Spectrophotometry

Spectrophotometer

Wavelength: 400 nm

Absorbance (A): 0.000

Transmittance (T): 100.0%

Intensity (I/I₀): 1.000

Absorption Spectrum (A vs λ)

Standard Curve (A vs c)

Parameters

Molar Absorptivity (ε): 1000 L·mol⁻¹·cm⁻¹

Path Length (b): 1.0 cm

Concentration (c): 0.001 M

Wavelength: 400 nm

Absorption Peak (λmax): 450 nm

Peak Width (σ): 50 nm

Sample Type:

Standard Solutions: 5

Deviation Factors

Chemical Deviation (Association/Dissociation)

Instrumental Deviation (Stray Light/Polychromatic)

High Concentration (> 0.01 M)

Beer-Lambert Law Equations

Beer-Lambert Law: A = ε·b·c

Absorbance: A = -log₁₀(T) = -log₁₀(I/I₀)

Transmittance: T = I/I₀ = 10^(-A)

Concentration: c = A/(ε·b)

Linear Range: A = 0.1-1.0 (ideal: 0.2-0.8)

Calculated Concentration: 0.00100 M R² (Linearity): 0.999

What is Spectrophotometry?

Spectrophotometry is an analytical technique used to measure the concentration of a substance in solution by measuring the amount of light absorbed at a specific wavelength. It is based on the Beer-Lambert Law.

Instrument Components

Light Source: Tungsten lamp (visible) or deuterium lamp (UV) provides continuous spectrum

Monochromator: Prism or grating selects specific wavelength

Sample Cell: Cuvette with known path length (typically 1 cm)

Detector: Photomultiplier tube or CCD converts light to electrical signal

Readout: Displays absorbance, transmittance, or concentration

Beer-Lambert Law

The Beer-Lambert Law states that absorbance is directly proportional to concentration and path length: A = ε·b·c. This linear relationship holds for dilute solutions (< 0.01 M) where solute molecules act independently.

Standard Curve Method

To determine unknown concentration, prepare standard solutions of known concentration, measure their absorbance, and plot A vs c. The resulting calibration curve should be linear and pass through the origin.

Deviations from Beer-Lambert Law

Chemical Deviations: Association, dissociation, or solute-solvent interactions change ε with concentration

Instrumental Deviations: Stray light and polychromatic radiation cause negative deviation

High Concentration: Refractive index changes and molecular interactions cause deviation

Applications

Biochemistry: Protein concentration (Bradford, BCA at 280 nm), DNA quantification (260 nm)

Environmental Analysis: Water quality, pollutant detection

Pharmaceutical: Drug purity, dissolution testing, quality control

Clinical: Blood glucose, cholesterol, enzyme assays

Food Industry: Color analysis, preservative concentration

How to Use This Visualization

Adjust Parameters: Use sliders to change ε, b, c, and λ

Scan Spectrum: Click 'Auto Scan' to generate absorption spectrum

Standard Curve: Calibrate with standards to see linear relationship

Explore Deviations: Enable deviation factors to see non-linear behavior