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February 2001
Vol. 10, No. 02, p. 47.
Focus: Separation Sciences

FEATURE

“Committeeing” to Calibrate

Table 1
Summary of calibration tests and acceptance criteria
HPLC Module Test Procedure Column* Acceptance Criteria
Detector PDA or UV–Vis Wavelength accuracy Measure lambdamax or maximum absorbance of anthracene solution (1 µg/mL). No 251 ± 3 nm 340 ± 3 nm
Pump Flow accuracy Run pump at 1 mL/min (65% acetonitrile/water) and collect 10 mL from detector in a volumetric flask. Measure time. Yes 1.00 ± 0.05 mL/min
Compositional accuracy Test all solvent lines at 2 mL/min with 0.1% acetone/ water, using step gradients of 10, 50, and 90%. Measure peak heights of respective step relative to 100% step. No ± 1% absolute
Autosampler Precision Determine the peak area Relative Standard deviation (RSD) of ten 10-µL injections of ethylparaben (20 µg/mL). Yes RSD < ± 0.5%
Linearity Determine the coefficient of the linear correlation of one injection of 5, 10, 40, and 80 µL of ethylparaben solution. Yes R > 0.999
Carryover Determine the carryover of peak area by injecting 80 µL of mobile phase following an 80-µL injection of ethylparaben. Yes < 1%
Sampling accuracy Determine gravimetrically the average volume of water per injection withdrawn from a tared vial after six 50-µL injections. No 50 ± 2 mL
Column Oven Temperature accuracy Check actual column oven temperature with validated thermal probe. No 35 ± 2 °C
* Waters Symmetry C18 (150 × 3.9 mm i.d., 5 µm)

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