Wavelength Calibration Procedure

When

If application requires, or see Wavelength Calibration Steps.

Tools required

Qty.		p/n	Description
1			Laboratory balance

Parts required

Qty.	p/n	Description
1	G7121-68001	FLD Calibration Kit contains parts below
1	5063-6597	Calibration Sample, Glycogen
1	5190-1539	Syringe 5mL with Luer-Lock
1	9301-0407	Syringe, External Valve adapter, SST
1	5190-5111	Syringe filter, 0.45 µm, 100/pk
1	0100-1516	Finger-tight fitting PEEK, 2/pk

NOTE

New instruments are shipped with a flow cell installed and have a factory wavelength calibration.

If a flow cell is used other then the installed/shipped flow cell:

verify that the installed flow cell is fixed very tight,

perform a wavelength calibration using Glycogen (part of FLD Calibration Kit (G7121-68001)).

Preparation of the Glycogen Calibration Sample.

NOTE
The calibration solution needs to be prepared freshly. Once dissolved, Glycogen is only useable for 24 hours. Using water for calibration is not supported!
1. Final concentration needs to be 1 mg/mL. To prepare 25 mL of the calibration solution, you have to use 25 mg of the Glycogen sample (a tolerance of ±20% is not critical).
1. Weigh in the Glycogen into a volumetric flask.
1. Fill 20 mL of distilled water (fresh, clean) into the volumetric flask and shake.
1. Sonicate for 15 minutes, wait 5 minutes, fill up to 25 mL with water and shake again.
Preparation of the Flow Cell.
1. Flush the flow cell with water.
1. Remove the inlet capillary from the flow cell.
1. Remove the outlet tubing from the flow cell and install a short (less than 5 cm) waste tubing instead. This helps avoiding unwanted syphoning effects.
1. Take the syringe and fix the needle to the syringe adapter.
1. Suck about 2.0 ml of the calibration sample into the syringe.
1. Keep the syringe in a horizontal position.
1. Remove the needle.
1. Add the filter to the syringe and eject the calibration sample to waste (through the filter). This will ensure removing any particles from the filter.
Figure: Syringe with Sample Filter
1. Remove the filter.
1. Take the syringe and fix the needle to the syringe.
1. Suck about 4 mL of the calibration sample into the syringe.
1. Keep the syringe in a horizontal position.
1. Remove the needle.
1. Add the filter to the syringe and fit the needle to the filter.
1. Lift the needle tip and carefully eject approximately 0.5 ml to remove air out of the syringe.
1. Add the PEEK fitting to the needle tip and fix both at the flow cell inlet. Avoid injecting any air bubble.
NOTE
Do not inject the calibration sample without the sample filter.
1. Slowly inject about 2 ml and wait for about 10 seconds to inject another 0.5 ml. This will assure that the cell is filled properly.
Additional hints for calibration:
- Do not forget to reset the grating calibration in LabAdvisor.
- Check that the cell has been fully inserted and fixed tightly.
- Prepare a little more calibration solution, so all glassware can be rinsed thoroughly prior to using.
- Never fill the syringe with the filter on.
- Never empty the syringe completely.
- With a 1mL glass syringe with Luer-lock, two to three injections of Glycogen might be required. Using a 5 mL glass syringe with Luer-Lock simplifies the procedure drastically lowers the risk of injecting air.
- After the final injection, leave the syringe in the in-port and keep it in a horizontal position.
Wavelength Calibration.
1. From the user interface start the FLD wavelength calibration.
- Agilent Lab Advisor (preferred option): Under Instrument Control > Special Commands press the Reset grating calibration button, then go for Service & Diagnostics > Wavelength Calibration
- Instant Pilot (G4208A): Maintenance > FLD > Calibration
NOTE
If the wavelength calibration process fails, refer to Wavelength Calibration Failed.
1. If a deviation is displayed, press Yes to adjust to new values. The history table will be updated.
Figure: Wavelength Calibration (Agilent Lab Advisor)

Figure: Calibration History (Agilent Lab Advisor, under Module Info)

NOTE
Restore flow connections and flush with fresh water at 1.5 mL/min - 2 mL/min for at least 15-20 min. Prolonged flushing (e.g. overnight flushing) at low flow rates will not be sufficient.
Verification using the "Wavelength Accuracy Test".
1. Refit the capillary to the flow cell.
1. Follow the procedure "Wavelength Accuracy Test".

21-Jun-2025