HPLC Lamp Energy check is the most important aspect of Liquid Chromatography. Lamp energy is also called HPLC Lamp Intensity Test or HPLC Reference Energy verification.
Monitoring of HPLC Lamp Energy (D2 Lamp energy)
It can also be called HPLC Reference Energy (D2 Lamp energy monitoring).
To ensure the performance and accuracy of the analysis, the D2 lamp shall be monitored on a scheduled basis more frequently (Monthly or Bimonthly). The observed energy details shall be documented as part of “Good Laboratory Practices”. To determine the HPLC reference energy, a brief procedure is illustrated for reference purposes.
Before performing the Lamp energy of HPLC, ensure the proper maintenance of the instrument. Follow the below general maintenance procedure.
Remove the suction filters, and line filters, and check the valves. Then dip in a beaker containing 6N Nitric acid, and heat at about 45°C on a hot plate for 15 minutes. If required, sonicate the beaker.
Discard the Nitric Acid and sonicate with 50:50 IPA: Water (Milli-Q) mixture for 15 minutes and finally with Water for about 15 minutes.
Purge the Injector port and Seal Wash by using proper rinsing solvent (Use water or a mixture of 50:50 IPA: water).
Wash the flow lines with hot water (Approx. 45°C) by connecting the union in place of the column.
Check the pressure of the system without a column by connecting the union using 50:50 IPA: water. In case of high pressure is observed, proceed for line purge with hot water followed by IPA at constant high flow to remove the blocks.
Washing shall be ensured with a suitable ratio of water, acetonitrile, and methanol for sufficient time.
Before measuring, the lamp energy hot water washing shall be given to the instrument for a minimum of 1hr.
HPLC Reference Energy Measurement
Measure the HPLC Reference energy (HPLC Lamp Energy) at 230nm with water at laboratory temperature and record the details. The acceptance criteria to replace a lamp in terms of reference energy (Lamp Intensity) for different HPLC Detectors / Instruments shall be as tabulated below.
| Model | Acceptance criteria |
| Waters 2487 | > 15 nA |
| Waters 2489 | > 15 nA |
| Waters 2998 | NLT 5000 Counts |
| Prominence / Class VP series | > 400 mV |
| Shimadzu LC2010 CHT | > 200 mV |
| HPLC (Agilent Make 1200) | NLT 200 Counts |
| HPLC (Agilent Make 1260 Infinity II) | NLT 6400 Counts |
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In cases of Agilent making HPLCs where a Lamp Energy monitoring facility is not available, the lamps shall be replaced once in six months. If any error is observed, the lamp shall be re-placed and ensure calibration.
In case of Low Reference energy, the energy shall be verified by placing a new lamp.
If the lamp is faulty, placing a new lamp the energy will drastically increase. A significant increase in Reference energy with the new lamp confirms that the previous lamp needs replacement.
If no increase in the Reference energy is observed even after placing a new lamp, the performance of the Mirror and Optics shall be evaluated with the help of an Internal/External Service engineer.
The lamp replacement can also perform in cases of Ignition failure, Error/problem-related to the lamp, Baseline Noise or Drift even after flow cell cleaning.
The specific calibration shall be performed in case of a D2 lamp change although the change is Moderate in nature. The detector wavelength accuracy shall be performed and evaluated for conformance.
In case the reference energy is not meeting the acceptance criteria, the analyst shall inform the superior for replacing the new lamp.
The lamp replacement details shall record in the instrument usage log book / History card.
HPLC Instruments washing procedure
Purge the Injector port and Seal Wash by using proper rinsing solvent. (Use water or a mixture of 50:50 IPA: water).
Washing shall be ensured with hot water (Approx. 45°C) by connecting the union in place of the column.
After the above washing, perform with Methanol and followed by IPA for sufficient time.
The final washing of the HPLC System shall ensure with Methanol and Water, for sufficient time.
HPLC system washing procedure is essential to perform more frequently.
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