This topic covers the principles behind spectrophotometry, including the interaction of light with matter, Beer-Lambert Law, and the use of spectrophotometers in analyzing samples in a laboratory setting.
Principles of Spectrophotometry
A concise, organized guide for quick reference and deeper study
| Aspect | Description |
|---|---|
| Definition | Quantitative measurement of the amount of light absorbed (or transmitted) by a sample as a function of wavelength. |
| Core Idea | Molecules absorb light at specific wavelengths; the intensity of absorption is proportional to the concentration of the absorbing species. |
| Typical Output | Absorbance (A) vs. wavelength spectra; single‑wavelength absorbance values for quantitative analysis. |
| Concept | Equation / Principle | Key Points |
|---|---|---|
| Beer‑Lambert Law | ( A = \varepsilon , b , c ) | • A = absorbance (unitless) • ε = molar absorptivity (L mol⁻¹ cm⁻¹) – intrinsic to the analyte at a given λ • b = path length (cm) – usually the cuvette width • c = concentration (mol L⁻¹) |
| Absorbance vs. Transmittance | ( A = -\log_{10}(T) ) , ( T = \frac{I}{I_0} ) | I₀: incident light intensity; I: transmitted intensity. |
| Molar Absorptivity (ε) | Determined experimentally; high ε → strong absorber, low detection limits. | |
| Linear Range | Region where A ∝ c (typically 0.1 – 1.0 AU). Outside this range, deviations occur due to stray light, detector saturation, or chemical effects. | |
| Selectivity | Depends on wavelength choice; select a λ where the analyte has a strong absorbance and interferents have minimal absorbance. |
| Component | Function | Typical Variations |
|---|---|---|
| Light Source | Emits continuous or discrete radiation covering the UV–Vis region (≈190–1100 nm). | Deuterium lamp (UV), Tungsten‑halogen lamp (visible‑NIR), LEDs, lasers. |
| Monochromator / Filter | Isolates a narrow band of wavelengths (Δλ ≈ 1–5 nm). | Diffraction grating, prism, interference filters. |
| Sample Holder (Cuvette) | Holds the liquid/solid sample; defines path length b. | Standard 1 cm quartz cuvettes (UV–Vis), glass (visible only), plastic (visible only). |
| Detector | Converts transmitted light into an electrical signal. | Photomultiplier tube (PMT), photodiode, charge‑coupled device (CCD). |
| Readout / Data System | Calculates absorbance, displays spectra, stores data. | Analog meters, digital displays, PC‑based software. |
| Reference Beam (optional) | Compensates for source intensity fluctuations. | Dual‑beam spectrophotometers have sample and reference channels. |