Dear Members.
For the quantification of diphenhydramine hydrochloride (pka = 9). I use column C18 with mobile phase Acetonitrile/Water (50/50) (pH around 5_, I got a pic with a tailing. But when i added Triethylamine and some drops of acetic acid to have pH = 6.5 to the mobile phase i got a sharp pic. Could someone explain this situation.
Sincerely yours.
In our laboratory we determine diphenhydramin HCl by HPLC with following conditions:
Column CN (Merck, 25 mm, 5 micro); mobile phase : Acetonitrile/Buffer TEA (40:60, v/v ); detection 254 nm, flow 1 mL/min. Buffer TEA (Water 360 mL +TEA 3 mL, mixed, adjust pH to 6.0 +/- 0.05 by acetic acid. If it is needed you can adjust the mobile phase by ACN/Buffer TEA (20:80, v/v)
https://www.chem.agilent.com/Library/eseminars/Public/Validated%20HPLC%20Methods.pdf
Try this from Agilent
Dear Dr Indrayanto,
I would like to thank you for you response.
Maybe i was not clear, but i would like to know if you can explain to me why the pic is sharp in the mobile phase ( ACN /H2O 50/50, pH = 6.5) and it is not at this mobile phase (ACN/H2O, pH around 5), we know that pka for diphenydramine HCl is 9.
Sincerely yours.
From the data off the Agilent's presentation, the higher the pH of the mobile phase, the long the retention time and may be the sharper of the peak. Looks like it bahaves like reversed phase mechanism. The higher the pH, the less the analyte is in the ionized form. This is true when you try to separate acid analyte by HPLC, you need to suppress ionization by lowering the pH.
Dear Dr Narong,
Thank you for your response
At the level of this pH (5 or 6.5 ) Diphenydramine has a salt form. and at this presentation there is no significant variation in the tailing with the pH.
Dear Dr. Zakaria,
You have sharp peak at pH 6.5 because you have added TEA in you mobile phase; TEA can be used as ion paired reagent . This also has been described in slide 17 of presentation from Agilent (that described by Dr. Narong above)
Both Dipheyldramamine and TEA are the amine compounds. I don't know what would TEA will form the ion-paire with?
Yes, Dr. Narong is correct. The term 'ion paired" is not correct . Actually, I mean TEA can act as competing base, reducing the availability of stationary phase silanols and interaction of the analyte with silanols.
Peak tailing could be caused by interaction of analyte (positive charge) with negative charge of silanols on the surface of the silica stationary phase.
You can also use better stationary phase such as Eclipse plus C18, that have lower silanol activities.
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