Unknown PEAK in HPLC EC

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

2 posts Page 1 of 1
I have been trying to run HPLC with electrochemical detection for serotonin in serum but have been getting an unknown peak that interferes with serotonin. I have been unable to get rid of this peak and still can't figure out where it's coming from. Details about the protocol and steps are listed below. Any help in getting rid of the unknown peak will be much appreciated!

Electrochemical detector Chromsystems CLC 100
Autosampler Chromsystems CLC 220
Agilent C8 column (100x2.1)

Mobile phase is 150 mM NaH2PO4, 4.8 mM Citric acid, 3mM SDS, 50 uM EDTA, 11 % MeOH, 17% ACN, pH 5.6 with HCL
Electrode set at 550 mV
Flow rate is 0.4 mL/min

Sample preparation:
100 ul Serum + 150 ul ACN, 0.01M HCL
15 min on ice
Centrifuge 13200 rpm, 10 min

The unknown peak occur before peak of serotonin in 0.6 min. (peak of serotonin 4.2 min, unknown peak 3.6 min). Height the unknown peak the same of serotonin peak.

I have taken serum without serotonin. I have got the unknown peak again.
I changed amount of ACN (1/4).

What am I doing incorrect? Maybe I must take another precepitation buffer...?
I don't see the problem here, it seems feasible that other compounds in your sample (which is blood, urine,..?) give a response in your detector. Given that the peaks are 0.6min apart and you perform UPLC, they shouldn't interfere unless the peaks are rather wide.

Anyway: does the peak occur in a (water) procedure blank?
2 posts Page 1 of 1

Who is online

In total there are 17 users online :: 0 registered, 0 hidden and 17 guests (based on users active over the past 5 minutes)
Most users ever online was 599 on Tue Sep 18, 2018 9:27 am

Users browsing this forum: No registered users and 17 guests

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry