Chromatography Forum

In reply to DR,
No, I actually haven't been lurking at all--yesterday was the first time I've looked at this forum.
I really don't have any long-term data yet, and nothing on the number of injections before seeing changes, primarily because I work in an upstream technology organization (in a big consumer products company), and I focus on capabilities and method development rather than on analysis. I'm eagerly awaiting reports from my colleagues who are routinely using these columns. None have reported any problems yet with column stability, but we do remain guarded in our optimizism until the data are in.
Like many other participants, I am greatly restricted in what I can divulge in forums like this. I'm sorry, but anything beyond vague generalities will be off limits for me.

All multies do this. We are repeating. Time to step out.

Just to make it clear:

It is not that I do like or dislike this technology and I do not have any problem with (most of) what have been said but the way these were said.

I have not used these columns yet so I can say nothing positive or negative.

supercritical,

As you mention, many companies have contributed to the technology of mixed mode columns, although not always with user forewarning. For example, Dionex has been selling polymer based mixed mode columns since 1989. But as you say technology continues to advance. The most recent example is the new silica based mixed mode column that Dionex is introducing at Pittcon (Paper 980-9) next week: the Acclaim Surfactant column. As the name suggests, it was developed specifically for separating surfactants, providing superior selectivity, efficiency and peak shape for anionic, cationic and nonionic surfactants. In fact, you can separate all three surfactant classes in a single separation without intermingling of classes in the chromatogram. However, thus far we haven’t done a lot of testing a lot of testing with pharmaceuticals so at the moment I wouldn’t want to make any claims as to the utility of this column for non-surfactant applications. The stationary phase is unique relative to previous mixed mode columns in several ways. First, the hydrophobic ligand (which contains a polar embedded functionality) and the weak anion exchange ligand used in the construct the phase are attached in “reversed geometryâ€

Cool! Thanks for the preview and nice description. I enjoy checking out new column technologies. Obviously the column is the "heart" of an LC system. I look forward to the launch of the product.

Have done a fair amount of prep LC and knowing how difficult it can be to simply pack a good column, I certainly appreciate all the work done over the years by column manufacturers in terms of manufacturing/packing columns, and researching new stationary phases, bonding technology & such.

My $0.02 on the Primesep columns: I've used them successfully for almost 2 years. Example: re-developed couple of finicky ion-pair methods that were a pain in the back to validate and transfer. The columns are quite rugged and take a lot of beating with junky samples, so a good choice for quick and dirty in-process methods.

I'd definitely recommend to give them a try when dealing with polar, hard to retain compounds -- alone or together with hydrophobic species.

Dimitre

Another advantage of mixed mode is that it might help you in separation of proteins by using two interactions. Combination of two interactions provides significantly higher selectivity than each individual interaction alone. We recently developed a method for one of our customers, who was struggling with separation of two insulins. Both insulins (Humalog and Humulin) have almost identical structure (52 amino acids). The only structural difference is in position of two amino acids in the side chain: Lys-Pro in Humulin vs. Pro-Lys in Humalog. I believe (but I might be wrong) that this is the first separation of these two peptides without chopping them apart. We presented this application at Pittcon and I can send reprint to anybody who would like to see it.

i am the one who post 'HILIC for small molecule', that SIELC listed as useful to his column,
so far i have tried a bit with HILIC columns, though i got some nice separations i would say they suck as a general purpose column for drug discovery, and i would think mixed-mode column will suck too.
the best results i got from HILIC is from, guess what, phenomenex luna amino column, even the claimed special HILIC column, like the waters HILIC silica performs horribly on my randomly picked samples from my daily work.
my feeling for such mixed mode is they are hard to manipulate on a fast paced industry like pharmaceuticals, i don't have the luxury to spend weeks on any one individual method, i have to get a working method in 2-3 days with a automated screening scheme(no buffer, no pH change for discovery compounds since the stability is mostly unknown) so the regular c18 or normal phase are the only answer because they are totally predictable and simple.
the last thing is i also work in a big pharmaceutical company and i know you can publish as long as you don't reveal the detailed molecule structure to public. so if supercritical wants to prove he is right he can tell us more details, like the type of the compounds, what kind of functional groups make it hard for regular column...
btw, SFC is a much better way to polar/hydrophilic compounds if supercritical is also concerned about prep part. i have routinely use it everyday and the resolution/throughput is GREAT. no buffer is needed...
too bad i can't find a forum just for SFC.
(maybe i can set up one, what do you guys think?)

Right now, SFC is lumped in with GC, mainly as a "critical mass" issue (if there are too few posts on a board, then no one reads it, so no one posts, so . . . ).
Is there enough SFC-specific interest to break it out as a separate area, or is the technique better served by leaving it in with GC where more people will see it?

Based on a success rate of automatic screening system you can see that about 30% compounds are not retained during these screening runs and that's where we see mixed mode chromatography can help. Thirty percent is approximate number we get as feedback from our customers. Controlling multi mode interaction boils down to the same as single mode chromatography. If you know nature and laws of interactions then you have no problem controlling it. In separation science it is all about laws of interactions, physical and chemical nature of all components of separation. Unless somebody tries something either on theoretical or practical level, it is hard to make an educated conclusion. It is strange for me to see words like "feelings" (outside fine arts, music and theater) as an argument for/or against any approach. SFC had a lot of skeptics a few years back and not every company has SFC.Until you see SFC unit next to Agilent 1100 it might be wrong to ignore new developments in liquid chromatography.

sorry for any misunderstanding here,
in my case where the environment is drug discovery, the non-retaining compounds only accounts about a few percent, so the ion-exchange mode is not that attractive to me( but i am still trying to do sth on HILIC because of its high organic content, which is good for the prep), the major problem in discovery/medicinal chemistry is the resolution since most are synthesized compounds whose precursor/starting material are very similiar in terms of chromatography, that's where i find SFC(packed column) a better tool.

The biggest brealthough?

Hmm... take for example sulphonated hypercrosslinked polystyrene (5 mkm or so).

1. It can act like reversed phase in water-rich mobile phases.
2. It can act like ion-exchange phase in water-rich mobile phases.
3. It can act as ion exclusion phase.
4. ... as HILIC phase.
5. .. for "ion ratardation".
6. It can act like normal phase in non-polar mobile phases to separate polar adsorbates.
7. It can act in quasi-normal phase mode in non-polar mobile phases to separate aromatic adsorbates (pi-interactions).
8. ... as SEC phase.

So.. I don't see any reason to get wonder and post many of "!!!!!"

If this thread keeps going long enough the question I posed a long time ago may eventually get answered, which is, "how does it work"? Am I correct that this column is a combination of reversed phase and cation exchange mechanisms accomplished by the inclusion of some cation exchage functionality in the ligand?

I think that Chris's post on the Dionex column should be the template for others wishing to describe new technology. The post contained sufficient information about the column to enable the reader to understand how it worked and begin to predict how it could be used. As such, it sounded like a scientific discussion rather than an advertisement.

Constantin,

I am sure that polymeric phase with sulphonated fragment are a good choice for a lot of applications. Primesep is not competing or undermining any other phase – just feeling a gap between reverse phase and ion-exchange phase. There are a lot of differences between Primesep and the phase you are talking about:
- Primesep is silica based and has higher mechanical stability
- There is no “memoryâ€

First I want to apologize for the following lengthy message-just trying to answer Bills’ question.

Bill,

Here are few words on our technology, you can go on our website and learn everything about the mechanism of interaction". I don't know how to post pictures, but we have several graphs on the website explaining how chemistry on the surface works
Mixed Mode with Reverse Phase and Cation-exchange Properties:

"SIELC Technologies provides several different columns ( http://allsep.com/Technology_CationExchange.php) with negatively charged functional groups due to the embedded anionic ion-paring reagent. Primesep A is the strongest acidic column, while Primesep C is the weakest acidic column. The Primesep 100 and Primesep 200 columns are in between. Difference in the functional group acidity allows selecting the most appropriate column for a particular set of basic compounds that differ in their pKa value.

The embedded acidic functional group can be in an ionized form, or in a non-ionized form, depending on the pH of the mobile phase. In order to get retention by ion-exchange mechanism on Primesep® columns, the pH of the mobile phase should be selected close to, or above, the transition value of embedded acidic groups. Below the transition pH value, the column behaves as a regular reverse-phase column with an embedded non-ionized polar group.

The column name corresponds to pH value of the mobile phase where acid residue on the column switches from ionize form to neutral one.


Cation-exchange
Column Type About 50% of embedded acidic groups ionized at transition pH value Separates basic
compounds
Primesep C pH = 3 strong bases, dibases
Primesep 200 pH = 2 strong bases, dibases
Primesep 100 pH = 1 weak, medium bases, AA, metals
Primesep A ionized in all working pH weak bases, AA, metals

Mixed-Mode with Reverse phase and anion-exchange properties
“…. two different B-type columns with positively charged functional groups (http://allsep.com/Technology_AnionExchange.php)
Primesep B is a strong basic column. The recommended pH range is from 0. 5 to 4 created by the addition of trifluoroacetic, phosphoric or perchloric acids to the mobile phase. Primesep B2 is a weak basic column that offers an extended pH range from 0.5 to 7, suitable for the ammonium acetate buffered mobile phase.

The columns provide two main interactions with analytes: the reverse-phase interaction and the anion-exchange interaction for neutral compounds. The reverse-phase interaction is sufficient to retain analytes, and the presence of the charged group in the alkyl chain provides additional selectivity uncommon for typical reverse-phase columns. Acidic analytes can be retained by both anion-exchange and reverse-phase mechanisms. To control retention and selectivity, there is a broad selection of the mobile phases with concentration of organic modifier in the 0-100% range. The concentration and the type of an acid will also significantly affect the retention of anionic compounds. Basic compounds can be retained only by the reverse-phase mechanism, but the presence of positively charged groups on both analyte and stationary phases produces the unique selectivity due to the ion-exclusion phenomena."