UPLC Contamination Issue
Posted: Fri Mar 22, 2013 2:04 am
Im new to the forum so hello all!
My lab have purchased a UPLC system. Following my training course i have been assigned a method redevelopment on uplc.
My problem is this:
Started a method development for an analyte. I quickly developed an isocratic method which is fine however lacking the sensitivity for my target LOQ (0.2ppb!) i decided to explore gradient elution however my attempt at a scouting gradient has failed. The gradient was eluting large interferring peaks over the entire course of the gradient. The highest peak fluorescing at 300EU! So investigating this i have ran the gradient without an injectionand obsereved the output - same profile. Keeping in mind this is a brand new system and column! I therefore suspect one of the two mobile phases (ACN and 0.1% formic acid). I prepared fresh mobile phases, flushed the system and column in MeOh. Still no different. I subsituted the formic acid for TFA and the contamination lowered significantly and continued to decrease with each successive column condition. The interference is swallowing my peak.
Is this a normal occurance using fluoresence detection and gradient? All reagents are hplc grade and the two modifiers are above 99% pure. The water is from a milli-q system and im starting to think its the water that is contaminated? I have asked for some water to be ordered but it wasnt met with approval.
Hope someone can help!
Conor
My lab have purchased a UPLC system. Following my training course i have been assigned a method redevelopment on uplc.
My problem is this:
Started a method development for an analyte. I quickly developed an isocratic method which is fine however lacking the sensitivity for my target LOQ (0.2ppb!) i decided to explore gradient elution however my attempt at a scouting gradient has failed. The gradient was eluting large interferring peaks over the entire course of the gradient. The highest peak fluorescing at 300EU! So investigating this i have ran the gradient without an injectionand obsereved the output - same profile. Keeping in mind this is a brand new system and column! I therefore suspect one of the two mobile phases (ACN and 0.1% formic acid). I prepared fresh mobile phases, flushed the system and column in MeOh. Still no different. I subsituted the formic acid for TFA and the contamination lowered significantly and continued to decrease with each successive column condition. The interference is swallowing my peak.
Is this a normal occurance using fluoresence detection and gradient? All reagents are hplc grade and the two modifiers are above 99% pure. The water is from a milli-q system and im starting to think its the water that is contaminated? I have asked for some water to be ordered but it wasnt met with approval.
Hope someone can help!
Conor