The molar extinction coefficient calculator is utilized to determine the ability of a substance to absorb light at a specific wavelength, also referred to as the molar attenuation coefficient or molar absorptivity.
For instance, in protein analysis, the molar extinction coefficient at 280 nm is often used to estimate protein concentration without the need for time-consuming and destructive assays.
Consider a biochemist working with a novel protein. By using the molar extinction coefficient calculator, they can predict the protein’s absorbance based on its amino acid sequence, particularly the number of aromatic residues like tryptophan, tyrosine, and phenylalanine.
This prediction allows for rapid and non-destructive concentration measurements, which is invaluable in experimental design and quality control processes.
Molar Extinction Coefficient Calculator
| Sample | Concentration (M) | Absorbance | Path Length (cm) | ε (L mol⁻¹ cm⁻¹) | Conversion Equation |
|---|---|---|---|---|---|
| Protein X | 1.0 × 10⁻⁵ | 0.45 | 1 | 45,000 | ε = A / (c * l) |
| DNA Fragment | 2.0 × 10⁻⁶ | 0.132 | 1 | 66,000 | ε = A / (c * l) |
| Small Molecule Y | 5.0 × 10⁻⁴ | 0.75 | 0.5 | 3,000 | ε = A / (c * l) |
| RNA Sample | 1.5 × 10⁻⁶ | 0.0924 | 1 | 61,600 | ε = A / (c * l) |
| Peptide Z | 2.5 × 10⁻⁵ | 0.1875 | 1 | 7,500 | ε = A / (c * l) |
Molar Extinction Coefficient Formula
The molar extinction coefficient (ε) is mathematically expressed using the Beer-Lambert Law. The formula is:
A = ε c l
Where:
- A is the absorbance (dimensionless)
- ε is the molar extinction coefficient (L mol⁻¹ cm⁻¹)
- c is the concentration of the absorbing species (mol L⁻¹)
- l is the path length of the sample (cm)
If a solution of a compound has an absorbance of 0.5 at a concentration of 1.0 × 10⁻⁵ M and a path length of 1 cm, the molar extinction coefficient would be calculated as:
ε = A / (c l) = 0.5 / (1.0 × 10⁻⁵ M 1 cm) = 50,000 L mol⁻¹ cm⁻¹
This high value would indicate a strongly absorbing compound at the measured wavelength.
Molar Extinction Coefficient Units
| Unit | Description | Common Usage |
|---|---|---|
| L mol⁻¹ cm⁻¹ | Liters per mole per centimeter | Standard unit in biochemistry and chemistry |
| M⁻¹ cm⁻¹ | Inverse molar per centimeter | Equivalent to L mol⁻¹ cm⁻¹ |
| cm² mol⁻¹ | Square centimeters per mole | Used in some physical chemistry contexts |
| (μg/mL)⁻¹ cm⁻¹ | Inverse micrograms per milliliter per centimeter | Often used for nucleic acid quantification |
| mL mg⁻¹ cm⁻¹ | Milliliters per milligram per centimeter | Sometimes used in protein science |
How do you calculate the molar extinction coefficient?
Calculating the molar extinction coefficient involves several steps and requires careful measurement:
Prepare a series of solutions with known concentrations of the compound of interest.
Measure the absorbance of each solution at the desired wavelength using a spectrophotometer.
Plot absorbance vs. concentration on a graph. The resulting plot should be a straight line passing through the origin.
Calculate the slope of this line. The slope represents the product of the molar extinction coefficient and the path length (ε * l).
Divide the slope by the path length to obtain the molar extinction coefficient.
Let’s say we prepare solutions of a compound at concentrations of 1 × 10⁻⁵, 2 × 10⁻⁵, and 3 × 10⁻⁵ M. We measure their absorbances at 350 nm using a 1 cm cuvette and obtain values of 0.2, 0.4, and 0.6, respectively. Plotting these points and calculating the slope yields 20,000 cm⁻¹. Since the path length is 1 cm, the molar extinction coefficient is 20,000 L mol⁻¹ cm⁻¹.
This method ensures accuracy by using multiple data points and accounting for potential experimental errors through linear regression.
What is the molar extinction coefficient at 280 nm?
The molar extinction coefficient at 280 nm is particularly important in biochemistry and protein science. At this wavelength, aromatic amino acids (primarily tryptophan and tyrosine) absorb strongly, making it useful for estimating protein concentrations.
For pure proteins, the extinction coefficient at 280 nm can vary widely, typically ranging from about 5,000 to 300,000 L mol⁻¹ cm⁻¹, depending on the protein’s size and composition. This variation is due to the different numbers of aromatic amino acids present in different proteins.
For example:
- Bovine Serum Albumin (BSA) has an ε₂₈₀ of approximately 43,824 L mol⁻¹ cm⁻¹
- Lysozyme has an ε₂₈₀ of about 36,000 L mol⁻¹ cm⁻¹
- Immunoglobulin G (IgG) has an ε₂₈₀ around 210,000 L mol⁻¹ cm⁻¹
These values demonstrate the wide range of extinction coefficients possible at 280 nm, reflecting the diversity of protein structures and compositions.
What is the Molar Extinction Coefficient at 260 nm?
The molar extinction coefficient at 260 nm is primarily used for nucleic acid quantification, as DNA and RNA absorb strongly at this wavelength due to their nitrogenous bases. The extinction coefficient at 260 nm varies depending on the type of nucleic acid and its sequence.
For double-stranded DNA, a commonly used average extinction coefficient is 50 (μg/mL)⁻¹ cm⁻¹, which corresponds to approximately 6,600 L mol⁻¹ cm⁻¹ per nucleotide pair. However, this can vary based on the GC content of the DNA.
For RNA, the average extinction coefficient is slightly higher, around 40 (μg/mL)⁻¹ cm⁻¹, corresponding to about 7,700 L mol⁻¹ cm⁻¹ per nucleotide.
Single-stranded oligonucleotides have more variable extinction coefficients, strongly dependent on their specific sequence. Online calculators are often used to determine precise values for specific sequences.
How to calculate molar extinction coefficient from amino acid sequence?
Calculating the molar extinction coefficient from an amino acid sequence is a common practice in protein science. This method, known as the Edelhoch method, uses the following formula:
ε₂₈₀ = (nTrp × 5,500) + (nTyr × 1,490) + (nCys × 125)
Where:
- nTrp is the number of tryptophan residues
- nTyr is the number of tyrosine residues
- nCys is the number of cysteine residues
This formula assumes that the protein is denatured in 6 M guanidinium hydrochloride. For native proteins, the actual extinction coefficient may differ slightly due to the effects of protein folding on the local environment of the aromatic residues.
To use this method:
Obtain the complete amino acid sequence of the protein.
Count the number of tryptophan, tyrosine, and cysteine residues.
Apply the formula above.
For example, if a protein has 2 tryptophans, 3 tyrosines, and 1 cysteine, its estimated extinction coefficient would be:
ε₂₈₀ = (2 × 5,500) + (3 × 1,490) + (1 × 125) = 15,595 L mol⁻¹ cm⁻¹
This method provides a good approximation for most proteins and is widely used due to its simplicity and reliability.
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