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Calibration of

Amit Arkad

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

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

A small deviation in wavelength, especially for analytes with a steep slope

in the absorption profile, will cause a large change in the absorbance value
of the measurement, and the accuracy of the quantitation will be affected
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.
+-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 + Is
I0 + 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

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 the

wavelength axis, and therefore inaccurate quantitation results.

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

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

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
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.

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 normalize
the light intensity measurement and the spectral output at different wavelengths
throughout the spectral range.
Air is scanned in the absorbance mode. The highest and lowest deflections in
the absorbance unit are recorded.

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

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.

The deflection is typically less than 0.002 AU/h.
7. Linearity
The linear dynamic range of the measurement is limited by stray light at high
absorbance 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.

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
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.

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.

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

Standard Values for the BP/EP Potassium Dichromate Solution

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

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