difference between polar encapped and polar embededed column

[PHOBIUS]

Can anyone tell me the difference between polar encapped and polar embededed columns?

Are polar embedded columns more resistant to IP agents contamination or which column should have the longest lifetime?

I was using a polar encapped column to a HPIPC aplication with 10 mM DBAA and after cca 200 injections, it went irreversibly contaminated.

One more question: Has anyone tried UPLC column from GRACE? (HT vision or HT vision polar?)

thanks for any suggestion.

[Rob Burgess]

Endcapping is a process to reduce the residual Si-OH silanol groups on the surface of the silica after the initial bonded phase coverage. For instance CH3 groups can be reacted/bonded to these silanols to stop unwanted polar interaction in reveresed phase HPLC. Polar endcapping I guess is the addition of polar groups to these residual silanols to afford better chromatography somehow.

Polar embedded groups on the other hand is where the polar functionality (carbamate / amide) actually forms a part of the bonded ligand attached to the silica.

I hope this makes some sense...

[Uwe Neue]

Many "polar endcapped" columns are not endcapped at all, and the "polar endcapping" is nothing but plenty of residual silanols.

Packings with an embedded polar group also come in several different categories. The packing can be prepared in a single step, or in two steps. If the embedded polar group is an amide, a two-step reaction means that you have plenty of amine on the surface, which creates similar issues as silanols on the surface. Packings prepared with single-step reactions are the better choice. Among the polar embedded groups, the ones that work best are amide groups, carbamate groups and urea groups. I do not have data on the newer sulfonamide packings to give an opinion. Packigns with just some either functions do not show the properties that are typical for EPG packings.

[PHOBIUS]

thanks for replies.

Should these polar embedded columns have a longer lifetime than the encapped ones when using IP agents?

[Uwe Neue]

A well designed surface has a better lifetime than one that is not well designed. A packing with residual amino groups on the surface will slowly bleed this amino group and die. A packing that is not endcapped will slowly loose ligand and die. Both are not primarily a function of using IP reagents.

A well designed EPG column should function as well as a well designed C18 with IP reagents.

[PHOBIUS]

Could you advise me some well designed columns suitable for IP aplications that do not "die" after 200 injections?

I was using the same column than these guys here, but it is too expensive to buy a new coulmn 2-monthly:

Development and validation of an assay for the quantification of 11 nucleotides using LC/LC–electrospray ionization–MS
Klawitter J et al. ANALYTICAL BIOCHEMISTRY 365 (2): 230-239 JUN 15 2007

Simultaneous determination of multiple intracellular metabolites in
glycolysis, pentose phosphate pathway and tricarboxylic acid cycle
by liquid chromatography–mass spectrometry
Luo B et al. Journal of Chromatography A, 1147 (2007) 153–164

[PHOBIUS]

and one more thing: I suggest, that the IP agent (DBAA) does not really contaminate the MS spectrometer, but the HPLC machine. How could I remove this DBAA from the system? Should I rinse it with water or rather with some organics?

[Bryan Evans]

To clean your HPLC system - disconnect the column (replace with zero dead volume fitting) and disconnect the LC from the detector. Then you can flush the system with high % aqueous eluent (e.g. 90:10 water:acetonitrile).

About the column. What particle size are you currently using? Did you notice an increase in pressure after 200 injections? Loss in k'?
More peak tailing?

[PHOBIUS]

thanks for advice, I am using 4 um particle size column,
pressure elevation was not observed but peak tailing - absolutely, especially with diphosphates. The original peak width of diphospate was 0,8 - 0,9 min, now it is about 4 minutes (it is terrible)

[PHOBIUS]

and I did observed loss of retention too.

[Bryan Evans]

Loss in k' and peak broadening in this case is most likely ligand desorption / dissolution of packing material.

What is the pH of your mobile phase? Under neutral mobile phase conditions,
you'll want to make sure your column of choice is very well endcapped (to protect against
dissolution of the silica packing material).

Below are nucleotides analyzed on Unison UK-C18 (3um) and Unison US-C18 (5um):

http://www.imtakt.com/TecInfo/TI084E.pdf
http://www.imtakt.com/TecInfo/TI133E.pdf

[Uwe Neue]

All the information that you gave tells me that the surface of your packing is slowly dissolving, which is not unexpected for a packing with a low coating and many silanols. My suggestion would be to try the Atlantis T3 column for this application, which I know. It is a fully endcapped C18 that can be run in 100% aqueous solutions. Alternatively, you can look at a true single-step EPG column on a pH stable hybrid packing, such as XBridge Shield RP18.

It is also not impossible that part of the problem is the sample prep of samples of biological orgin. Can't teel from the quick glance at the papers, if the sample prep method is good enough.

[Uwe Neue]

All the information that you gave tells me that the surface of your packing is slowly dissolving, which is not unexpected for a packing with a low coating and many silanols. My suggestion would be to try the Atlantis T3 column for this application, which I know. It is a fully endcapped C18 that can be run in 100% aqueous solutions. Alternatively, you can look at a true single-step EPG column on a pH stable hybrid packing, such as XBridge Shield RP18.

It is also not impossible that part of the problem is the sample prep of samples of biological orgin. Can't teel from the quick glance at the papers, if the sample prep method is good enough.

[PHOBIUS]

Biological samples (mainly extracts of leukemia cells) were only a small part of those 200 injections (max10%) and the extraction buffer was the same as the initial mobile phase of the gradient so I think, the samples were not the problem here.

Mainly (90%), I was injecting standards (trying to validate a method by measuring intrabatch and interbatch reproducibiliy, LOD etc.). The pH of the buffer was 7,00. The pH stability of the column (Synergi Hydro C18) is 1,5 - 7,0. The local dealer told me, that using pH about 7 is absolutely safe. well, the reality prove him wrong.

The next time I use rather a Synergi Fusion column, which also can be run under 100% aqueous conditions and has a pH stability 1.5 to 10.
I hope, this Synergi fusion sustain more than the Hydro one.

Thanks for your advices.

To Bryan Evens: What is the pH stability of the Unison UK-C18 (3um) and have you tested how much injections can you make without loss of retention or peak broadening using exactly the same conditions as here:

http://www.imtakt.com/TecInfo/TI133E.pdf

And are these columns more or less expensive than Synergi Hydro/Fusion?

[Bryan Evans]

The column lifetime should be fine under those conditions.
I'd recommend a guart cartridge / holder though - there could be
some impurities from the IP reagents / buffer that can be sticking
to the packing material (especially under isocratic conditions).