Chromatography Forum

Hi I am having a doubt here, hope some of u can help.

I want to perform the HPLC of a very hydrophilic compound (log P = 0.14)
However the compound gets eluted very rapidly.
Can someone suggest how I can increase the retention time (apart from by reducing flow rate or increasing column length)

I tried increasing the proportion of ACN to water thinking that since my solvent has become less polar, the drug will partition out in the mobile phase less rapidly.
However, I have noticed that on increasing the proportion of ACN the drug is eluting even faster,
Can someone explain this to me please.
Thankyou very much.

thankyou for your reply.
Although i dont think i can change the column, since i am a student with limited resources.
I am using a Luna C18 column as of now

What you saw was not suprising. What you are doing is "reversed-phase" chromatography. That means that the stationary phase (column) is less polar, while the mobile phase (solvent) is more polar. In that situation, the more polar the solvent (i.e., the more water it contains), the *weaker* it is as a mobile phase. This often seems counterintuitive, since we usually think of water as a strong solvent, but that's why it's called "reversed-phase chromatography".

So, by adding more ACN, you were going the wrong way and making your solvent stronger when you wanted it weaker.
Try *decreasing* the ACN content of your solvent. You can go down to about 3-5% without any problems. If you still don't get retention, your options will depend on the detailed chemistry of your compound, per the suggestions made by DawnRazor.

Reducing the flow rate won't help, because it increases the retention time of all compounds by the same proportion (you really don't gain much, you just take longer to get the same result). Making the column longer can help if you have at least some retention, but it's a very inefficient way to do it (the quality of a separation is a function of the square root of column length).

With Luna C18 you can go down to a very small percentage of organic content without dewetting; try running in 2% methanol, 98% water (and ideally a buffer at a pH that won't dissolve the column but will keep your molecule as neutral as possible). If this doesn't give enough retention, DawnRazor's suggestion of an ion pair reagent is good. But do remember that your column may never be exactly the same again! It can be very hard to remove an ion pair reagent entirely from a column on which it has been used.

An additional note on the idea of an ion-pair reagent: that will only work if your compound is ionic. If it's hydrophilic but neutral (a carbohydrate or polyol, for example) then adding an ion-pair reagent may actually decrease retention slightly.