I have a polar compound (Benzamidine) and want to retain/analyze it by HPLC using Discovery C18 or C8 column. I tried many eluents (e.g., ACN, MeOH, EtOH, phsphate buffer pH 7, water) with these columns but could not seperate it. Is there any suggestions? Provided that polar compounds are difficult to retain by these conventional columns because they make an electrostatic bond with the stationary phase of the column.
Did you try with HILIC? like 10mM AmmAcetate pH5, water in ACN gradient 0 to 60%. Keep us posted.
Thank you for the responses.
I did not use amm.acetate as a mobile phase but if you believe it will work then I can try.
Actually I have these two types of columns I mentioned beside a C8 amide column. I have many solvents to use beside those ones I mentioned.
Maybe you're using very polar mobile phase, I recommend using a gradient that started with less polar solvent such as ethyl acetate, and gradually you increase polarity to reach acetonitrile, because if you put a polar mobile phase, column fails to retain your analyte of interest, since the affinity of the same with the mobile phase is high. If this does not give return results, you can try a column shift to a more polar, or last change of reverse phase chromatography, and normal phase.
We analyse daily 300 basic compounds with a column X-Bridge Waters and a gradient of H20 + 5 mM Ammonium bicarbonate buffer at pH:9.2 / Methanol in HRMS (Exactive orbitrap technology) to separate in 25 min and all of the retention of the 300 basic compounds elute between 11 to 23 min .We are in neutral form and it is the hydrophobic part of the basic compound which can interact with the C18 column.
Hello I agree with Yves Moulard.
In fact you Benzamidine is a basic compound and in the conditions you mention, you won't retain the molecule on a C18 because of the basic site protonation (you should observe a large peak in your void volume : your molecule is charged so too polar)
Try to use the conditions of Yves but take care to use a specific column that can sustain the basic pH : The discovery C18 won't ! (pH range is 2-8)
Do not use a high pH mobile phase. In the long run it is neither good for your column or your detector. Use the Cogent DH stationary phase from Microsolv. You can do this analysis at low pH, formic acid, and get reproducible results and very fast equilibration times. Not always possible to get these results with a HILIC column
I would use the Cogent Diamond Hydride phase (Microsolv Technology) for this analysis. First you do not need to use a high pH buffer that will be detrimental to your column and your instrument. This can be done using formic acid. Second it is more reliable than HILIC giving reproducible results and faster equilibration.
Not sure if you can switch columns easily but I have had good experiences with the Agilent SB-Aq column which is still reversed phase but has amide functional groups that make it good for bio-molecules (read polar) and can handle higher pH's (such as 7 where your molecule might be neutral and therefore stickier and easier to manage).
Thank you all. Now I can understand that with my current columns I will not be able to analyse my compound, instead I need to have a different type column "Hydride form" so that my compound won't interact with it.
I found this on the internet and it has ion-pair method see on page 4 of the document.
Thank you Narong. I know there are some other solutions with different columns but my question was about available methods using ordinary silanol C18 or C8 columns.
The column suggested in the paper is a reversed-phase column c18.
HPLC Method for benzamidine
quantification - The separation was carried out
isocratically by 10 mM 1-octylsulfonate sodium
salt and 17% acetonitrile (v/v) (pH not adjusted)
by a LiChroCART® 250-4 HPLC-Cartridge with
LiChrospher® 60 RP-select B (5 μm) and a
LiChroCART® 4-4 guard column (Merck KgaA,
Darmstadt, Germany) at a flow rate of 1.0
ml/min.
What was the retention time you obtained in different mobile phases. If its not retained wee, use plain water as mobile phase in order to get highest retention time. U can definitely separate the compound. Msure
I am also facing problem for HPLC analysis of hydroxycitric acid with C18 column. I tried different modifiers including sulphuric acid but column is not able to retain the same analyte.
You are taking the wrong approach for polar compounds. You need to operate in the aqueous normal phase mode using high organic (acetonitrile) in the mobile phase. The best column for this is Microsolv's Cogent Diamond Hydride. On the Microsolv web page you can look at the application page and click on the DH column to find many examples of polar compound analysis
Yes, this is call HILIC, Hydrophilic Interaction Chromatography. The water modifies the stationary phase to attract polar molecules causing retention.
No the column I am referring to is not HILIC. The separation mode is aqueous normal phase that is different from HILIC. There is almost no water layer on an ANP stationary phase. Separation is by an adsorption mechanism that is much more versatile than HILIC. Very reproducible and fast equilibration times because of the absence of the water layer. Also if using MS detection, very low additive concentrations in the mobile phase are needed. Additive effect is the opposite of HILIC: higher retention with lower additive concentration where in HILIC you need more additive to increase retention.
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