Instrumentation for spectrophotometry
• single-beam and double-beam spectrophotometers
• light sources
Instrumentation for UV-Vis absorption measurements
• all the cells, windows, lenses and dispersive element must be transparent to the radiation used
◦ visible: silicate glass O.K.
◦ UV: fused silica or quartz (silicate glass absorbs UV)
Which depicts the correct order for the parts in the schematic of a general scanning
a) Light source, wavelength selector, sample compartment, light detector, and read-out device.
b) Light source, sample compartment, wavelength selector, light detector, and read-out device.
c) Light source, sample compartment, wavelength selector, and light detector
Types of instrument
• spectrophotometer-based instruments
◦ use a monochromator (with grating)
▪ allows continuous λ selection
▪ MOST WIDELY USED
• photometer-based instruments
◦ use a filter
▪ constructive or destructive interference of light waves
• solvent (blank) and sample must be measured separately
◦ cannot measureAdirectly
• if source intensity and detector response fluctuate in time: error inA
• simple, inexpensive and easy to maintain
◦ stable source required Procedure with single-beam instruments
1- 0% T calibration
• sample compartment is empty and the shutter blocks all light (no light reaches the detector)
• adjust T reading to 0%
2- 100% T calibration In theory: measure all samples after single
• cell contains pure solvent in sample compartment calibration
• adjust T reading to 100% In practice: frequently recheck 0% and
100% T calibration for maxiumum
accuracy (~5-10 sameples)
3- Sample analysis
• cell contains sample
• read T (orA)
Can easily be used for
recording ofA vs
wavelength (can't w/
attenuator:adjusts so light is the same
• signal from 2 beams is compared electronically and converted to T orA
◦ ≈ 2 simultaneous measurements
◦ compensates for changes in source intensity and detector response with time and
Which statement is correct for a double-beam spectrophotometer?
a) Light passes through the sample and reference, directed by a fixed mirror, chopper.
b) Light alternatively passes through the sample and reference, directed by a rotating mirror,
c) Light alternatively passes through the detector, directed by a rotating mirror, chopper.
Precautions with double- beam instruments
• path length through 2 cells must be as identical as possible
◦ use matched cells (ex: as identical in dimensions, wall thickness, etc.)
• same amount of light must pass through
◦ adjust attenuator to read 100% T (or 0A) with solvent present in both reference and sample
Precautions with any spectrophotometer
• adjust analyte concentration so that 0.695% of all analyses are done by spectrophotometry in Health Sciences alone Precautions with cells
• always position the cells the same way
◦ variations in path length and reflection losses → appreciable error
• avoid scratches, fingerprints, grease or other deposits on the cell windows
◦ do not touch the windows
• never dry a cell in an oven or over a flame
◦ distortion in glass
◦ change in path length
A cuvet should be
a) thoroughly rinsed and dried with an absorbent towel before use.
b) rinsed and allowed to drain before use.
c) rinsed and oven dried before use.
A) select λ
• for maximum sensitivity: λ corresponding to a peak
◦ change in Aper unit concentration is greatest
◦ absorption curve is flat at a maximum
▪ good adherence to Beer’s law
▪ reproducing exactly the same λ is less critical
B) Identify all variables
• nature of solvent → coose conditions where small, uncontrollable
• pH of solution variations ofthese variables will not affect A
• electrolyte concentration
• interfering substances present?
C) Clean and handle cells correctly
• use good quality matched cells
D) Calibration curve
• measureA for a series of standard solutions, including the reagent blank, encompassing the
expected concentration(s) in the samples.