Home Analytical Chem Operating an HPLC: Part 1
Steps
  1. 1 Understand HPLC principles and components 00:10
  2. 2 Gather filtration equipment and supplies 01:16
  3. 3 Set up the filtration apparatus 01:56
  4. 4 Start vacuum filtration process 02:27
  5. 5 Complete filtration and decontamination 02:56
  6. 6 Finish filtration and prepare for HPLC 03:31
Analytical Chem YouTube (Curated Tutorials)

Operating an HPLC: Part 1

Protocol
Difficulty
intermediate

Steps

1
Understand HPLC principles and components

Learn that HPLC (High Pressure Liquid Chromatography) detects individual compounds in complex solutions. Identify the main components needed: mobile phases, columns, and filtration equipment.

▶ 00:10
2
Gather filtration equipment and supplies

Assemble all required materials for mobile phase filtration: mobile phase solution, funnel, sidearm trap, tubing, vacuum pump, and membrane filters (0.45 μm or 0.22 μm).

▶ 01:16
3
Set up the filtration apparatus

Select a membrane filter and insert it into the membrane filter holder. Tightly secure the funnel on top of the filter holder. Position the mobile phase solution nearby for pouring.

▶ 01:56
4
Start vacuum filtration process

Turn on the vacuum pump and verify there are no bubbles in the tubing. Slowly pour the mobile phase into the funnel to begin filtration into the side flask.

▶ 02:27
5
Complete filtration and decontamination

Wait for the filtration process to finish. Note that a sonicator water bath is recommended for buffers to decrease contamination. The trap removes bubbles and contaminants from the filtrate.

▶ 02:56
6
Finish filtration and prepare for HPLC

Turn off the vacuum pump once no more mobile phase flows into the sidearm. Open the funnel and transfer the filtered mobile phase into a clean HPLC-ready container.

▶ 03:31
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