Chromatography Forum

Hello

I am totally new in LC-MS, and just started working in this field. My question is the following:

I got a Agilent Zorbax Eclipse XDB-C18 and a Zorbax SB-C18 (both are 0.3 mm X 150 mm X 5 µm). I used them with a Agilent 1100 Series Capillary LC system coupled with a Agilent LC/MSD TOF (ESI). The max flow rate for the Cap pump is 20 µL/min. I am now trying to use this system to study the metabolite extracted from a mouse brain tissue.

I just want to ask what is the appropriate injection volume and flow rate for this kind of column/MS system to obtain a good separation ? I am expecting a lot of metabolite comes out form the tissue. I noticed from the description of the column that the guideline is to inject a volume of 0.05 - 0.1 µL on a 0.3 mm I.D. column and 0.1 - 0.2 µL on a 0.5 mm I.D. column. This injection volume is much less than I usually used (5 µL). I just want to make sure that I am optimizing my column performance. Thanks !

Hello,

Injection volume on your HPLC column is related to the dimensions, in fact to the amount of stationary phase. As you are using small ID columns , so less amount off stat phase , so less amount of sample to be loaded on your column, obviously the injection volume wil be smaller as you indicated.
So use these small(er) injections. If not you could overload your column with could results in less effecient chromatography and separation: peak distortion, overlap,...
Of course by injecting less volume , your amount of sample will be smaller , but using the appropriate detection technique that should not be a to great problem.

the same for flowrate, the smaller the ID the smaller the flow rate (refer to H/U curve)

succes

philippe