CALIBRATION OF UV & IR

INSTRUMENTS
BY
Dr.A.S.CHARAN
Analytical Instrument Qualification
• A large variety of laboratory equipment, instruments and computerized
analytical systems, ranging from simple nitrogen evaporators to complex
multiple-function technologies, are used in the pharmaceutical industry to
acquire data to help ensure that products are suitable for their intended use.

• AIQ is the collection of documented evidence that an instrument performs

suitably for its intended purpose. Use of a qualified instrument in analyses
contributes to confidence in the validity of generated data.
CALIBRATION OF UV
INSTRUMENTS
Calibration of UV-Visible Spectrophotometer
List of tests to be performed
1. Spectral slit-width (if applicable)
2. Wavelength accuracy
3. Wavelength precision (for mechanically set wavelengths)
4. Photometric accuracy (Control of absorbance)
5. Photometric linearity
6. Limit of stray light
7. Baseline noise
8. Photometric drift
9. Resolution/ Repeatability
10. Absorption of cells
Slit Width
• Effect of spectral slit width on absorbance fluctuation (performed with pure
solvents).
• Measure the width of the peak (in nanometres) at half the height of the peak.
• This represents the spectral bandwidth and should be within ± 10% of that
selected via the computer.

Slit width Slit width Slit width Slit width Difference Theoretical Test status
5.0 nm 2.0 nm 1.0 nm 0.05 nm in Abs. Values
Cyclohexane
0.016 0.015 0.015 0.015 < 0.001 < 0.010 Passed
Ethanol
0.020 0.019 0.019 0.019 < 0.001 < 0.010 Passed
Methanol
0.005 0.005 0.005 0.005 0.00 < 0.010 Passed
WAVELENGTH ACCURACY
Method:
• Verify the wavelength scale using the absorption maxima of holmium
perchlorate solution, the line of a hydrogen or deuterium discharge lamp or
the lines of a mercury vapour as shown below.
Limits:
• ± 1 nm in the UV range
• ± 3 nm in the Visible range
Absortion Observed
Acceptance
maxima of Reading nm
criteria(nm)
Holmium(nm)
241.15 ±1
287.15 ±1
361.50 ±1
536.30 ±3
WAVELENGTH PRECISION (for mechanically set wavelengths)
Method
• Carry out 6 measurements of the absorbance maxima. For this test, the same
materials of the previous test can be used.
Limits:
• Repeatability: the relative standard deviation of the absorbance maxima
should satisfy the manufacturer’s specifications.
• The difference between the 6 individual peaks should comply with the
manufacturer’s specifications (e.g. < 0.5 nm).
 PHOTOMETRIC ACCURACY (CONTROL OF ABSORBANCE)
Method:
• Check the absorbance using the solution of potassium dichromate R or
the certified filters at the wavelengths indicated in the table below,
which gives for each wavelength the exact values and the permitted
limits of the specific absorbance.
• Limits
Maximum
Wavelength (nm) Specific absorbance
tolerance
235 124.5 122.9 to 126.2
257 144.5 142.8 to 146.2
313 48.6 47.0 to 50.3
350 107.3 105.6 to 109.0
430 15.9 15.7 to 16.1
PHOTOMETRIC LINEARITY
Method
• Measure the net absorbance of the solutions of potassium dichromate
against a blank at 235, 313, 257 and 350 nm.
• Make 3 measurements for each solution. Calculate the mean value and
the linearity of the concentration of each solution against the measured
absorbance at the control wavelengths.
Limits:
• At each wavelength: r2 ≥ 0.999
LIMIT OF STRAY LIGHT
Method:
• The absorbance of a 12 g/l solution of potassium chloride R in a 1 cm cell
increases steeply between 220 nm and 200 nm.
• When available, use the in-built software of the instrument.
Limits:
• A > 2.0 at 198 nm, when compared with water as compensation liquid.
Alternatively:
• Method: Check stray light at 220 nm by using a 20 g/l NaI solution.
• Limits: according to manufacture’s specifications (e.g. Transmittance ≤ 0.1 %).
BASELINE NOISE
Method 1:
• Make 61 absorbance measurements with an integration time of 1 second at
a wavelength of 500 nm, with no sample in the sample chamber, and
calculate the mean.
Limits:
• Mean ± 0.002 Absorbance units
Method 2:
• Record the absorbance for 60 seconds at 200, 300 and 400 nm with a highly
pure, synthetic Quartz block. The measurement is made against air.
Filter Wavelength (nm) Maximum tolerance
Quartz block 200 0.049 ± 0.01
(against air) 300 0.033 ± 0.01
400 0.031 ± 0.01
PHOTOMETRIC DRIFT
Method 1:
• As routine test, the drift is measured at 250 nm over a period of 2 hours by
using the Time Scan mode of the instrument, with no sample in the sample
chamber.
Limits: ± 0.001 Absorbance units/h

Method 2:
• Record the baseline for 60 minutes at 500 nm and compare the absorbance
with the initial value.
Limits: ± 0.002 Absorbance units/h
SYSTEM SUITABILITY TEST OF THE METHOD
• - REPEATABILITY (for quantitative analysis)
• - RESOLUTION
Method:
• Criteria such as repeatability or resolution are usually given when test
methods are performed according to Ph. Eur., MAH dossier or a suitably
validated in-house method.
RESOLUTION (if required for qualitative analysis)
Method:
• When prescribed in a monograph, measure the resolution of the
apparatus as follows: record the spectrum of a 0.02 per cent V/V
solution of toluene R in hexane R. Alternatively, record the spectrum of
the reference material.
Limits:
• The minimum ratio of the absorbance at the maximum at 269 nm to that
at the minimum at 266 nm is stated in the monograph or in the test
method description.
Absorbance of 0.02%
Wavelength Absorbance Ratio Acceptance
S.No w/v toluene in
nm 269/266nm criteria
hexane
1 266
NLT 1.5
2 269
ABSORPTION CELLS
Method:
• Fill the cell with distilled water and measure its apparent absorbance
against air at 240 nm for quartz cells and at 650 nm for glass cells. With
recording instruments it is desirable to scan over the optical region of
interest. The apparent absorbance should be not greater than 0.093 for
1 cm quartz cells (UV region) and 0.035 for 1 cm glass cells (Visible
region). Rotate the cell in its holder (180°) and measure the apparent
absorbance again.
Limits:
• Rotating the cells should give an absorbance difference not greater than
0.005.
 Parameter to be checked Materials used in testing Typical tolerance limits
1. Spectral slit-width (if Cyclohexane, ethanol, methanol ± 10 %
applicable)
2. Wavelength accuracy Holmium Perchlorate sloution ± 1 nm for the UV range
± 3 nm for the Visible range
3. Wavelength precision Holmium Perchlorate sloution See manufacturer’s specifications
(for mechanically set
wavelengths)
4. Photometric accuracy Potassium dichromate As per defined limits
(Control of absorbance)
5. Photometric linearity Potassium dichromate r2 ≥ 0.999
6. Limit of stray light Potassium Chloride/Sodium Iodide A > 2.0 at 198 nm
7. Baseline noise Empty cuvttes/quartz blocks ± 0.002 Absorbance units (500 nm)
or
± 0.01 Absorbance units (200, 300, 400
nm)
8. Photometric drift Empty cuvettes ± 0.001 Absorbance units/h (250 nm)
or
± 0.002 Absorbance units/h (500 nm)
 Typical tolerance
Parameter to be checked Materials
limits
1. System suitability 0.02% v/v toluene and According to
check of the method hexane Pharmacopoeia or
-e.g. Repeatability validated in-house
-e.g. Resolution (if method
required for qualitative
analysis)
2. Absorption cells Distilled water Absorbance
difference ≤ 0.005
CALIBRATION OF IR
INSTRUMENTS
List of tests to be performed
1. Wave-number scale
2. Detector energy ratio
3. Signal/Noise ratio
4. Resolution
5. Zero test
6. Contamination check (only for ATR instruments)
7. Throughput check (only for ATR instruments)
8. System suitability check
WAVE-NUMBER SCALE-ACCURACY
Method and Limits:
• The wave-number scale may be verified by recording the spectrum of a
polystyrene film, which has transmission minima (absorption maxima) at
the wave numbers (in cm-1) shown in the table below:
Transmission minima Acceptable tolerance (cm-1)
(cm-1) Monochromator Fourier-transform
instruments instruments
3060.0 ± 1.5 ± 1.0
2849.5 ± 2.0 ± 1.0
1942.9 ± 1.5 ± 1.0
1601.2 ± 1.0 ± 1.0
1583.0 ± 1.0 ± 1.0
1154.5 ± 1.0 ± 1.0
1028.3 ± 1.0 ± 1.0
WAVE-NUMBER REPEATABILITY
Method and Limits:
• Difference between the wave numbers of two successive scans is not more
than ±1cm-1:
Scan Wave number accuracy
Standard Wave Repeatability
S.No
number cm-1 (diff NMT ± 1 cm-1)
1 2 Observed cm-1 Limit cm-1
1 3060.0 ± 1.5
2 2849.5 ± 1.5
3 1942.9 ± 1.5
4 1601.2 ±1
5 1583.0 ±1
6 1154.5 ±1
7 1028.3 ±1
DETECTOR ENERGY RATIO
Method:
• Record the minimum energy ratio value for at least one of the following measurement
points and compare it to the vendor’s specifications:
• Energy at 3990 cm-1 / energy at 2000 cm-1
• Energy at 4000 cm-1 / energy at 2000 cm-1
• Energy at 3400 cm-1 / energy at 1300 cm-1
• Energy at 2000 cm-1 / energy at 1000 cm-1

Limits:
• Energy ratio test specifications vary for each spectrometer configuration.
• Refer to the manufacturer’s specification.
SIGNAL/NOISE RATIO
Method:
• Record the maximum noise level for each of the following regions:
• Peak-to-peak noise between:(systems with DTGS detector only)
• 4050 cm-1 and 3950 cm-1
• 2050 cm-1 and 1950 cm-1
• 1050 cm-1 and 950 cm-1
• 550 cm-1 and 450 cm-1
• RMS (root mean square) noise between:(systems with DTGS detector only)
• 4050 cm-1 and 3950 cm-1
• 2050 cm-1 and 1950 cm-1
• 1050 cm-1 and 950 cm-1
• 550 cm-1 and 450 cm-1
Limits (% T):
• Noise level test specifications vary for each spectrometer configuration. Refer to the
manufacturer’s specifications.
RESOLUTION
Method:
• For instruments having a monochromator, record the spectrum of the polystyrene film.
• For Fourier-transform instruments, use suitable instrument resolution with the appropriate apodisation prescribed
by the manufacturer. The resolution is checked by suitable means, for example by recording the spectrum of a
polystyrene film approximately 35 µm in thickness.
Limits:
• Monochromator instruments:
• Difference x between the percentage transmittance at the transmission maximum A at 2870 cm-1 (3.48 µm) and
that at the transmission minimum B at 2849.5 cm-1 (3.51 µm) > 18.
• Difference y between the percentage transmittance at the transmission maximum C at 1589 cm-1 (6.29 µm) and
that at the transmission minimum D at 1583 cm-1 (6.32 µm) > 10.
• Fourier-transform instruments:
• Difference between the absorbances at the absorption minimum at 2870 cm-1 and the absorption maximum at
2849.5 cm-1 > 0.33.
• Difference between the absorbances at the absorption minimum at 1589 cm-1 and the absorption maximum at
1583 cm-1 > 0.08.
ZERO TEST
Method:
• When using a polystyrene film of approximately 35 µm in thickness as
standard at the wavelength of 2925 cm-1 and 700 cm-1, almost complete
absorption of the irradiated energy can be observed. With this test, the
remaining transmission is measured. As the maximum absorption can be
observed at 700 cm-1 negative values may be observed. The objective of
the test is to evaluate if, despite the fact that there is almost complete
absorption, energy is still detectable.
• Non-valid results are an indication of a non-linear behaviour of the
detector and the electronic system.
Limits (%T):
• See manufacturer’s specification.
 Parameter to be checked
1. Wave-number scale
2. Detector energy ratio
3. Signal/Noise ratio
4. Resolution
5. Zero test
6. Contamination check (only for ATR
instruments)
7. Throughput check (only for ATR
instruments)
8. Spectral library
9. System suitability check
Material used Typical tolerance limits
Polystyrene film of 35 µm See procdure
thickness
- See manufacturer’s
specifications
- See manufacturer’s
specifications
Polystyrene film of 35 µm See procdure
thickness
Polystyrene film of 35 µm See manufacturer’s
thickness specifications
- See procdure
- 80 %
- See procdure
- According to Ph. Eur. or MAH
dossier or validated in-house
method
THANK YOU