Primesep 100 and Primesep 200 columns

[Victor]

These columns are recommended for the analysis of basic drugs at low pH using something like TFA in the mobil ephase to adust the pH.

I understand that the retention of drugs is likely to be higher on the Primesep 100 column, because the embedded ionic group has a lower pKa i.e. it remains ionised unless very low pH values are used. Conversely, the ion interaction properties of the Primesep 200 column can be "switched off" at a higher pH than the 100 column, because the pKa value of the group is higher in the 200 column. Is this right?

However, are the numbers of ionic groups in the two phases about the same or different, when the groups on both types of column are fully ionised? i.e. is the ion exchange capacity of the two columns the same or different?

[SIELC_Tech]

Victor,

The number of groups acidic groups is almost the same. We did some experiments at pH-6-7 where all our acidic columns are fully ionized and got close retention.
In order to fully switch off Primesep 200 column you need to go below pH-2. The easiest column to switch is Primesep C column. pKa value on the columns are 1, 2 and 3 (Primesep 100, Primesep 200 and Primesep C).

There is no need to use TFA for Primesep columns. You can use any additive/buffer with creates ion-strength (sulphuric acid, phosphoric acid, formic acid, ammonium formate, ammonium acetate, phosphate and sulphate buffers).

ou can go to our website www.sielc.com and under Technology find section on method development (step-by-step, or based on your compound, buffer or detection technique).

I will try to see if I can find our study at higher pHs (amount of acid on the column)


Regards,

Vlad

[Victor]

Vlad,

Thank you for this helpful reply.

Can I ask you about the pH stability of Primesep columns? Usually with bonded silica columns there is concern about loss of the bonded group below about pH 2.5. This is especially true when endcapping groups like trimethylsilyl groups are present on the phase, because these appear to be lost first. If the pKa of Primesep 200 is 2, and the pKa of Primesep 100 is 1 , isn't there a danger of cleaving off the whole ligand (or the endcapping if it is endcapped) while trying to control the ionisation of these groups? Clearly it would not seem sensible to try "turning off" the ionic group in Primesep 100, because Primesep 200 could be used instead. But in the case of Primesep 200? I believe Primesep C is not so closely related in structure to Primesep 100 and 200, so might not provide an equivalent lower retention substitute for Primesep 200.

[SIELC_Tech]

Victor,


All three columns (Primesep 100, 200 and C) have different acids on the surface, but in most cases they within the same pattern. The only difference or Primesep C column is that in addition to reverse phase and ion-exchange properties it provides complex formation with metals and primary and secondary amines. Primesep C column is easier to switch from reverse phase mode to mix mode and to ion-exchange, due to pKa value (3). If can find a lot of mobile phase within pH-2-7 to play the switch mode. It is harder to do with Primesep 200 (pH below 2) and almost impossible for Primesep 100, but there is no need to do that because one of these three will provide you with the retention. It is the matter of right mobile phase (buffer concentration and nature, pH).
Personally my favorite column is Primesep C. You can use three mechanisms to separate your ionizable compound. pH of the mobile phase will drastically effect your separation and you can move compounds (ionizable) along the column any way you want.
(Please check few technical notes and methods for the influence of pH on Primesep C column).
http://www.sielc.com/pdf/SIELC_PrimesepC_Column.pdf

http://www.sielc.com/application_079.html

http://www.sielc.com/application_052.html

http://www.sielc.com/application_089.html


Primesep C column is very good for hydrophobic, hydrophilic basic compounds with pKa-7-14, as you can easily control retention. You have to keep in mind ionization state of your compounds at different pH. For example for amino acids Primesep c might not give you best results as at 3-7 you will se effect of acidic fragment on the acid, but n this case you have Primesep 100 and Primesep 200 which will provide you with the same mechanisms. The advantage of Primesep C is that you can analyze polyamines. In the method below three amines are separated on 50 mm Primesep C column with K prime of 5-25. Primesep 200 will give you much stronger interaction and you need only 10 mm column to separate three amines.

http://www.sielc.com/compound_140.html

Regarding stability: Primesep columns have the same stability as many columns with polar embedded groups. It is not simple acid with pKa of 1 or 2 on the surface, there are some other groups which stabilize the phase.

[JA]

when would one want to switch off the ionic functionality in Primesep 100, 200, C, by going below their pKas?

If there are cases, is there any data showing the stability of retention of a neutral molecule in a mobile phase pH low enough to neutralise the surface charge? With the 3 columns above wouldn't we be talking about pH 0, 1 and 2, respectively.

The catalogue states the Primesep silica is stable at pH 1. Are there any comparisons to some leading C18 columns?

[SIELC_Tech]

We do not recommend to use column for prolong time in pH-1 and you don't need to do that. You can choose one of Primesep columns and operate at usual pH=2-7 and retain acidic, basic hydrophobic and hydrophilic compounds. We do not have comparison data with leading brand C18, but we have columns in the lab which are almost 1 year long and they perform fine.

You might need to shut down ion-exchange properties when you are analyzing compounds with different hydrophobicity and basicity, compounds with multiple charge. Very good example is NyQuil cough medication when you have 4 different compounds with totally different properties (acetaminophen, pseudoephedrine, doxylamine and dextromethorphan). Tow are very hydrophilic (acetaminophen and ephedrine) two are very hydrophobic and basic. With Primesep C you can run it at pH-3 in isocratic mode. Check the link below, regular C18 column will give you three peaks close to void and the last one at 17 minutes. With Primesep C all four are spread "evenly" with total run time of 10 minutes:

http://www.sielc.com/compound_116.html


regards,

Vlad

[JA]

Hello

To shut down the ion exchange capacity, as you put it, isn't it necessary to go perhaps 1 pH unit below the "switching" pKa? I think this is where we were headed in terms of column bed stability with the earlier questions. If the ligands are neutralised, isn't it fair to say it's a little bit like working with a polar-embedded stationary phase?

Are separations reproducible when we work at the pKa of an analyte, or in this case, the stationary phase?

[SIELC_Tech]

That is exactly what we have. If you are above pKa value of the column your acid is ionized and you are separating compounds based on reverse phase and ion-exchange mechanism. If you are below pKa value then you have only reverse phase interaction and column behaves as RP column with polar embedded group. If you use high ACN (80-90%) and you can operate it in HILIC mode or in ion-exchange mode (depending on pH, concentration, etc.). Here is the poster on universal nature of Primesep (RP, ion-exchange, HILIC and mixed mode):

http://www.sielc.com/pdf/SIELC_Universa ... yPhase.pdf


In terms of reproducibility at pH close to pKa value: it requires more accurate mobile phase preparation as you are very sitting on a very steep slope of pKa curve, but if you are preparing mobile phase accurately every time you should not have any problem. To enhance mixed mode interaction and it might make sense to stay 0.5 units above pKa value of the column.

[Chris Pohl]

SIELC_Tech,

You are greatly oversimplifying the situation when you describe a specific pKa associated with your surface groups. In fact, when you attach a ionizable groups to a surface, unless the surface density is extremely low, the pKa range of ionizable groups associated with the surface will be significantly broader than that of the same molecules in solution. Inductive effects weaken the acidity or basicity of neighboring ionizable groups while statistical effects result in increased acidity or basicity for some of the ionizable groups. So, if the underlying ligand pKa has a value of 1 then the real range of pKa values would be something like 0-5. Furthermore, in order to fully eliminate ionization of 99% of the ionizable groups you will need to operate two pH units lower, i.e. -2, so it doesn't seem very likely that you can operate with the ion exchange mode "shut down"

[JA]

After reading Chris' post, I wondered again about reproducibility on a column being operated in mixed mode. Seeing as column manufacturers are able to produce phases with good lot-to-lot variance of the physical properties, such as ligand density, I guess we could still take from this that when a Primesep phase is operated around it's "switching pKa" it should be reproducible, all other things being equal, from analysis to analysis or perhaps when a new column was required. This must be true, otherwise they wouldn't be marketed, right? I guess this applies to all other ion exchange phases too, not just Primesep..

[Chris Pohl]

JA,

I think you're reading a bit too much into my earlier post. Actually, I think the real situation in terms of a broadened pKa range relative to the pKa of the ligand in essence creates a circumstance not terribly dissimilar from a buffered stationary phase. Thus, unless there's a serious problem with reproducing surface functional density, there are no unusual implications in terms of reproducibility (and, of course, the entire discussion only relates to WAX and WCX ion exchangers, anyway, since SAX and SCX are 100% ionized at any practical pH).

[JA]

Yes, I was digressing a bit, sorry. It was really this trick of switching the ionic capacity of Primesep on and off which made me post. Like the SCX you mentioned in your last sentence, the Primesep 100, 200 and C phases must be, to some degree, ionised at any practical pH..

[SIELC_Tech]

I want to clarify statement about switching off ion-exchange. This switching also depends on the pKa of the amine you are analyzing and how strong it interacts with stationary phase by ion-exchange mechanism.
Hydrophilic amines with multiple charges will interact with Primesep C even at pH-2.5 or 2. If you take peptide with 4-7 amino groups you will have hard time eluting it even pH=2. But if you take benzylamine for example and run it at pH-2 (0.1% TFA) t will come in the void. If you do the same thing at pH 3 you will have more retention and at pH well above 3 retention might be so strong that you might need to increase buffer concentration. The "pure" experiment in this case would be with TEA phosphate (in order to avoid changing two parameters). In our presentations we actually comparing it with the dimmer switch rather then just switch. You can "dim" ion-exchange in specific pH range (2-7 for Primesep C column). You have to look at relative ion-strength in wide pH range an they state that IE is negligent.

[Chris Pohl]

SIELC_Tech,

Perhaps this might be considered a bit of a semantic argument but if you have an analyte retained via an ion exchange mechanism and you've adjusted the electrolyte type and concentration so that the analyte has no retention (as you suggest in your most recent post), this is not the same as "switching off" the retention mechanism (by eliminating the ion exchange sites through adjustment of the pH). The former is dependent not just on pH but also on ionic strength and the nature of the electrolyte where as the latter is solely dependent on pH. It seems like you need to be a bit more clear and avoid such a vague descriptions as "switching modes " when describing retention and selectivity control in a mixed mode system if you want users to be able to develop methods on their own.

[SIELC_Tech]

Chris,

I agree that from the ion-exchange equations our statements might have some flaws. But when we are talking about “switching offâ€