IP-RP-HPLC

[HW Mueller]

Bryan, what is the solution?

Vlad, if silanols counteract the positive charges via exclusion then how come the "added" negative sites don´t do it?

The data presented by Bryan makes me wonder why some regard TFA a IPA, but not acetic acid.

[Vlad Orlovsky]

"Vlad, if silanols counteract the positive charges via exclusion then how come the "added" negative sites don´t do it? "

Hans,

"Added" sites can do the same. This is true when your acidic analtyte is weaker or has same as acidic group on the surface. Both acidic groups are competing for the same basic site and that is why retention time is much shorter then in case of TFA or any other strong acid. In case of silanol and acetic acid they both have pKa around 5 (some of the silanols can be even stronger due to impurity inclusion in the silica)

[Bryan Evans]

Bryan, what is the solution?

To quote Mr. Yazawa:

"Several mixed-mode reversed-phase stationary phases consisting of alkyl and ion
exchange ligands have been introduced into the HPLC column market (Fig-1).
However, these mixed-mode stationary phases have the following disadvantages:
(1) Column designs that employ either RP/cation or RP/anion offer limited
solutions for unknown samples (difficult to choose which column to use) and (2)
The RP/anion/cation phase columns that are marketed today consist of only one
ligand structure. A balance between hydrophobicity and ionic strength cannot be
controlled when this surface structure is employed.

We have overcome these disadvantages with a revolutionary multi-mode ODS
phase structure"

A Development and Application Study for Anion Exchange + Cation Exchange + Normal Phase + Reversed Phase Multi-Mode ODS Column

Yazawa Itaru, Tachikawa Hiroshi
Imtakt Corp. / Kyoto / Japan
Separation Science Singapore 2009

[Vlad Orlovsky]

Brian, This still not answers Hans question....and for Mr. Yazawa statement I would say that SIELC introduced tri-modal column (hydrophobic with cation and anion-exchange sites and HILIC with cation and anion exchange sites) in 2006. SIELC has at least 3 phases with DIFFERENT tri-modal chemistries. Dionex introduced tri-modal columns in 2007 as prototype and in 2008 as commercial product, so the statement of "new" is at least three years old. Balance between hydrophobicity and ion strength is controlled by buffer pH, buffer concentration and amount of organic.
I did not want to participate in this discussion but after last statement decided to add my opinion about "mixed" phase vs. mixed-mode phases.
This opinion is based on common knowledge and 6 years of hands-on participation in creation of over 100 different mixed-mode phases (22 are commercialized by SIELC).

When you mix two stationary phases you are going against nature for several reasons.

1. Chemistry - change in selectivity. You have a variation in synthesis for both stationary phases. This spec might be very narrow, but this means that each time you either need to adjust the exact ratio between two stationary phases or keep the ratio the same but sacrifice selectivity of the column. For example, if the spec is set at 2% which is a reasonable spec even for C18 columns and your anion-exchange mixed-mode phase shows minus 2 percent of the spec and you cation-exchange mixed-mode shows plus 2% the resolution between cation and anion will change much more than 2%, it can easily be 4, 6 or even 8%, in addition to that hydrophobicity of the anion-reversed-phase ligand is slightly different than hydrophobicity of cation-reversed-phase ligand, so the variation in retention time for hydrophobic anion and hydrophobic cations will change even more. In mixed-mode chromatography you can often observe a synergy effect of two interactions, hydrophobic and ionic, so in a lot of cases interaction 1+1 is equal to 3 and sometimes even 4. If you have polycharged compounds (diamines, diquats, diacids, triacids, etc.) you will observe a magnifying effect of 2% variation. This is assuming that stationary phase is rock solid and does not deactivate or hydrolyze, but in real life this is not true. People can forget to store column appropriately or leave it at higher or lower pH, inject molecules which absorb irreversibly on one portion of the "mixed" phase. If you have polycharged cation (peptides, proteins, polyamines) it is not going to stick to anion-exchange part of the phase, but will stick to cation-exchange part, this is true for anion-exchange with multi charged anions. This absorption will change the ratio of cation-, anion and RP interactions. What works in advantage of "mixed" phases is that when you hydrolyze ligand it leaves the particle really fast (due to repulsion effect) and jumps into the oppositely charged particle where it remains until much stronger mobile phase is used. This mobile phase needs to be strong in terms of ions and RP properties. That is why when you use a weak mobile phase you might not see any bleed of stationary phase. This "migration" takes 2 sites from the equation, changing ion-exchange properties of the column, keeping reversed phase properties almost intact. If you decide to wash column with much stronger mobile phase there is a chance that you will elute hydrolysed pieces and change properties of the column again, but now it affected all three interactions (cation-exchange, anion-exchange and reversed phase)

2. 50/50 mixture - lower capacity/loadability: If you have a mixture of two silicas you automatically reduce capacity of the column which might be critical in terms of retention and loadability in prep chromatography. Hydrolysis/deactivation will reduce this even further.

3. Efficiency of the column - packing two differnet particles: Two oppositely charged particles will attract each other in a slurry forming clumps. You need to play with pH of the slurry and might need to go beyond stability range of the stationary phase in order to spread particles apart (for people not familiar with packing) In a slurry phase of packing you need to place particles apart in an "organized" manner and on packing stage you need to bring them as close and as organized as possible, in order to have perfect symmetry and good plate count. It is hard to do when you have particles which attract each other like two magnets with opposite sides. There are ways to fight this "artificially" like packing columns with 5 um silica which in real life is 4.3 um (or 2.8 silica instead of 3 um)

I represent SIELC and my opinion might be or is bias.

[HW Mueller]

I am used not to get answers.
Vlad, your 3rd reason, attraction of charges, is a bit hard to understand as there have to be counterions on these particles, it seems one can prevent lumping by using a high ionic strength in the slurry solvent. The molecules which you attach don´t lump together either?

[Bryan Evans]

3. Efficiency of the column - packing two differnet particles: Two oppositely charged particles will attract each other in a slurry forming clumps. You need to play with pH of the slurry and might need to go beyond stability range of the stationary phase in order to spread particles apart (for people not familiar with packing) In a slurry phase of packing you need to place particles apart in an "organized" manner and on packing stage you need to bring them as close and as organized as possible, in order to have perfect symmetry and good plate count. It is hard to do when you have particles which attract each other like two magnets with opposite sides. There are ways to fight this "artificially" like packing columns with 5 um silica which in real life is 4.3 um (or 2.8 silica instead of 3 um)

Yes, correct. The breakthrough for this technology was the blending technology. These particles are blended in a way
that forms a homogenous packing material. You can see a diagram of it in the poster.

Imtakt's data also shows that column efficiency is equivilant for both the multi-mode ODS (3um) and conventional ODS (3um).

[Vlad Orlovsky]

Hans,

it depends on the nature of counter-ions. If stationary phase has strong ligands then they will attract each other, you can solve this problem by packing columns in slurries containing ions and playing with pH. Slurry and pusher can be selected based on ionic/hydrophobic properties of the stationary phase.
The main concern in "mixed" phases is reflected in my first statement about reproducibility/selectivity and Brian did not address this issue in his response, only loadability and efficiency. What happens is that both phases are hydrolyzed at different rate, so by the time people move from method development to validation they have a completely different column in terms of selectivity and retention. People have tendency not to follow Column Care instructions very often -some times even throwing away all paper work when they open the column (from my 10 years of experience in big pharma). When they re-order new column retention/selectivity will be different from the column they used for method validation. If you decide to wash column with very strong mobile phase you will elute whatever hydrolyzed from the stationary phase. In mixed-mode chromatography 1+1 in terms of interaction is not 2 (synergy effect of two interactions).
When we started developing new mixed-mode in 2003 we explored "mixing" phases and learn a lot. Conclusion was simple "don't mix". It took us few months of extensive work to realize that all three functionalities suppose to be assembled on one ligand. Mixing/blending is the easiest way to create "mixed-mode"phase and I did not see too much success. Dionex also (according to their statements) did not "mix" several phases to get desired result.

[XL]

I would like to join the conversation at this point since mixed-mode chromatography is my favorite subject.

In my opinion, the biggest advantage of mixed-mode columns is "adjustable selectivity" which can mean many things, such as:
1. Retain and separate hydrophilic charged analytes without using ion-pairing agents, which is usually a difficult and/or tedious job for a RP column.
2. Selectivity complementary to RP columns
3. Elution order can be "tuned" according to the need.
4. Complementary selectivities can be achieved on the same column.

Since Dionex Trimode Mixed-Mode column was mentioned, I am providing some technical information as below:
Dioenx trimode column is called "Acclaim Trinity P1." It is based on high-purity, spherical, porous silica particles. The inner-pore area is functionalized with an organic layer that provides both reversed-phase and anion-exchange properties. The outer-pore area, conversely, is modified with cation-exchange functionality using proprietary nanoparticle technology. [b][u]This chemistry ensures spatial separation of the anion-exchange and cation-exchange regions so that they don't interfere each other. [/u][/b]As a result, this column simultaneously provides reversed-phase, anion-exchange, and cation-exchange retention so that neutral, acidic, and basic analytes can be separated in the same chromatographic analysis. [b](http://www.dionex.com/en-us/webdocs/707 ... 239-01.pdf)[/b]

Here is the link of a poster that summarizes pharmaceutical applcations of this column. including:
• Simultaneous separation of pharmaceutical counterions (both anions and cations)
• Simultaneous separation of drug substances and respective counterions
• Separation of mixtures of basic and acidic drugs with respective counterions
[b](http://www.dionex.com/en-us/webdocs/775 ... 169-01.pdf)[/b]

This column has much more applications that are demonetrated in the links above. Please feel free to contact me if you have any comments and.or questions.

Thanks for reading this message.

[HW Mueller]

OK, so I wonder what happens when Vlad´s and XL´s columns are mistreated and loose ligands. Does the silanol concentration increase?

[XL]

Hans, any silica based bonded phase will loose ligand upon use and silanol concetration will increase over time. The key point is how robust a column is towards such changes. It takes careful design of fthe column chemistryT and properly use. The features of the Dionex Trimode column lie on the following aspects:

1. Anion-exchnage functionality is located at the "tip" o fa n alkyl ligand where is far away from the surface silanol groups. The benefit of this arrangement include desirable selectivity for anionic analytes such as orgainc acids and high resistance to column aging compared to other version in which the anion-exchange group is at the proximity of the surface.

2. More importantly, Dionex Trinity column not only has both anion-exchange and cationi-exchange properties (n addition to RP propertiesi), but also these two functionalities are "separated" in two different locations: Anion-exchange is at inner pore area and cation-exchange is at outer surface area. They don't interact (interfere). This allows for independent control of both retention mechanism.

I would appreciate that you spend a few minute take a quick look at the information from the links I provided in my previous post. You might find something interesting to you.

I would say column manufacturers are doing their best to develop something useful to make chromaotgraphers life easier. But it is up to users to decide if such objective is fulfilled. Of course we don't want to mistreated our column.

Thanks,

[HW Mueller]

Well, I was after some info as to whether abuse or aging of the columns will change stat. ion ratios in all the mentioned columns or only in Bryan´s mix. So obviously all have that problem . Thanks.

As far as mixed, whatever, we have been through it before. In short, I think they are speciality columns. I still would like to have a silica reverse phase without any SiO- possibility. Just pure rp.

[Vlad Orlovsky]

Sorry, Hans, I did not mean to ignore your question – we're just busy with new products introduction for EAS.
Here is what happens in true mixed-mode columns:

Almost any column can undergo hydrolysis of stationary phase. In our case, the ligand contains all functionalities, so when stationary phase is hydrolyzed, the column loses all two or three groups (bi-modal or tri-modal columns). This exposes the silanols, whose pKa is about 5, while the pKa of acidic groups in SIELC columns range from 1 to 4, so even the weakest has 10 times stronger acid on the surface.
In the cases of Primesep 100 and Primesep 200, there is even bigger difference in the strength of the silanol groups and acid on the surface (10,000 and 1,000), so even if you lose 5% of the ligand and expose 5% of silanols, the impact of Si-OH on the overall change in acidity of the column is much less (0.005-0.5).

Another point is that when you operate at pH 2-4 (which is the most effective range for SIELC columns), silanols groups are partially suppressed and not ionized (due to high purity of modern bare silicas, you can neglect stronger pKa silanols groups which have inclusions nearby).

Even if you expose some silica, you are not going to see as much effect on retention as in case of “mixedâ€

[Bryan Evans]

Well, I was after some info as to whether abuse or aging of the columns will change stat. ion ratios in all the mentioned columns or only in Bryan´s mix. So obviously all have that problem . Thanks.

Not an issue - novel bonding chemistry takes care of all this.
But, alkali durability may be not be great. So, our instruction manual
says 1.5 - 7.

As far as mixed, whatever, we have been through it before. In short, I think they are speciality columns. I still would like to have a silica reverse phase without any SiO- possibility. Just pure rp.

Let's hope you never have to do LC-MS or purification of polar ionic compounds.
Many people do, and they are finding out that "fully endcapped ODS" is useless for these compounds.

[Uwe Neue]

Bryan, I beg to differ…

Ionic compounds can be run very well on modern packings under conditions useful for MS analysis. The many publications by David McCalley that have focused on ionic and specifically basic compounds are worth reading.

Preparative chromatography of ionic analytes is limited, due to ion repulsion between the ions. One can get a 10- to 50-fold higher loadability in the non-ionized state, which for basic analytes, means that you run the separation at pH 10 or higher. The following is a publication that explains this mechanism and demonstrates the effect mentioned above:

U. D. Neue, T. E. Wheat, J. R. Mazzeo, C. B. Mazza, J. Y. Cavanaugh, F. Xia, D. M. Diehl, “Differences in the preparative loadability between the charged and uncharged form of ionizable compoundsâ€

[HW Mueller]

Bryan, Why this contradiction:
"Not an issue - novel bonding chemistry takes care of all this.
But, alkali durability may be not be great."?
So your columns can be abused as well, and they are not chemically inert.

Also, I didn´t say that specialty columns are useless, and you don´t have to hope, . . . I am handling and have handled polar ionic compounds, just didn´t come across anything which would require specialty columns of the type discussed here.