Retention of polar compound

[HW Mueller]

mohan, could it be that a diol column is polar enough to do HILIC?

This pi-pi stuff is way overrated. Aromatics have been retained on practically anything for a 100? years. If there are no pi bonds in the stationary phase it is totally inproper to talk of pi-pi, maybe pi-sigma or pi-n, but not pi-pi.

To bad there is no counterpart in chromatography to Org. Syn. In a case like that above it would be instructive to have several independent people repeat experiments to see whether a k´= 0.5 was realistic or not.

[Bryan Evans]

It's possible for ODS phases to have pi-dipole interaction between
solute and silanols. That is one of the advantage of Cadenza CL-C18:

Pi-dipole interaction: http://www.imtakt.com/TecInfo/TI260E.pdf
Structural isomers: http://www.imtakt.com/TecInfo/TI265E.pdf
Positional isomers: http://www.imtakt.com/TecInfo/TI266E.pdf

[HW Mueller]

The amide group on a planar molecule, with an ortho ether in the same plane to boot, the inductive effect and mesomery of the benzene ring should be better candidates to explain the shifts in Bryan´s first link. But this is all conjecture.

[mohan_2008]

Hi Mueller,

-NH2 columns are pretty polar than diol columns. Eventhough, I did not have a chance to run RP on diol columns, I used -NH2 columns for regular RP separations and they work excellent.

It is still hard to believe that diols work as substitutes for Hilic columns.

[Vlad Orlovsky]

Mohan,

Just do a search on Google...and may be you will believe that diol is a HILIC column (170K references).
Dionex recently introduced HILIC/RP mixed-mode column which has hydrophobic chain and terminal diols, other regular diol columns are used for HILIC as a primary application.

[Uwe Neue]

Another important application of diols is the size-exclusion chromatography of proteins, which requires minimal interactions.

Back to the pi-pi stuff: those interested may want to read the publications by Sander and Wise who covered in detail the selectivity effects on C18s as a function of ligand density for aromatic hydrocarbons. They show pictures how one can fit a pie into a slot.

[Nest Group]

Mohan -

Since your thiourea isn't ionizable, you don't need buffer in your HILIC separation for it. But you could vary the amount of the buffer or the pH, to increase the polarity of your drug substance to move it away from the thiourea in your separation.

The choice of the column chemistry: silica, Diol, NH2, SO3, etc. influences the RT by electrostatic or hydrogen donor effects, in addition to the induced partitioning into the bound water layer from the ACN. So look at your drug and choose a surface that will strongly interact with it. We already know that the thiourea isn't particularly attracted to the hydroxyls, and it should be somewhat repulsed by an NH2 surface. So a SO3 surface will increase its retention. The question for you is what is going to happen relative to the functional groups of your drug?

Alternatively, you may be eluting your analyte because of the composition of your sample. You should reduce the amount of salt/buffer in your sample to 5-20mM and/or reduce the amount of water in the sample to 10% (in this case) to test this.

[XL]

Hi Gandhi, I think you should stay with HILIC approach. The column I would use first is the one you are using. If 90% ACN is not weak enough, try 95% as already suggested. This way you will get better retention. However, the problem here has more to do with selectivity than retention since the drug substrance interferes. Now, we need to figure out a way to place the drug substance somewhere else. Therefore, more information on the nature of drug substance is critical to solve the puzzle. Can you feed us the following infomation?
1. Is the drug neutral, basic, or acidic (ionizable or not)?
2. Is the drug hydrophobic or hydrophilic?
3. I assume the drug has UV response, right?

Even though you may not be allowed to disclose the drug itself, it would be very helpful if you can provide a model drug (say one of the over the counter drugs) that resembles your drug. Armed with this information, we can help you better.

[unmgvar]

Hi Gandhi,

i agree with XL,
stay in HILIC mode for now. we have a similar case where we decided to a Zic-HILIC column.

regarding your peak shape, in what do you dissolve your sample?

[kaganm]

Santosh,
I've just run a series of experiments (took me about 40 min) with thiourea on a Luna CN 2 x 150 mm column packed with 5 mkm particles using normal-phase mode of operation. I used 4-min linear gradients at 1 ml/min flow rate of the following two solvents: A - mixture of heptane and CH2Cl2 9:1 with 0.1% n-propylamine (NPA), and B - mix of CH2Cl2 and MeOH 8:2 with 0.1% NPA. The gradients were as follows: 10-50, 10-90, 20-90, 30-90, 40-90 and 50-90%, retention times obtained for thiourea were 3.4, 2.6, 2, 1.5, 1.1 and 0.9 min, with retention coefficients (K) 7,5, 5.5, 4.25, 2.75, 1.75 and 1.5, respectively. Under these conditions thiourea produces a nice sharp and symmetric peak that is easy to quantify and you have total control over whether you want to keep it on the column or elute it early. I suspect your drug substance will elute earlier than the thiourea. You can dissolve your sample in MeOH or CH2Cl2-MeOH or DMSO for the injection.

You need presence of a basic additive: without it or in the presence of acidic additive peak shape becomes broad and non-symmetrical, most likely, because of the shift towards larger percentage of tautomeric isothiourea. Most likely, you can also use any other CN column and of different size, just remember to keep gradient volume around ten column volumes. I think even your DIOL column will work, as they are usually slightly more retentive than cyano columns.

I'll be happy to send you a pdf file with all chromatographic traces obtained, my email: kaganm@wyeth.com

Michael Kagan,
Wyeth Research, Princeton, NJ

[Kazimierz]

I need method HPLC for hydrochlorothiazide in plasma or urine samples.
I have used Primesep-100, 150x3.2mm on Prostar UV/VIS detector 223 nm and mobile phase H2O/TFA 0.5% in MeCN (93:7). Retention time is, however, only 4.5 min at flow=500 ul/min. Conclusion: 250 mm length column of Primesep-100 could be accepted for HCTZ HPLC in biological material as well as extraction sample is also need for this column.


In HILIC using Rx-SIL 250x4.5 5um, I not able to obtain good retention for hydrochlorothiazide. Also similar with Altima HP HILIC column.

============================================
[i]SIELC_Tech wrote:
Maristela,

Based on the structures of your compounds you should not have any problems retaining losartan and valsratan. I expect the retention to be at least 10 min on 150 mm column at 50/50 ACN-water due to good hydrophobicity (couple benzene rings, alkyl chain, etc.).
Chlortalidone and hydrochlorthiazide a less hydrophobic or rather hydrophilic and you need a gradient to have reasonable retention for all four compounds. Going from 10% ACN to 70% might help you.

Hydrochlorothiazide has aniline fragment in and you can use weak basic properties of it to retein it by combination of ion-exchange and RP properties. For this purpose you can use Primesep 100 column.

here is application for some chlorsulphonamide structures similar to chlortalidone and hydrochlorthiazide

http://www.sielc.com/compound_084.html

I would advice to use a guard column to prolong the life of your column.

Contact us if you have questions,

regards,

Vlad
===========================================[/i]

[Vlad Orlovsky]

You are using way to much TFA, reduce TFA to 0.2 or 0.1. This should increase retention at least 2 folds. Switching to a weaker acid (phosphoric) will increase your retention too. Less ions in the mobile phase longer retention for basic analytes on Primesep 100 column.

Vlad

[seamoro]

hey man,
Check out Quinine Bisulphate or Quinine sulphate rs method in the BP, the method works for Thouriea. Thouriea is used in the system suit of the method