Chromatography Forum

I am using HPLC Ulitemate 3000 UV detector
i faced issue at wavelength 210 nm after i use formic acid as a mobile phase
- when i use the acetonitrilel/ H2O or methanol/buffer as mobile phase the problem disappear.
- when i use only H2O as mobile phase the problem appear again .

kindly notice i did the following :

1- the lens of flowcell has been replaced , update lamp intensity and calibrated grating motor .

2- the problem appear only when i use H2O at wavelength of 210 , so i don't think the water its the source of problem + we use same source of water in other devices and everything OK

The UV cutoff for formic acid is typically given as 210 nm so you are working on the "ragged edge" of suitability. A lower concentration or a fresh batch of formic acid might help.

tom jupille wrote:
The UV cutoff for formic acid is typically given as 210 nm so you are working on the "ragged edge" of suitability. A lower concentration or a fresh batch of formic acid might help.

When i wash the HPLC with acetonitrile the problem solved , but if i use H2O now at 210 nm its appear again , if i wash it with lower concentration formic acid it will be solved ?

You really didn't give nough detail to allow us to provide a specific answer.

When you said "buffer", *what* buffer? and at what concentration?
What concentration of formic acid are you using?

Search on-line and find the UV absorbance spectrum of formic acid. What you will find is that the absorbance increases steeply as you decrease the wavelength in the region below about 215 nm. The UV cutoff is sort of arbitrary; it is typically given as the wavelength below which the absorbance is > 1. That's why I said you were on the "ragged edge"; it doesn't take much additional junk in the formi acid to increase that absorbance. If you have a high concentration of formic acid, the problem will be worse.

If you are seeing a high baseline with pure water, try running a UV spectrum of the water in the mobile phase reservoir and also of the effluent from the system. If the mobile phase shows increasing absorbance around 210 and below, it's probably contaminated (possibly with formic acid previously in the jug!). If the mobile phase spectrum looks clean but you see absorbance in the effluent, then you are flushing junk out of your system.

tom jupille wrote:
You really didn't give nough detail to allow us to provide a specific answer.

When you said "buffer", *what* buffer? and at what concentration?
What concentration of formic acid are you using?

Search on-line and find the UV absorbance spectrum of formic acid. What you will find is that the absorbance increases steeply as you decrease the wavelength in the region below about 215 nm. The UV cutoff is sort of arbitrary; it is typically given as the wavelength below which the absorbance is > 1. That's why I said you were on the "ragged edge"; it doesn't take much additional junk in the formi acid to increase that absorbance. If you have a high concentration of formic acid, the problem will be worse.

If you are seeing a high baseline with pure water, try running a UV spectrum of the water in the mobile phase reservoir and also of the effluent from the system. If the mobile phase shows increasing absorbance around 210 and below, it's probably contaminated (possibly with formic acid previously in the jug!). If the mobile phase spectrum looks clean but you see absorbance in the effluent, then you are flushing junk out of your system.

thank you for help , I am sorry because of lack of information , i tried to do what u advised about pure water and its still highly absorbance at 210 nm ( invalid baseline data) so its contaminated cause of previous formic acid , HOW I CAN SOLVE THIS PROBLEM OR CLEAN HPLC ?
note : when i used acetonitrile the problem solved at 210 nm
kindly help me .