Two questions about HPLC of neurotransmitter (NE/5-HT/DA)

[hachoo]

I'm a beginner in HPLC technique. Please forgive my questions if they seem too simple... I'm wondering if anyone here has experienced similar problems before.

1. We collected all of our samples through microdialysis probe implanted in the rat. Will the KCl in aCSF (artificial cerebrospinal fluid) cause the huge dip, when the samples are going through the column and cell? It seems the other components of aCSF doesn't cause any peaks or dips at all.

2. How to control the time when peaks of individual chemical appear, given a certain column? It would be ideal if the NE peak could appear later than 4th minute, so that it won't be influenced by that strange dip caused by aCSF.

Thank you!

[tom jupille]

They are simple (but, hey, we all had to start somewhere!)

Will the KCl in aCSF (artificial cerebrospinal fluid) cause the huge dip, when the samples are going through the column and cell? It seems the other components of aCSF doesn't cause any peaks or dips at all.

More details about your separation would be useful here, but assuming you're using a UV detector, it's not unusual to see "system peaks" (which can be either positive or negative) when you inject a large excess of something that doesn't absorb UV. UV detectors also respond somewhat to refractive index shifts; I suspect that's what you're seeing.

2. How to control the time when peaks of individual chemical appear, given a certain column? It would be ideal if the NE peak could appear later than 4th minute, so that it won't be influenced by that strange dip caused by aCSF.

Again, "the devil is in the details". I'll assume you're doing "reversed-phase" chromatography, in which case you can increase retention by decreasing the concentration of organic solvent in the mobile phase. As a rule of thumb, dropping the organic solvent percentage by 10% approximately triples retention for small molecules.

There are a lot of good on-line tutorials on basic HPLC. Click on the FAQ link near the upper left of the screen and drill down to "Where to Obtain Additional Information" for some suggestions.

[Mark Tracy]

Typically, these neurotransmitters are analyzed with ion-pairing conditions with octansesulfonate or similar. A large slug of KCl can perturb the equilibrium of the column, and that sends a wave of disequilibrium that appears as waves and dips in the detector. The KCl itself will elute in the void.