Summary
Determines how much light of a given wavelength a sample absorbs, linked to concentration by the Beer-Lambert law.
In depth
Beer-Lambert law: A = ฮตcl. DNA is quantified at 260 nm, proteins at 280 nm; ratio 260/280 of about 1.8 indicates pure DNA. NADH absorbs at 340 nm so can be used to follow dehydrogenase reactions.
Absorbance is directly proportional to concentration within a limited range. Cuvettes must be clean. Dilute samples that are too concentrated.
Example: 260 nm absorbance measures DNA concentration.
A spectrophotometer passes light of a selected wavelength through a sample and measures absorbance (A) or transmittance. The Beer-Lambert law states A = epsilon x c x l, so absorbance is proportional to concentration (c) and path length (l), where epsilon is the molar absorptivity.
It is more flexible than a colorimeter, as scanning wavelengths gives an absorption spectrum. DNA and RNA absorb at 260 nm and proteins at 280 nm, so purity can be checked by A260/A280 (about 1.8 for pure DNA). It is used for enzyme kinetics, cell density (OD600) and pigment analysis.
Example: chlorophyll absorbs mostly blue and red light, shown by its spectrum.
Common mistake: reading values where absorbance is above about 1.5, where linearity fails.
Frequently asked questions
What is Spectrophotometry?
Determines how much light of a given wavelength a sample absorbs, linked to concentration by the Beer-Lambert law.
Which area of biology covers Spectrophotometry, and how advanced is it?
Spectrophotometry is one of the 51 topics in Lab Methods & Biostatistics. It is rated Intermediate, so it suits learners who already know the core vocabulary. Related topics in this area include Colorimetry, ELISA, Magnification and Resolution.
Can you give an example of Spectrophotometry?
Bacterial growth is monitored by optical density at 600 nm.
What should I learn before Spectrophotometry?
Work through the Beginner topics in Lab Methods & Biostatistics first, such as Scientific Method, Hypothesis, Variables, then return to Spectrophotometry. Intermediate topics assume the Beginner vocabulary.
What topics are related to Spectrophotometry?
The closest topics to Spectrophotometry are Colorimetry, ELISA, Magnification and Resolution, Hypothesis. Comparing them side by side is a good way to see how the ideas connect.
How can I check that I understand Spectrophotometry?
Read the summary and the in-depth section, then use the quick check on the interactive lesson for Spectrophotometry. To practise the whole of Lab Methods & Biostatistics, use the Lab Methods & Biostatistics quiz.
How is Spectrophotometry different from Colorimetry?
Spectrophotometry: Determines how much light of a given wavelength a sample absorbs, linked to concentration by the Beer-Lambert law. Colorimetry: Measures the colour intensity of a solution to estimate concentration. Read both side by side and note what each one explains.
What is a common mistake about Spectrophotometry?
Reading values where absorbance is above about 1.5, where linearity fails.
How should I study Spectrophotometry step by step?
First, read the summary and write the definition in your own words. Second, read the in-depth section and note any example. Third, compare it with Colorimetry and explain the difference aloud. Finally, take the Lab Methods & Biostatistics quiz and come back to this page for any question you miss.