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- Posts: 15
- Joined: Mon Apr 11, 2022 8:09 am
Instrument: Shimadzu Prominence-i LC-2030C Plus (UV detector)
Other detectors: Shimadzu RID-20A
Column: Phenomenex Kinetex 2.6 µm C18 100Å, LC Column 150 x 4.6 mm, Core-shell silica, R Phase (00F-4252-E0)
Guard: Phenomenex SecurityGuard UHPLC C18 4.6mm AJ0-8768 with AJ0-0000 Holder.
Mobile Phase: H20:MeOH:Formic acid (80:20:0.16) Analytical grade chemicals and HPLC grade filtered H20.
UV Signal: 265 nm
Oven Temp: 45 C
RID Temp: 45 C
Flow Rate: Started at 0.5 mL/min and slowly increased to 1.0 mL/min over 24 hours. Trying to wash off/out the column, UV cell, and everything.
Pressure: 25.6 MPa at 1.0 mL/min
I'm an chromatography novice. I did it 25 years ago with glass plates, silver stain, sampling with capillary tubes from packed silca gel column samples only.
Before I installed the column, I washed the system with HPLC filtered water for about 32 hours. Then I washed with the H20:MeOH:FA for about a day. I installed the column with the guard end first and let the column purge before I connected it inline. Attached the column in the oven. And slowly increased the flow rate of the mobile phase as described above.
I finally have everything looking good in the LabSolutions v5.97 software. During the mobile phase/washing the UV signal and RID signal were very jumpy. The HPLC has not been used in months or possibly a year or so. I let the mobile phase just run at 1.0 mL/min and 25.6 MPa. This morning the noise levels of the baseline are very low, but both signals (UV and RID) have been dropping at about -0.1 every 15 minutes. The flow mode is isocratic.
I would appreciate any thoughts. The temperatures in the log seem to be steady. The mobile phase seems to be mixed well.