To run a Delay Calibration, you must create a method that identifies all modules to include in the calibrated system and contains the following information:
Sample information (location, name, etc.)
Setup information and run for each of the modules (Sampler, Pump, Detector, etc.)
The Delay Calibration procedure should be performed with method settings as close to the target application as possible to avoid potential issues. It is advisable to use the same Flow Modulator settings (split mode) as in the target application to avoid unexpected issues. In case the signal is too small in the detector(s) of the split flow, the split mode must be adjusted to a value closer to Mode 1.
Important: Delay time(s) and volume(s) that were measured for detectors in split flows will be valid only for this combination of main and make-up flows. Only calibrated delay time(s) and volume(s) for detectors in the main flow will be recalculated depending on the actual flow rate.
Calibration using the delay and checkout calibrant cannot be performed isocratically. Some dyes in the mixture will not elute properly from the column when a high percentage of organic solvent is used. This is a specific effect of sulfonated dyes being separated with high surface area columns, which are typical for preparative chromatography.
Before the first run, flush the column with three column volumes using 95% organic channel B (around 2 minutes).
The table below lists the suggested default injection volumes of the Agilent Delay and Checkout Calibrant (part number 5190-8223) for different column diameters. If needed, adjust the injection volume in case the final response in the detector or fraction collector delay sensor are saturated or too low.
Column Inside Diameter | Calibrant Injection Volume |
|---|---|
3.0 mm | 0.5 μL |
4.6 mm | 1 μL |
around 10 mm | 10 μL |
around 20 mm | 50 μL |
30 mm | 100 μL |
50 mm | 300 μL |
If the system contains a 1290 Infinity II Preparative Open-Bed Sampler/Collector (G7158B), switch the operation mode to Preparative in the sampler method. From the method presets list, pick the preset for Non-polar Sample Matrix. In the plug settings on the right, provide a location for a vial of 25% methanol in water, which serves as post-sample plug solvent. Leave all other parameters at their default setting.
The LC system has been delivered with at least one capillary kit designed for a certain flow rate range. Depending on the installed capillaries, the following combinations of columns and flow rates are recommended.
Column Inside Diameter | Flow Rate Range | Column Delivered with System |
|---|---|---|
3.0 mm | 0.4–0.6 mL/min | 3.0 × 50 mm, 2.7 μm (699975-302) |
4.6 mm | 1.0–1.5 mL/min | 4.6 × 100 mm, 2.7 μm (695975-902) |
around 10 mm | 4–8 mL/min | 10 × 50 mm, 5 μm (446905-802) |
around 20 mm | 15–40 mL/min | 21.2 × 50 mm, 5 μm (446905-702) |
30 mm | 40–80 mL/min | 30 × 50 mm, 5 μm (446905-302) |
50 mm | 80–200 mL/min | 50 × 50 mm, 5 μm (446905-502) |
If the passive split from the tubing kit is included in the plumbing before the nebulizer, the suggested make-up flow is 1.5 mL/min.
If the passive split from the tubing kit is not used, the suggested make-up flow should be 0.5 mL/min. The maximum flow rate for the ESI source is 0.8 mL/min.
The following gradient is recommended to be used with the 50 mm long columns included in the preparative-scale system capillary kits. If longer columns are used for calibration, extend the slope time of the gradient by an appropriate factor. The gradient is suitable for any inside diameter of the column, provided that the flow rate is adjusted accordingly.
Time | Composition A | Composition B |
|---|---|---|
Preconditioning time: 1.5 min | ||
0 min | 90% | 10% |
0.5 min | 90% | 10% |
4.0 min | 5% | 95% |
6.0 min | 5% | 95% |
6.1 min | 90% | 10% |
Stop time: 7.0 min | ||
In case the chromatogram shows spikes or spikes joined into a peak-like band, try to increase the starting composition from 10% to 20%.
UV | Use a wavelength of 600 nm (no reference wavelength) to monitor the blue calibration dye of the delay and checkout calibrant. |
MS | Visible ions for the blue calibration dye:
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