Spectrophotometry

Awareness level only. This lesson explains the ideas so you recognise them. It does not qualify you to do this work. Before you use lab equipment or handle chemicals, biological materials or patient samples for real, you need the training your employer or institution requires, and you must work under supervision until they sign you off.

Why it matters

Spectrophotometry is one of the most used measurements in science. Labs use it to measure protein and DNA concentration, follow enzyme reactions, run colour-based clinical tests and check water quality. The instrument is simple to operate. Getting a trustworthy number depends on understanding the law behind it and building a good standard curve.

Absorbance and transmittance

A spectrophotometer shines light of one wavelength through a sample in a cuvette and measures how much gets through.

• Transmittance (T) is the fraction of light that passes through: T = I / I0.
• Absorbance (A) is defined as A = -log10(T).

Absorbance is used because it is proportional to concentration.

The Beer-Lambert law

A = epsilon x b x c

• epsilon (molar absorptivity) is how strongly the compound absorbs at that wavelength, in L/(mol·cm).
• b is the path length, usually 1 cm for a standard cuvette.
• c is the concentration in mol/L.

Worked example. NADH absorbs at 340 nm with epsilon of about 6,220 L/(mol·cm). A reading of A = 0.311 in a 1 cm cuvette gives c = 0.311 / (6,220 x 1) = 5.0 x 10^-5 mol/L, or 50 µmol/L.

Choosing a wavelength and a blank

• Measure at the compound's absorbance maximum (lambda max), where sensitivity is highest and small wavelength errors matter least.
• Blank the instrument with a cuvette of everything except the analyte (the solvent or reagent blank). This sets A = 0 and removes the absorbance of the solvent, the reagents and the cuvette.

The standard curve

Epsilon is not always known, and real assays include reagents that change it. Labs therefore build a standard curve (calibration curve):

1. Prepare standards of known concentration that bracket the expected unknowns.
2. Measure the absorbance of each, after blanking.
3. Fit a straight line: A = slope x c + intercept.
4. Measure the unknown and solve for concentration: c = (A - intercept) / slope.

Worked example. Standards of 0, 2, 4, 6, 8 and 10 mg/L read 0.002, 0.101, 0.199, 0.304, 0.398 and 0.502. A least-squares fit gives a slope of about 0.0499 per mg/L and an intercept of about 0.0013. An unknown reading 0.250 is (0.250 - 0.0013) / 0.0499 = about 4.98 mg/L.

If you diluted the unknown 1 in 5 before reading it, multiply by the dilution factor: 4.98 x 5 = 24.9 mg/L in the original sample.

Stay in the linear range

Beer-Lambert holds only over a range. At high absorbance (often above about 1 to 1.5, depending on the instrument) very little light reaches the detector, stray light dominates and the curve bends. If an unknown reads above your highest standard, dilute it and re-read; do not extrapolate the line.

Cuvette handling

• Hold cuvettes by the frosted or ribbed sides; fingerprints on the clear faces absorb light.
• Use quartz cuvettes below about 340 nm (UV); plastic and glass absorb UV light.
• Wipe the outside with lint-free tissue, check for bubbles and insert in the same orientation every time.

Common mistakes

• Forgetting to blank, or blanking with the wrong solution.
• Extrapolating beyond the highest standard.
• Forgetting the dilution factor.
• Reading at the wrong wavelength.
• Using plastic cuvettes for UV work.

Quick recap

• A = -log10(T), and A = epsilon x b x c.
• Blank first, measure at lambda max, keep readings in the linear range.
• Build a standard curve that brackets the unknowns; c = (A - intercept) / slope, then multiply by any dilution factor.

Try it

Open the Do tab and finish a Python standard curve. The fit is done for you with statistics.linear_regression; you write the line that turns an absorbance into a concentration.