Elution profile

[Chandrima]

Hello Everyone,
Before posting my questions, i must acknowledge that i am new in working with HPLCs and do not have much training. My work mostly involves visualizing (Chromatographs) metabolism of radio-labelled (C14) herbicides and their metabolites in HPLC. I have been given this elution profile "The elution profile was as follows: 0 to 2 min, 0 to 20% (of eluent B) linear gradient; 2 to 10 min, 20 to 40% linear gradient; 12 to 17 min, 40 to 70% linear gradient; 17 to 19 min, 70 to 90% linear gradient (19 min total) followed by; 19 to 22 min, 90 to 50% linear gradient; 22 to 25 min, 20% isocratic hold to re-equilibrate the column for the next sample injection (25 min total)." .
Previously, our lab was using reverse-phase High-performance Liquid Chromatography (HPLC) (System Gold, Beckman Coulter, Pasadena, CA, USA) but recently we bought Agilent Infinity 1260. The HPLC is connected to Berthhold radio-detector as we are working only to visualize compounds with C14 present in them. The eluents i am using for the separation are : eluent A (water with 0.1% TFA) and eluent B (acetonitrile with 0.1% TFA).
I have few questions regrading this :
1) What should be the A:B ratio between 10-12 minutes?
2) I am not sure how much pressure i should be using for the separation? It goes till 600 bars in the new machine.

I appreciate any help regrading this. Also, if you need any more information. Feel free to ask.

[tom jupille]

1. 40%
2. Pressure is not something you set; it is a dependent variable. If you use the same column / mobile phase / flow rate / temperature on both systems, the pressure should be approximately the same.

[lmh]

… but with the proviso that if you take a method that's running at fairly high flow-rates, like 1mL/min plus, from an old-fashioned system that was designed for that sort of flow rate, and stick it on a modern UHPLC that's been optimized for very short runs (1260's not a bad instrument), then you may get an increase in pressure because it may have been set up with much narrower tubing than the old system. It's simple to test this: just replace your column with a union, run the flow-rate you're using, and see what the pressure is. This is the background system pressure created just by the tubing of the HPLC, and if it's already rather high, then that explains why your method is running at a higher pressure (if it is!)

[James_Ball]

Difficult to trouble shoot the problem without knowing a few more pieces of information.

Column dimensions, length, diameter and packing size in microns plus the flow rate of you mobile phase in ml/min.

Depending on the column and flow rate 600 bar could be either high or low versus what it should be.