HPLC separation of Phosphatidyl inositol Phosphates

[ssankella]

Hello everyone
I am trying to separate Phosphatidylinositol (PI) and its phosphates using a HPLC-ELSD detector.

Method:
Mobile Phase A:chloroform:methanol:ammonium hydroxide (80:19.5:0.5)
Mobile Phase B:chloroform:methanol:water:ammonium hydroxide (60:34:5:0.5)

Gradient:
0-14 min: 100% A to 100% B linear gradient
14-25 min: hold 100% B
25-30 min: 100% B to 100% A linear gradient
30-45 min: hold 100% A
Flow Rate:1ml/minute

So far I have been able to detect PI, but none of the phosphates are being detected.
Can someone please suggest a method which would separate all the PI isomers.

Thanks
Sankella

[neelams]

Hello All,
I would like to use Hplc for Phosphatidyl inositol Phosphate separation/ analysis. Pls let me know if u have tried this before . Does the analysis require (ELSD)detector. We have a multiple wavelength detector??
pls suggest

[ssankella]

Neelam can you start a new topic instead posting it here.

[Busear]

Hello everyone
I am trying to separate Phosphatidylinositol (PI) and its phosphates using a HPLC-ELSD detector.

Method:
Mobile Phase A:chloroform:methanol:ammonium hydroxide (80:19.5:0.5)
Mobile Phase B:chloroform:methanol:water:ammonium hydroxide (60:34:5:0.5)

Gradient:
0-14 min: 100% A to 100% B linear gradient
14-25 min: hold 100% B
25-30 min: 100% B to 100% A linear gradient
30-45 min: hold 100% A
Flow Rate:1ml/minute

So far I have been able to detect PI, but none of the phosphates are being detected.
Can someone please suggest a method which would separate all the PI isomers.

Thanks
Sankella

Maybe all compounds have the same retention time,so you cannot see other esters

[Andy Alpert]

Looks like you're using normal phase chromatography for this. Try HILIC. It should be more sensitive to the position of the phosphates as well as their number, and you have more options concerning eluting the more strongly retained PI phosphates.