Chromatography Forum

in acclaim trinity p1 which chemical substance is used when we determine t0.
I am using UV detection.

t0 measurements are a "can of worms" because of complications due to size exclusion, ion exclusion, and slight retention. For most purposes, simply looking for the first baseline excursion after injection suffices (assuming that your sample diluent is not identical to the mobile phase). That works because most UV detectors will respond somewhat to refractive index shifts.

If you need something more precise, your best bet would be to contact Dionex tech support and ask them what *they* use.

In my work I prefer the approach that Tom suggests for the same reasons listed below. On the other hand, I believe the following method should be adequate and accurate enough.

Mobile Phase: MeCN/ammonium acetate buffer (anywhere between 5 and 100 mM and between pH 4 to 5.5) v/v 50/50
Probe: Uracil (0.1 mg/mL in H2O)
Detection: UV at 210 nm and 254 nm (or full spectrum if using PDA or DAD)
Flow rate: 0.45 mL/min (can be 0.4 or 0.5 mL/min, too)

This way, you can determine the t0 using either the disturbance in UV spectrum or the retention of uracil. Just be consistent.

Many times I have seen small peaks ahead of tm (tO) due to a pressure shock created by the Rheodyne, so I fully agree that determining tm is like a can of worms. For a long time I used to think using T2O was perfect until I looked at it carefully. If there is low enough H2O in the mobile phase used with NP columns one gets retention. With both NP and RP one can get exclusion at the right pH. Luckily tm needs not to be very accurate for most applications.