HPLC of Phospholipids-unable to get the right retention time

[ssankella]

my coulmns are novapak C18 4 um, 4.6x250mm
Sunfire silica column, 5 um 4.6 x 150mm

[ssankella]

ya C 18 are reverse phase but I have seen few papers which used C18 for normal phase separation.
I also tried a Silica column which is made for normal phase

[jwkim6]

I also do phospholipids using ELSD. Are you familliar with ELSD? One critical aspect of this detector is that the drain and exhaust tubes need to be positioned properly. Both tubes should not have bends and should point down so that liquids and gases can exit the detector. Drain tube should goto liquid waste but no in the liquid.

[jwkim6]

I am using the Waters 1520 pump with 2420 ELSD detector,
gradient elution with Hexane:IPA:water
Buffer A 40:52:4
Buffer B 40:58:4
Flow rate:1ml/min

Your mobile phase compositions dont add up. Your %IPA seems high in A. This mobile phase might elute all your stds right away.

I use this, approximately
A- hexane:IPA 80:20 (w/ 1% acetic acid/TEA)
B- IPA:water 85:15 (w/ 1% acetic acid/TEA)

[ssankella]

thx for the input jw
I have never worked on elsd, but read a lot about before I got started.

[ssankella]

my mobile phase is
(Buffer A 40:52:8 (V/V/V) and Buffer B 40:58:2 (V/V/V)

[phospholipi]

What kind of phospholipids do you want to analyze? Why don't use DFG Standard Methods Section F - Minor Components of Lipids F-I 6a (07) HPLC of Phospholipids (Determination with Light Scattering Detector)?

[ssankella]

I tried an isocratic elution with Buffer B which is Hexane:IPA:H20 40:58:2.
So finally I can see the peaks of the phospholipid standards. But I still have a problem with the retention time, the stds are eluting 4-5 min earlier than the std retention time.
Any suggestions will be greatly appreicated.

Regards
Sankella

[phospholipi]

What standards are you using?

PC, LPC, PI, PE, PA ? Something else?

[ssankella]

I am using PA, LPA, PC, PI, PE and PS.
I am able to elute all the stds but some how none of them is at the right retention time

[phospholipi]

Another time I want to recommend to use:

DFG Standard Methods Section F - Minor Components of Lipids F-I 6a (07) HPLC of Phospholipids (Determination with Light Scattering Detector)

If you are not able to get the DFG-Methods, I can send it to you, if you provide me your email-adress.

It's gradient hplc with diol phases.
Buffer A: n-Hexan / 2-Propanol / ethanoic acid / triethylamine
81,42 / 17,00 / 1,50 / 0,08

Buffer B: 2-Propanol / water / ethanoic acid / triethylamin
84,42 / 14,00 / 1,50 / 0,08

The mixtures should be derived via weigth in from their density.
All running conditions are named in this DFG Method (DFG stands for "Deutsche Gesellschaft für Fettwissenschaft")