Chromatography Forum

Hi,
I am using following mobile phase (MP) with gradient for RP-HPLC using ELSD.
Column= C18, 150mm x 4.6 mm, 5um,100 A
A= Methanol:Water:acetic acid (75:25:0.4)
B= Acetonitrile:Methanol:THF:Acetic acid (50:37:13:0.4)

0-3 min= 90%A, 10% B
3-5 min= 90 to 0 % A
5-13 min= 100 % B
13-15 min= 100 to 10% B
15-20 min= 90%A, 10% B
I have found that after 10 mins I can see a very broad (lasting for 2-3 mins) M shaped peak. The peak also appears when I run my samples at the same time with same intensity. What could be the possible reason(s) for this broad peak. The MP phase contains volatile solvents and temp. of drift tube (95oC) and nebulization (54oC) are high enough to evaporate the MP completely.

Which THF (Supplier and Cat-No) do you use?

THF from Duksan Reagents, 109-99-9

Hi,
You failed to state your samples. What did you inject?
Why did you use a steep gradient: 3-5 min= 90 to 0 % A.
The M shape peak may be just a split peak due to poor connections at the fittings.
Try: reconnect the tubings; increase the temp of the evap tube to > 90C to achieve better baseline.

BTW, are you trying to adapt an existing method to your new product matrix? Or, are you using an established methods (validated) and encounter problems?

Alfred

Hi, have you solved you problem yet? Can you tell me the reason, because I seemed to have met the similar problem.

Hi, @Alfred88
Sorry for late reply. I got busy in some other experiments stuff.
I was trying to separate a lipid with different substitutions. My aim was to determine the degree of modification of the lipid.
The connections of the system seemed OK. I used this steep gradient as mentioned in literature for the separation of lipid mixture . I used temp up-to 95 degree.

Hi, @Caroline91
Nope. I couldn't solve the problem.
Good luck...