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Diagnostics Tips and Tricks for Retention Time in HPLC

What is the retention time in HPLC?
Retention time (RT) is a measure of the time taken for a solute to pass through a chromatography column. It is calculated as the time from injection to detection. The RT for a compound is not fixed as many factors can influence it even if the same HPLC and column are used.

How do you increase retention time in HPLC?
Analyst can change its retention by changing the polarity of the mobile phase (the mobile phase is competing with the solute). Thus a more polar mobile phase will increase retention, and a more non-polar mobile phase will decrease retention.

How do I reduce retention time in HPLC?
As temperature is increased, retention will decrease. If the room experiences wide temperature fluctuations, the HPLC retention times will probably be affected. The best solution is to run analyses at a temperature that can be controlled by using an oven.

What factors affect retention time in HPLC?
For a particular compound, the retention time will vary depending on:
  1. the pressure used (because that affects the flow rate of the solvent)
  2. the nature of the stationary phase (not only what material it is made of, but also particle size)

Retention Time

  1. Retention time changed to a new constant value
  2. Erratic retention time
  3. Increasing retention time
  4. Decreasing retention time

Retention time changed to a new constant value: 
  • Check column, mobile phase, temperature, method, flow rate

Increasing/decreasing retention times: 
  • Column contaminated, degraded
  • System not equilibrated
  • Mobile phase contaminated

Erratic retention times: 
  • Check if system also has erratic pump pressure  or pressure fluctuations
  • System not equilibrated
  • Check for leaks
  • Improper solvent blending
  • Temperature fluctuations

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