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Spectrophotometry
Introduction
Introduction
What is spectrophotometry? (Why are some solutions coloured?)
Spectrophotometry in the lab
The Beer-Lambert Law
Introduction to the Beer-Lambert law
Deriving the Beer-Lambert Law Equation
The All Important Equation: A = ε c l
What is optical density (OD)?
What is the extinction coefficient?
The units of the extinction coefficient (ε)
When the Beer-Lambert Law does not apply?
Summary for this section
Test your understanding of the Beer-Lambert law
Standard Curve
Introduction
The Beer-Lambert Law
Setting up the standard curve in the lab
How to draw a standard curve
Deriving the extinction coefficient from a standard curve
Deriving unknown concentrations of samples from a standard curve
Standard curves: Concentration v. Amount
Direct v. Indirect measurements
Test your knowledge of standard curves
Direct and in-direct measurement assays
Introduction
The Beer-Lambert Law
Direct v. Indirect measurements
Advantages of Direct v. Indirect measurements
Disadvantages of Direct v. Indirect measurements
Why can't I extrapolate standard curve if the Beer-Lambert Law and I'm using an indirect assay?
Calculating Concentrations using the Beer-Lambert Law - worked examples
Introduction
Example 1: Absorbance 1, extinction coefficient of 0.1 mM
^{-1}
cm
^{-1}
Example 2: Absorbance 0.1, extinction coefficient of 100 M
^{-1}
cm
^{-1}
Example 3: Absorbance 0.1, extinction coefficient of 100 µg
^{-1}
ml cm
^{-1}
Example 4: Absorbance 0.125, extinction coefficient of 50 mg
^{-1}
ml cm
^{-1}
Test your knowledge of calculating concentrations using the Beer-Lambert Law
Calculating the Extinction Coeffecient using the Beer-Lambert Law - worked examples
Introduction
The Beer-Lambert Law
Calculating the extinction coefficient
Example 1: Concentration 0.1 M and Absorbance of 1
Example 2: Concentration of 100 mM, Absorbance 0.75
Example 3: Concentration 0.1 mg/ml, Absorbance of 1
Example 4: Concentration of 0.01 g/ml, Absorbance of 1
Summary
Test your understanding of calculation the extincting the coeffecient
When the Beer-Lambert Law does not apply?
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