How to measure LC system dispersion for SEC analysis - WKB83286

OBJECTIVE or GOAL

Determine the LC system dispersion for size-exclusion chromatography method transfer and/or troubleshooting using a caffeine sample

ENVIRONMENT

• ACQUITY H-Class Bio
• ACQUITY Arc Bio
• Alliance HPLC
• SEC columns

PROCEDURE

1. Replace the column with a Zero-Volume Union (700002636).

2. Purge all solvent, wash, and purge lines with 50:50 water/acetonitrile.

3. Create an instrument method with the following parameters:

• Run time of 1 minute
• Flow rate of 0.5 mL/min with 100% solvent A (isocratic)
• Column heater Off
• Set wavelength to 273 nm with a sampling rate of 40 points/s or greater

4. Inject three times 0.5 µL of mobile phase blanks (50:50 water/acetonitrile).
5. Inject five times 0.5 µL of caffeine with a concentration of 0.160 mg/mL in 50:50 water/acetonitrile (for example, caffeine from the ACQUITY UPLC/UV Qualification Standards Kit (700002642; Solution 7).
6. To calculate dispersion:

• Measure the caffeine sample peak width in minutes at 4.4% peak height.
• Multiply the peak width by the flow rate to determine peak volume in mLs, and then multiply by 1000 to determine peak volume in µLs.

ADDITIONAL INFORMATION

The impact of LC system dispersion on SEC of proteins can be found here: https://www.waters.com/waters/library.htm?cid=511436&lid=134996381&locale=en_BE