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HPLC · Calibration procedure

Detector linearity

Checks that detector response is proportional to concentration across the absorbance range your methods use.

Photo of a HPLC
Photo: Fernando Frick, CC0 (cropped), via Wikimedia Commons
PURPOSE

Why this is checked

Quantitation against a single standard assumes a linear response. Linearity confirms results are reliable at both low and high concentrations.

Frequency

Typically every 6 months, and after major repair or relocation, as defined in your calibration SOP.

Learn how the HPLC works
YOU WILL NEED

Standards & tools

  • A series of caffeine solutions covering the working range (at least five concentrations, e.g. from about 10 to 100 µg/mL)
BEFORE YOU START

Prerequisites

  • System equilibrated with a suitable column and mobile phase
  • Detector at 273 nm
PROCEDURE

Step by step

  1. 1Prepare at least five caffeine solutions across the range.
  2. 2Inject each solution (in duplicate if your SOP requires).
  3. 3Plot mean peak area (or absorbance) against concentration.
  4. 4Calculate the correlation coefficient and check the residuals.
RECORD

Example observation table

Concentration (µg/mL)Peak area
10301,250
25752,180
501,503,900
752,254,600
1003,009,800
CALCULATION

How to calculate the result

Linear regression y = mx + c; report the correlation coefficient r
  1. Using the five points above
  2. r = 0.99999 (passes r ≥ 0.999)
Use the Standard curve (linear regression) calculator
ACCEPTANCE CRITERIA

Pass if

  • Correlation coefficient r not less than 0.999 over the tested range.
IF IT FAILS

What to do

  • Check lamp energy and flow-cell cleanliness
  • Repeat with freshly prepared solutions
  • Service the detector if it still fails
MORE

Other HPLC calibrations

In LimsPulse · Calibration Management

LimsPulse schedules this calibration, records the result against limits, and blocks the instrument everywhere if it fails or goes overdue.

See how