Unit 1: Spectrophotometry, colorimetry and photometry
Analytical Biochemistry notes · PTU syllabus (BMLS302-18)
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Unit summary
Most clinical chemistry tests measure how much light a coloured solution absorbs or a flame emits. This unit covers the theory of spectrophotometry and colorimetry, Lambert's and Beer's laws and their applications, flame photometry and atomic absorption spectroscopy.
After this unit you can
- State and apply Beer–Lambert's law
- Describe the components of colorimeters and spectrophotometers
- Explain flame photometry and its limitations
- Explain atomic absorption spectroscopy
PTU syllabus topics
- Theory of spectrophotometry and colorimetry
- Lambert's law and Beer's law
- applications
- general principles
- limitations
- instrumentation and applications of flame photometry
- atomic absorption spectroscopy principle and applications
Absorbance
A = ε c l
ε
Molar absorptivity
c
Concentration of the solution
l
Path length (usually 1 cm)
Unknown concentration
(A test / A standard) × concentration of standard
Topic 1
Theory of colorimetry and spectrophotometry
- A coloured solution absorbs light of particular wavelengths; the amount absorbed is proportional to the concentration of the coloured substance. Colorimeters select wavelengths with filters; spectrophotometers use a monochromator (prism or diffraction grating) and can work in the UV (190–380 nm) and visible (380–800 nm) range.
Lambert's law
Absorbance is proportional to path length (thickness) of the solution
Beer's law
Absorbance is proportional to concentration
Combined
A = ε c l (ε = molar absorptivity, c = concentration, l = path length, usually 1 cm)
Absorbance and transmittance
A = −log T = 2 − log %T
Calculation
Concentration of test = (A test ÷ A standard) × concentration of standard
Example
Glucose: A standard (100 mg/dL) = 0.40, A test = 0.62 → test = 0.62 ÷ 0.40 × 100 = 155 mg/dL.
- Limitations: the law holds only for dilute solutions, monochromatic light, and when the substance does not associate, dissociate or fluoresce.
Topic 2
Instrumentation
- 1
Light source
Tungsten lamp (visible); deuterium lamp (UV)
- 2
Wavelength selector
Filters (colorimeter); monochromator (spectrophotometer)
- 3
Slit
Narrows the beam
- 4
Cuvette
Glass or plastic (visible); quartz (UV); 1 cm path
- 5
Detector
Photocell, phototube or photomultiplier tube
- 6
Readout
Meter or digital display of absorbance
- Blank: a reagent blank zeroes the instrument so only the analyte's colour is measured.
- Applications: glucose, urea, creatinine, cholesterol, proteins, bilirubin, enzymes (kinetic assays at 340 nm for NADH), haemoglobin (540 nm).
Topic 3
Flame photometry
- Principle: a solution sprayed into a flame excites metal atoms; as electrons return to the ground state they emit light of a characteristic wavelength; emission intensity is proportional to concentration.
Sodium
589 nm
Yellow
Potassium
767 nm
Violet
Lithium
671 nm
Red (used as internal standard)
Calcium
622 nm
Brick red
- 1Atomiser (nebuliser) sprays the sample
- 2Burner with fuel gas (LPG or propane) and air
- 3Filter selects the element's wavelength
- 4Photodetector
- 5Readout compared with standards
- Limitations: only alkali and alkaline earth metals; interference from other ions and viscosity; flame stability; now largely replaced by ion-selective electrodes for Na⁺ and K⁺.
Topic 4
Atomic absorption spectroscopy
- Principle: ground-state atoms in a flame or graphite furnace absorb light of their own characteristic wavelength from a hollow cathode lamp made of that element; absorbance is proportional to concentration.
- Applications: trace metals — lead, copper, zinc, magnesium, calcium, iron, mercury — in blood, urine, water and food. More sensitive and specific than flame emission.
Measures
Light emitted by excited atoms
Light absorbed by ground-state atoms
Light source
Flame itself
Hollow cathode lamp
Elements
Na, K, Li, Ca
Many metals including trace elements
Sensitivity
Moderate
High
Key terms
- Absorbance
- Log of incident to transmitted light intensity
- Monochromator
- Device selecting a narrow band of wavelengths
- Cuvette
- Container holding the sample in the light path
- Flame photometry
- Measuring light emitted by excited metal atoms
- Hollow cathode lamp
- Element-specific light source in AAS
Quick revision
- Beer's and Lambert's laws; A = εcl; A = 2 − log %T; test ÷ standard calculation.
- Colorimeter vs spectrophotometer; components; blanks; applications.
- Flame photometry: emission, Na 589 nm, K 767 nm; limitations; ISE.
- AAS: absorption, hollow cathode lamp, trace metals.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.State Beer's law.
- Q2.Convert 50% transmittance to absorbance.
- Q3.Why are quartz cuvettes used in the UV range?
- Q4.What is the role of a blank?
- Q5.What colour does sodium give in a flame?
- Q6.Name two elements measured by AAS.
Long-answer questions
- Q1.Explain the Beer–Lambert law and its limitations.
- Q2.Describe the instrumentation of a spectrophotometer.
- Q3.Explain the principle and instrumentation of flame photometry.
- Q4.Explain atomic absorption spectroscopy.
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