This Application Note describes the separation of a trypsin-digested protein sample, using the Agilent 1290 Infinity LC System with Agilent ZORBAX Eclipse Plus C18, 150 mm × 2.1 mm, 1.8 µm particles column. Peak capacity variation with change in flow rate and time for various gradients were evaluated. Increase in flow rate from 0.2mL/min to 1.2mL/min effectively improved the peak capacity value to double and increase in gradient time from 5minutes to 50 minutes enhanced a 3.4 fold improve- ment in peak capacity value. The unique design of the Agilent 1290 Infinity LC System tolerates 1200 bar column back pressure, allowing higher flow rates, and reducing the run time.
This document describes the separation of a trypsin-digested protein sample using the Agilent 1290 Infinity LC System with Agilent ZORBAX Eclipse Plus C18 columns. The study evaluates how peak capacity varies with changes in flow rate and gradient time.
The results indicate that increasing the flow rate from 0.2 mL/min to 1.2 mL/min effectively doubles the peak capacity value. Additionally, increasing the gradient time from 5 minutes to 50 minutes enhances the peak capacity value by 3.4-fold. The unique design of the Agilent 1290 Infinity LC System tolerates 1200 bar column back pressure, allowing for higher flow rates and reduced run times.
| Column | Agilent ZORBAX Eclipse Plus C18, 150 mm x 2.1 mm, 1.8 pm |
|---|---|
| Column oven temperature | 50 °C |
| Detection wavelength | 214/4 nm |
| Reference wavelength | 400/60 nm |
| Peak width response time | 0.062 s |
| Data acquisition rate | 80 Hz |
Increasing the flow rate from 0.2 mL/min to 1.2 mL/min effectively improves the peak capacity value to double.
Increasing the gradient time from 5 minutes to 50 minutes enhances the peak capacity value by 3.4-fold.