How do I perform an HPLC injection accuracy test? - WKB8833

OBJECTIVE or GOAL

Perform an HPLC injection accuracy test.

ENVIRONMENT

• Alliance HPLC System
• 2695 Separations Module
• 2695D Dissolution Separations Module
• 717plus Autosampler

PROCEDURE

Injection Volume Accuracy when using the standard 250µL syringe.To improve the accuracy of this test, handle the vial with forceps to ensure that the vial is as clean as possible.

1. Fill a standard vial with methanol or water.
2. Seal the vial with a Teflon Septum and cap.
3. Zero the analytical balance, carefully weigh the vial, and record the weight.
4. Place the vial in the carousel and set the flow rate to 1.0mL/min.
5. Program the system to make six 50µL injections from the vial. (Use a run time of approximately one minute or less)
6. Weigh the vial after the last injection and record that weight.
7. Using one of the following equations (depending on the solvent used), calculate the average amount injected per injection: ((W1-W2) /6) x (1/density of solvent) = µL injected
   • Using Methanol
     • Density of Methanol is 0.7890
     • ((W1-W2) /6) x 1.2687 = µL injected
   • Using Water
     • Density of Water is 1.0000
     • ((W1-W2) /6) x 1.0000 = µL injected
8. RESULTS: 50µl +/- 1µL

ADDITIONAL INFORMATION

This is commonly referred to as a 'lift test' by Waters engineers.

Specifications differ based on syringe size, check the instrument specifications and PM protocols for additional information.