HPLC of Phospholipids-unable to get the right retention time

[ssankella]

Hi everyone

I am trying to reproduce HPLC results for phospholipids (PL) which was performed 2 yrs back in my current lab. All the parameters were standardized by my colleague.

When I tried to run the PL standards, whichever std I ran, the retention time (RT) is 3 min.
I have cleaned and regenerated the column, equilibrated it with the gradient solvents, checked the flow rate, degassed the mobile phase but nothing helped.
Plz suggest me wat cud be the prob for this drift in RT.

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
nebulizer temp 30C, tube temp 75C.

Thanks
sankella

[ssankella]

My phospholipids are in Chlorofom. When I injected pure chloroform on to HPLC, I observed the same 3 min peak. So does this mean my sample are degraded or do I still have a problem with my HPLC?

[RAD]

I would wonder if a nebulizer temp of 30C would be sufficient to completely evaporate your chloroform injection solvent and what you are actually seeing is a solvent front of chloroform at 3min rather than your phospholipid stds.
What concentration are your standards?
Are they in high enough concentration that you are certain you would see them with the ELSD?

[ssankella]

RAD thx for the reply.
I am following the same parameters used by my colleagues which were standardized.
She had used 10-550 ug of the samples, I tried with 100, 200 & 250 but none has worked.

[JGK]

What is the gas flow rate of the ELSD?

[ssankella]

N2 gas pressure 30 psi
nebulizer temp 30-35C
drift tube temp 75-80C
flow rate 1ml/min
Sample vol 100-200ul

I have increased the temps by 5C but I still see the CHCL3 peak at 3min.

[tom jupille]

What size column? If you have a 250 x 4.6 mm column at a flow rate of 1 mL/min, the dead time will be around 2.5 minutes, so it could be that your compounds simply are unretained (a dead column perhaps?).

[ssankella]

I have tried 2 columns,
5um, 4.6x150mm and 4um, 4.6x250mm
Both give me the same 3 min RT

[bisnettrj2]

How about dead standards? Are you using the same standards from the last time this analysis was run? (just throwing it out there... )

[ssankella]

no I have tried different stds, made fresh working stocks too.

[tom jupille]

Your peaks are essentially unretained. Go back and re-test the column using the manufacturer's test procedure.

[bisnettrj2]

My phospholipids are in Chlorofom. When I injected pure chloroform on to HPLC, I observed the same 3 min peak. So does this mean my sample are degraded or do I still have a problem with my HPLC?

Tom - shouldn't he be seeing a bigger peak with his standards than with a solvent blank, if everything is unretained? I'm not very familiar with ELSD, but I'm wondering if this may point to a problem with the detector?

[ssankella]

I have tried running my standards on another HPLC which is connected to a UV detector. I tried with my columns as well as other C 18 columns from another lab.
I still see the 3 min peak and none of my samples were eluted at the expected RT which is around 10-15 min.
From this I could understand that nothing is wrong with my columns & instrument.
I also tried an isocratic run with the buffers instead of gradient.
But nothing seems to work
Plz help me out guys

[bisnettrj2]

On your UV-detector equipped system, you can do the following (a trick I learned here): Remove the column from the system, and connect the detector inlet to the autosampler or column compartment outlet. Inject a standard and record the area count. That should represent the total area count in your standards. If you get nothing, or an area count equivalent to your chloroform peak, you're injecting something that doesn't have UV-absorbance at the wavelength you're monitoring - e.g. dead standards. If you get a significant area count, greater than your chloroform peak, you're not eluting your compounds from your column using the mobile phase that your colleague developed. I'm tending toward non-elution at this point.

[Alex Buske]

Hi,

What is your column Phase? Your solvents (actually no buffer) look like normal phase, C18 columns are reversed phase.

Alex