Peak separation problem in HPLC

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Su Mon Thwe
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Peak separation problem in HPLC

I'm trying to develop HPLC method for blood cyclosporine assay.
But I have difficulty in peak separation just after injecting 50uL of cyclosporine A standard dissolved in mobile solvent.
HPLC conditions are as below
Shimazu rp-HPLC with UV detector
Nova-Pak phenyl column (3.9mm x 150mm, 4um particle size)
Mobile - Acetonitrile: 0.04M Potassium dihydrogen phosphate (65:35) , pH - 2.5, & also tested with 3.9 and 5.0
Flow rate -1ml/min
Column temperature - 75'C and also with 65'C
Here's the photos of peaks -
(Black line- mobile alone, green -1ug/ml, brown-2.5, blue-5 & pink-10)
pH-5, Temperature-75
Cyclosporine retention at 4minutes.
The problem is that there are 2mobile peaks at 3.8 & 4minutes & these 2peaks interfere with cyclosporine peak.
Please kindly give your suggestions. Thanks.


Right now I am unable to see the chromatogram, but I want to know the sample prepareation, Either thhe sample is dissolve in the initial composition of mobile phase?????? II nd thing is that what is wavelength u r using in the analysis. Use higher wavelenght if ur sample hase suffivient UV absorbance