Uwe Neue]

You have enantiomers, and you react them with something to generate two different products. This is not a separation, but the reaction needs to be followed by a separation step for the two products.

In principle, this is rather similar to the reaction of enantiomers with an enantiomeric reagent to form diastereomers, which can be separated with standard chromatography.

The chemical reaction does not generate a separation. It only enables a separation.

[hplc.chem]

Many thanks for both of you Tom and Uwe.

A more plausible use would be for pre-column derivatization.

What do you think about using such a reagent as a mobile phase additive for chiral separatopn?

[Fiz]

Dear all,
One of my colleagues, who is an organic synthesis chemist, has synthesized an organic material with the potential to separate enantiomers. He asked me, as a HPLC analyst, if we can work together to develop a HPLC column based on this material as the stationery phase.

That sounds pretty similar to a project I had years ago.

However, as a HPLC USER, I was not sure how to develop such a column. I read some articles in which the workers developed new stationary phases by a series of chemical reactions, but I am not sure that the method described there can fit other materials.
Can you help me to know the basic principles behind preparing new stationery phases?
What experience and equipment are required for a chemist to prepare a new stationary phase?

First you need think how to immobilize your chiral selector to the stationary phase. And think which stationary phase meets your demands best.

Then you should do a prove of concept. Pack a column and use a set of chiral compounds to test the enantioselective behaviour of your material. I suggest that you read some articles from Yoshio Okamoto who did an excellent work in the development of chiral stationary phases.

One main problem I was facing right from the beginning was to synthesis enough material for the column packing. So I decided to pack capillary columns (75 µm i.d.) for testing my stationary phases which was nicely working.

I think optimizing packing of analytical columns just make sense if you really decide to market them. Because column packing is more art than science.

[tom jupille]

What do you think about using such a reagent as a mobile phase additive for chiral separatopn?

I think the various replies have been interpreting your posts in different ways, so please correct me if I'm misinterpreting

If what you have is other enantiomeric compounds irreversibly to form diastereomers, then:

A. If the reaction is quantitative and extremely fast (goes to completion in milliseconds), then adding the reagent to the mobile phase is feasible. The main concern would be the effect on the detector (e.g., if the reagent is UV absorbing).

B. If the reaction is incomplete or slow, then adding reagent to the mobile phase would not work, and the derivatization reaction would have to be done before injection (this is what I was thinking of in my earlier post).

If what you have is a compound which reacts with other enantiomeric compounds *reversibly* to form diastereomers, then either immobilizing may be feasible (depends on the speed of reaction and on the equilibrium constant). Beta cyclodextrin, for example, has been used both ways. The downsides to mobile phase addition are detectability (per above) and the fact that you are throwing away the expensive reagent.