A spectrophotometer is a medical laboratory instrument that measures how much light a sample absorbs. This matters because many tests in medicine use color or light absorption to estimate the concentration of a substance in blood, urine, or another fluid. Examples include measuring glucose, hemoglobin, enzymes, proteins, and drug levels.
By turning light measurements into concentration data, the device helps clinicians diagnose disease and monitor treatment.
Key Facts
- Absorbance is defined as A = log10(I0 / I), where I0 is incident light intensity and I is transmitted light intensity.
- Beer-Lambert law: A = εlc, where ε is molar absorptivity, l is path length, and c is concentration.
- Higher concentration usually means higher absorbance if the sample follows Beer-Lambert law.
- A typical cuvette path length is l = 1.00 cm.
- Transmittance is T = I / I0, and percent transmittance is %T = 100T.
- A blank sample is used to set a baseline so the instrument measures the analyte rather than the solvent or cuvette.
Vocabulary
- Spectrophotometer
- A device that measures how much light a sample absorbs or transmits at selected wavelengths.
- Cuvette
- A small transparent container that holds the sample in the light path.
- Absorbance
- A logarithmic measure of how much light is absorbed by a sample.
- Wavelength
- The distance between matching points on a light wave, often measured in nanometers.
- Calibration curve
- A graph made from known standards that is used to find the concentration of an unknown sample.
Common Mistakes to Avoid
- Forgetting to blank the instrument first is wrong because the solvent, cuvette, and background light can add to the reading.
- Touching the clear sides of the cuvette is wrong because fingerprints can absorb or scatter light and change the measured intensity.
- Using the wrong wavelength is wrong because the sample may not absorb strongly there, making the result less sensitive and less accurate.
- Assuming absorbance is always linear at high concentration is wrong because very concentrated samples can exceed the useful range of Beer-Lambert law.
Practice Questions
- 1 A spectrophotometer measures I0 = 100 units and I = 25 units. Calculate the absorbance using A = log10(I0 / I).
- 2 A sample has absorbance A = 0.600 in a 1.00 cm cuvette. If ε = 1500 L mol^-1 cm^-1, calculate the concentration c using A = εlc.
- 3 A lab technician measures a colored medical sample without using a blank first. Explain how this could affect the reported concentration and why.