The daily calibration and validation of UV visible spectrophotometers are key to ensuring data accuracy, mainly including wavelength calibration, absorbance calibration, baseline calibration, and performance validation. The specific methods are as follows:
1、 Wavelength calibration
Use standard lamp: calibrate using the characteristic emission spectral lines of mercury or deuterium lamps (such as 253.7nm and 365nm of mercury lamps). Set the instrument to a specific wavelength, place a calibration lamp, adjust the light path to pass through the sample cell, observe the absorption peak and record the actual wavelength value, compare it with the standard wavelength, and correct any deviation through the built-in function of the instrument or manually adjusting the grating.
Use standard filter: Use Ho ³ ⁺ doped glass filter, scan its characteristic absorption peaks (such as 418nm, 450nm), and correct the wavelength reading. This method does not require complex light source switching, but requires regular replacement of filters (recommended once a year).
2、 Absorbance calibration
Transmittance standard solution method:
Visible area: Use copper sulfate solution (CuSO ₄ · 5H ₂ O, λ=640nm, A=0.5).
Ultraviolet region: Use potassium dichromate solution (K ₂ Cr ₂ O ₇, λ=350nm，A=0.5）。
Preparation requirements: Use analytical grade reagents and primary volumetric flasks, with a shelf life of no more than 7 days for the solution. Using a 1cm quartz colorimetric dish and air as a reference, the sample cell needs to be rinsed three times with reference solution before measurement. Require 3 consecutive measurements with RSD ≤ 0.3%, otherwise refill the liquid.
Neutral filter method: When the absorbance is greater than 1.5AU, a combination of neutral filters (such as OD=0.5+1.0 superposition) is introduced to correct the Beer's law deviation through multi-point fitting.
3、 Baseline calibration
Measure the absorbance using blank solvent (such as distilled water) as a reference. Adjust the baseline adjustment device of the instrument to make the absorbance value of the blank solvent zero. Repeat the measurement several times to ensure baseline stability.
4、 Performance verification
Accuracy verification: Prepare a series of standard solutions with different concentrations, measure the absorbance of each standard solution separately, calculate its average and standard deviation, and evaluate the accuracy of the instrument.
Repeatability verification: Using the same standard solution, measure continuously multiple times, calculate the absorbance value of each measurement, calculate its average and standard deviation, and evaluate the repeatability of the instrument.
Linear validation: Prepare a series of standard solutions with different concentrations, measure the absorbance of each standard solution separately, draw the absorbance concentration curve, calculate the linear correlation coefficient of the curve, and evaluate the linearity of the instrument.