HPLC recommendations needed!

[mixmixi]

Hello,

I am running a mixture sample of 3 pharms compounds of paracetamol, ibuprofen and carbamazepine on HPLC-UV. I used a semi-gradient mobile phase as sequence and a windowed wavelength programme (215, 240, 250) for each of these compounds for detection. My mobile phase was a proportion of HPLC grade water, ACN and MeOH with more water percentage and less MeOH. My result was not satisfactory as I had very small peaks for my compounds and 3 big peaks which didn't belong to my compounds. I would like to ask for some suggestion to improve my method for having a better peak in HPLC. (I am new to this system and not experienced, so I apologise if I have any technical mistake in my question)

[DR]

To an extent, you will have to consider the ratios of APIs in your samples. I suspect you may find that your greatest concentration is for the API with the biggest extinction coefficient (paracetamol will produce flat-topped peaks at <mg injection amounts on most detectors). If your carb. is at very low levels, you may need to prepare 2 different dilutions.

If carb. has a local maximum at a higher wavelength than the other components, you may benefit from running at that wavelength, thus attenuating the signal from paracetamol in particular.

Without more detail, we can't address the separation issues.

[mixmixi]

Thank you for your answer. I may add the concentration for all the compounds were 50 micrograms/ml. The HPLC programme wavelength and water:ACN:MeOH proportion was as below:

1- from 4-10 min, with 250 wavelength and 80:20:10 v/v/v
2- from 10-17 min, with 260 wavelength and 50:30:20 v/v/v
3- from 17-22 min, with 215 wavelength and 80:20:10 v/v/v

I ticket the "balance" option in the wavelength setup as well.

If you require any more information, please tell me so I could add. I am looking to hear more please.

[DR]

Steps 1 and 3 (which do not add up to 100) are the same. This is not normal.
Where did you get these conditions?
Expected retention times for each compound?
Column being used?
Injection volume?
Temperature?

[mixmixi]

Dear DR,

You mean I'd better increase the water percentage to 100% in the step 3? The conditions are concluded by reading some papers and especially a paper published by Baranowska (2010). On that paper she worked on 15 different pharms which I have three of them here. Based on that the expected retention time for paracetamol, Ibuprofen and Carbamazepine are to be expected at 8, 14, 20 mins. My column is SunFire C18 Column, 4.6 x 250 mm. The injection volume is 20 micro litre in room temperature (25c).
I'd be appreciated if you may suggest any better mobile phase sequence for the conditions I have here or any recommendations to help my work achieves better result.

Best,

[JGK]

Dear DR,

You mean I'd better increase the water percentage to 100% in the step 3? The conditions are concluded by reading some papers and especially a paper published by Baranowska (2010). On that paper she worked on 15 different pharms which I have three of them here. Based on that the expected retention time for paracetamol, Ibuprofen and Carbamazepine are to be expected at 8, 14, 20 mins. My column is SunFire C18 Column, 4.6 x 250 mm. The injection volume is 20 micro litre in room temperature (25c).
I'd be appreciated if you may suggest any better mobile phase sequence for the conditions I have here or any recommendations to help my work achieves better result.
Best,

No, Look at the ratios in lines 1 and 3

1- from 4-10 min, with 250 wavelength and 80:20:10 v/v/v (total =110 rather than 100 which is more usual)
2- from 10-17 min, with 260 wavelength and 50:30:20 v/v/v
3- from 17-22 min, with 215 wavelength and 80:20:10 v/v/v (total =110 rather than 100 which is more usual)

either it is a typo (70:20:10?) or the Author thoght the presentation would not be as neat as 73:18:9 v/v/v

[mixmixi]

My apologies, sorry! I just realized it after checking. Both 1 and 3 are 80:10:10 v/v/v. That was a typo error as I was in rush typing everything.

[Hollow]

but beside of the 100% error that was, it is still not common to go back to a weaker solvent strength during a gradient run
(unless step 3 is the reconditioning for step1)