Calibration of UV-Spectrophotometer

byAmit Arkad

PERFORMANCE ATTRIBUTES

1. Wavelength Accuracy

2. Stray Light

3. Resolution

4. Noise

5. Baseline Flatness

6. Stability

7. Linearity

8. Photometric Accuracy

1. Wavelength Accuracy

Wavelength accuracy is defined as the deviation of the wavelength reading at an absorption band or emission band from the known wavelength of the band.

Most UV–Vis assays will specify that the measurements are to be taken at λmax.

A small deviation in wavelength, especially for analytes with a steep slopein the absorption profile, will cause a large change in the absorbance value of the measurement, and the accuracy of the quantitation will be affected

Tests.Wavelength reference standard:

Deuterium lamp,

Mercury vapor lamp,

Holmium oxide filter, and

Holmium oxide solution

Emission spectrum of deuterium lamp

Emission spectrum of mercury vapor lamp

Absorption spectrum of holmium oxide filter

Absorption spectrum of holmium oxide solution.Acceptance. +-1 nm in the UV range (200 to 380 nm) and +-3 nm in the visible range (380 to 800 nm). Three repeated scans of the same peak should be within +―0.5 nm.

Wavelength Standard Comparison

Wavelength Assignment: 4% Holmium Oxide in 10% Perchloric Acid

2. Stray Light

Stray light is defined as the detected light of any wavelength that is outside the bandwidth of the wavelength selected.

The causes for stray light are scattering, higher-order diffraction of the monochromator, or poor instrument design.

Stray light causes a decrease in absorbance and reduces the linear range of the instrument.

Absorbance(A) = −log transmittance (T )

T = I + IsI0 + Is

Changes in absorbance due to different levels of stray light at 1% transmittance

The stray light problem causes a deviation from linearity at high absorbance.

Effect of stray light on absorbance measurement

Absorbance values ranging from 0.3 to 1 AU are less susceptible to stray light and noise problems and therefore become the preferred absorbance range for UV–Vis analyses.

Stray Light Measurement

Acceptance. The transmittance of the solution in a 1-cm cell should be less than 0.01, or the absorbance value should be greater than 2.

3. Resolution

Insufficient resolution leads to a decrease in the extinction coefficient across thewavelength axis, and therefore inaccurate quantitation results.

The spectral changes from scans of a 0.02% v/v toluene-in-hexane solution using slit width

Tests. A solution mixture of 0.02% v/v toluene in hexane (UV grade) Scanned from 250 to 300 nmAbsorbance values of the λmax at 269 nm and the λmin at 266 nm are recorded.

Acceptance. The ratio of the absorbance at λmax (269 nm) and absorbance at λmin (266 nm) should be greater than 1.5

4. Noise

Noise in the UV–Vis measurement originates primarily from the light source and electronic components.

Photon noise from the light source affects the accuracy of the measurements at low absorbance.

Electronic noise from the electronic components affects the accuracy of the measurements at high absorbance

Tests. Air is scanned in the absorbance mode for 10 min. peak-to-peak noise is recorded at 500 nm. The root mean square (RMS) noise is then calculated.

Acceptance. The RMS noise should typically be less than 0.001 AU

5. Baseline Flatness

The flat baseline test demonstrates the ability of the instrument to normalizethe light intensity measurement and the spectral output at different wavelengths throughout the spectral range.

Tests. Air is scanned in the absorbance mode. The highest and lowest deflections in the absorbance unit are recorded.

Acceptance. The deflection is typically less than 0.01 AU

6. Stability

Variations in lamp intensity and electronic output between the measurements of the reference and the sample result in instrument drift.

The stability test checks the ability of the instrument to maintain a steady state over time so that the effect of the drift on the accuracy of the measurements is insignificant.

Tests.

Air is scanned in the absorbance mode for 60 min at a specific wavelength (typically, 340 nm). The highest and lowest deflections in the absorbance unit are recorded.

Acceptance. The deflection is typically less than 0.002 AU/h.

7. LinearityThe linear dynamic range of the measurement is limited by stray light at highabsorbance and by noise at low absorbance.

For routine measurements involving samples and the related reference chemical standards, the accuracy of the quantification of the sample depends on the precision and linearity of the measurements.

Tests. A series of potassium dichromate solutions of concentration 20, 40, 60, 80, and 100 mg/L in 0.005 M sulfuric acid can be used to test the linearity of the system. First, 0.005 M sulfuric acid as reference is scanned and then the potassium dichromate solutions at 235, 257, 313, and 350 nm. The absorbance values at various wavelength are plotted against the conc. of the solutions and the correlation coefficients are calculated.

Acceptance. Correlation coefficient r ≥ 0.999.

8. Photometric Accuracy

It is very important in the accurate measurement of the extinction coefficient that is used to characterize an analyte and to ensure that transmittance and absorbance data are comparable between spectrometers in different laboratories.

Photometric accuracy is determined by comparing the difference between the measured absorbance of the reference standard materials and the established standard value.

Tests. Either the neutral-density filters or potassium dichromate solutions are used.

Standard Values for the BP/EP Potassium Dichromate Solution

Acceptance. Typical accuracy +-0.01 AU, or the specific absorbance value falls with the limit outlined

Reference:

ANALYTICAL METHOD VALIDATION AND INSTRUMENT PERFORMANCE VERIFICATION

Edited byCHUNG CHOW CHAN

PERFORMANCE VERIFICATION OF UV–VIS SPECTROPHOTOMETERS