Hi Emmanuel,

I am glad you posted this question! I get asked questions like this all of the time... the first question that needs to be answered for purification is:

What flow rate can your HPLC system handle?

There are systems for analytical, systems for semi-prep, and systems for preparative... which all depends on the size of your pump heads. Once you have decided the flow rate that your system can handle, you will then want to choose a suitable diameter size column. For instance, if your system can only handle 30 ml/min maximum, then you will want to use a 21.2mm diameter prep column.

I did some research on alkaloids (in general), and found a good alkaloid application using a C18 column. Attached is a file with this application and isocratic run conditions. If these alkaloids are structurally similar to the ones you are running, this should work for you. This application uses an ion pairing reagent:

87% 0.003 M Sodium pentane sulfonate (pH 2.0 with phosphoric acid) : 13% acetonitrile

If this application doesn't work, then a 10-90%B generic gradient on a C18 column should give you a good elution profile using 10mM NH4HCO3, pH 10.5 as your A, and Acetonitrile as B.

From these chromatographic results, you can optimize your run depending on the elution.

Does this help?

Paul

Assuming a C18 column, I would use a water/methanol or water/acetonitrile solvent system, both containing 0.1% TFA (trifluoroacetic acid). 1% formic or acetic acid would work too. The exact ratios of your solvents depends on the alakloid(s) you are purifying. A gradient from water to organic solvent should give you a rough idea of the isocratic conditions you might use and also indicate if there are impurities to be washed off the column at the end of the purification.

The acid helps keep the basic alkaloid ionized to improve the peak shape. Note that more aqueous solvent conditions retard peak elution while increased organic solvents tend to reduce elution time for reversed phase columns such as C18.

Agree with that! You should use the water/methanol or water/acetonitrile solvent system if you are using a C18 column for your purification. The solvent ratios which you are going to employ would be determined upon the nature of alkaloids you are going to purify. For the purification, the isocratic elution condition depends on the gradient from the aqueous solvent (water) to organic solvents. The higher the ration of the aqueous solvent, the longer time would be taken for your peak elution and vice versa. Hope this would help! Cheers!

If youare talking about prep HPLC so i think phenyl hexyl column suitable for alkaloids separation using water and methanol combination because most of alkaloids having aromaticity hence phenyl hexyl column give better separation,separate individual peak but you could try with different mobile phase percentage (gradients or isocratic solvents) and flow rate for better separation.good luck

Yes, you can go for analytical RP-HPLC using C18 column and water, methanol and acetonitrile as solvents for the determination of alkaloids.

But if you want to obtain individual compounds as purified, then try preprative HPLC.

Yes, I would like obtained individual compounds purified.

Hi Emmanuel,

I am glad you posted this question! I get asked questions like this all of the time... the first question that needs to be answered for purification is:

What flow rate can your HPLC system handle?

There are systems for analytical, systems for semi-prep, and systems for preparative... which all depends on the size of your pump heads. Once you have decided the flow rate that your system can handle, you will then want to choose a suitable diameter size column. For instance, if your system can only handle 30 ml/min maximum, then you will want to use a 21.2mm diameter prep column.

I did some research on alkaloids (in general), and found a good alkaloid application using a C18 column. Attached is a file with this application and isocratic run conditions. If these alkaloids are structurally similar to the ones you are running, this should work for you. This application uses an ion pairing reagent:

87% 0.003 M Sodium pentane sulfonate (pH 2.0 with phosphoric acid) : 13% acetonitrile

If this application doesn't work, then a 10-90%B generic gradient on a C18 column should give you a good elution profile using 10mM NH4HCO3, pH 10.5 as your A, and Acetonitrile as B.

From these chromatographic results, you can optimize your run depending on the elution.

Does this help?

Paul

Good answer dear Paul.

Dear Emmanuel

Go for RP-HPLC with UV detector using C18 column, 5 m particle size and solvent system as mobile phase of methanol/0.1 M ammonium acetate, pH 5.5 (60:40) with a flow rate of 1.5 L/min and eluent monitoring at 245 nm . You may also try gradient elution of methanol-water, methanol-TEA solution, methanol-ammonium acetate buffer salt solution. Also two phase solvent system composed of MtBE–acetonitrile–water (4:0.5:5, v/v) with 10 mM TEA in the organic stationary phase and 5 mM HCl in the aqueous mobile phase is used to separate alkaloids.

All the best

@ Kiganda-

What have you tried already? What were the results?

I'd still try C18- most of them should work fine. They are likely to elute in more organic solvent. I'd still use the acidic modifier too.

Please note that using ALL-CAPITAL letters is the internet equivalent of shouting.

C18 coloumn with methanol-acetic acid-H2O as mobile phase

I Think C18 column and methanol \ ammonium acetate as mobile phase is used to separate alkoloids.

following