Spectrophotometry is a technique that measures how much light a substance absorbs at different wavelengths. Since many biomolecules absorb light in predictable ways, spectrophotometry is one of the most widely used tools in biochemistry labs .
Key insight: The amount of light absorbed is directly proportional to the concentration of the absorbing molecule. This allows us to quantify unknown concentrations by comparing to known standards .
The relationship between light absorption and concentration is described by the Beer-Lambert law:
Where:
Since path length is fixed (1 cm), and ε is known for many compounds, we can calculate concentration directly from absorbance:
Spectrophotometers measure the intensity of light before (I₀) and after (I) passing through a sample. Two related terms are used:
Absorbance is preferred because it's linearly related to concentration. Transmittance is exponential .
When ε is unknown, or when using colorimetric assays where the color intensity depends on reaction conditions, we use a standard curve.
To quantify unknown protein samples, you prepare BSA standards at 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL. After adding Bradford reagent and measuring A₅₉₅, you get a standard curve with equation y = 0.85x + 0.02 (R² = 0.99). An unknown sample gives A₅₉₅ = 0.45. What is its protein concentration?
Solution: 0.45 = 0.85x + 0.02 → x = (0.45 - 0.02)/0.85 = 0.51 mg/mL
Coomassie Brilliant Blue G-250 binds to proteins, shifting absorbance from 465 nm to 595 nm. Fast, sensitive, compatible with many reagents .
Concentrated H₂SO₄ hydrolyzes sugars to furfural derivatives, which react with phenol to form colored compounds. Measures total carbohydrates .
Phenols reduce phosphomolybdic/phosphotungstic acid in alkaline conditions, producing a blue color. Standardized with gallic acid .
Chlorophyll a and b absorb at specific wavelengths in 80% acetone. Equations allow calculation of concentrations .
2,6-dichlorophenolindophenol (DCPIP) reduction; color change from blue to colorless .
3,5-dinitrosalicylic acid reacts with reducing sugars to form colored compounds .
| Step | Procedure |
|---|---|
| 1 | Prepare BSA standards: 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL |
| 2 | Add 100 μL of each standard or unknown to labeled test tubes |
| 3 | Add 5 mL Bradford reagent to each tube, vortex |
| 4 | Incubate at room temperature for 5-10 minutes |
| 5 | Measure absorbance at 595 nm |
| 6 | Construct standard curve and calculate unknown concentrations |
Arnon's equations (in 80% acetone):
Chlorophyll a (mg/L) = 12.7 × A₆₆₃ - 2.69 × A₆₄₇
Chlorophyll b (mg/L) = 22.9 × A₆₄₇ - 4.68 × A₆₆₃
Total chlorophyll = 20.2 × A₆₄₇ + 8.02 × A₆₆₃
Procedure:
For accurate quantification, you should measure at the λmax (wavelength of maximum absorption). This provides:
To find λmax, you can perform a wavelength scan (if your instrument allows) or consult published data.
| Problem | Cause | Solution |
|---|---|---|
| Absorbance > 1.5 | Sample too concentrated; Beer-Lambert law may not hold | Dilute sample and repeat; ensure A < 1.0 for best accuracy |
| Negative absorbance | Blank has higher absorbance than sample | Check blank; might be contaminated or sample less than blank |
| Poor R² in standard curve | Pipetting errors, degraded standards, bubbles in cuvettes | Repeat with fresh standards; practice pipetting technique |
| Bubbles in cuvette | Scatter light, giving falsely high absorbance | Tap cuvette gently to remove bubbles |
| Cuvette orientation | Fingerprints or scratches on clear sides | Always handle cuvettes by the frosted sides; wipe clean with lens paper |
Key tip: Always zero the spectrophotometer with a blank between measurements, especially if you change wavelengths .
1. Beer-Lambert calculation: A solution of NADH has an absorbance of 0.45 at 340 nm in a 1 cm cuvette. The molar extinction coefficient of NADH at 340 nm is 6220 M⁻¹cm⁻¹. What is the concentration of NADH?
A = ε × c × l
0.45 = 6220 × c × 1
c = 0.45 / 6220 = 7.23 × 10⁻⁵ M = 72.3 μM
2. Standard curve: A BSA standard curve gives equation y = 0.85x + 0.02 (y = A₅₉₅, x = mg/mL). An unknown protein sample gives A₅₉₅ = 0.62. What is its concentration? If you diluted your sample 10-fold before assay, what was the original concentration?
0.62 = 0.85x + 0.02 → x = (0.62 - 0.02)/0.85 = 0.71 mg/mL (in cuvette)
Original concentration = 0.71 × 10 = 7.1 mg/mL
Many Ethiopian universities and research institutions have spectrophotometers, but they may be older models. Tips for success:
Researchers at an Ethiopian university are studying drought tolerance in teff. They need to measure chlorophyll content as an indicator of stress. Using a simple spectrophotometer and the acetone extraction method, they can quantify chlorophyll a and b. This data helps identify drought-tolerant varieties .
| Concept | Key points |
|---|---|
| Beer-Lambert Law | A = εcl; absorbance is directly proportional to concentration |
| Standard curves | Plot absorbance vs. concentration for known standards; use equation to find unknowns |
| Bradford assay | 595 nm; Coomassie dye binds protein; fast and sensitive |
| Phenol-sulfuric acid | 490 nm; measures total carbohydrates |
| Folin-Ciocalteu | 765 nm; measures total phenolics (gallic acid equivalents) |
| Chlorophyll | 647 nm and 663 nm; Arnon's equations for chl a and b |
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