Absorbance and the Beer-Lambert Law
You have just extracted DNA from a mosquito. How do you know how much is there?
One could simply dry down the DNA extract and weigh it right? But the mosquito is full of all kinds of the
4 basic biomolecules (carbohydrates, nucleic acids, lipids and proteins). And try as one may, it is literally
impossible to remove all the contaminants in any DNA isolation. So that won’t work.
It was discorvered many years ago that chemicals literally absorb light differently. To understand how
that works, one must first understand the nature of light.
What is light?
Visible light literally means electromagnetic radiation that one can see. In fact phyiscist will tell you that
light moves like a wave. It has peaks and troths just like waves in water.
peak
troth
Figure 1- peaks and troths in waves
The distance between the peaks is known as the wavelength, which is represented by the and is
measured in nanometers (nm).
(nm)
Figure 2- One wavelength is shown above by the arrow and each wavelength is measured in
nanometers (nm)
Light in the visible spectrum is a composite of many different wavelengths of light, which a prism will
show you as different colors as shown below (violet to red). There are other types of electromagnetic
radiation as well, like ultraviolet light (uv).
The shorter the wavelength the greater the energy the electromagnetic radiation has, so gamma rays (
rays in Figure 3, note the arrow) is really high energy radiation. This is the one that could fry someone in
a nuclear reactor. The red light in the visible light spectrum has much lower energy and much longer
wavelengths.
Figure 3- Spectra of Electromagnetic Radiation
(http://en.wikipedia.org/wiki/File:EM_spectrum.svg)
It turns out that molecules can absorb light at different wavelengths which makes it possible to
determine if one type of molecule is in a large mixture of molecules. For example, proteins tend to
absorb light with a wavelength of 280 nm (or we would say “absorb light at 280”). On the other hand
nucleic acids absorb light at 260nm. You will notice that the visible spectrum above ranges from about
380 nm to roughly 700 nm. So 260 nm and 280 nm are ultraviolet light (uv). After all ultra means “more
than” and ultraviolet light has shorter wavelengths and thus more energy than violet light.
If only one could measure absorbance! Well one can measure the absorbance of a solution in a
spectrophotometer. In a nutshell, a spec (short for spectrophotometer) can filter light from a bulb to
give the required wavelength of light. It shines the light so it passes through the sample which is usually
in a cuvette. The molecules in the sample will absorb some of the light. The rest will pass through and
hit a detector which in turn will measure the amount of light hitting it and give you a unitless number
from 0.0 to 2.0 (see Figure 4).
Figure 4- Light in a Spectrophotometer
(http://www.bing.com/images/search?q=spectrophotometer+and+picture&view=detail&id=555BCD6EEFEB61293BED8E4268F6AD438AA34
78E&first=1)
Absorbance Spectra
If you graph the absorbance of a protein sample stimulated with different wavelengths of light one will
see the following type of graph.
A
240 250 260 270 280 290 300 310 320
Wavelength (nm)
Absorbance Maximum
at 280nm
B
240 250 260 270 280 290 300 310 320
Wavelength (nm)
Figure 5- A Typical Absorption Spectra of Protein and DNA- Note the maximum absorbance at 280 m
(Panel A) which indicates protein in the sample. In Panel B the peaks at 260 nm and 280 nm indicate
nucleic acids and proteins in the sample.
Using Absorbance to Measure Concentration of Chemicals
Many years ago two people Beer and Lambert determined that the concentration of a chemical can be
measured with absorbance. In fact they were directly proportional, meaning that if concentration says
doubles, the absorbance will also double. They described this relationship in the Beer Lambert Equation.
A = ebc
absorbance = molar extinction coefficient* x length of light path** x molar concentration
extinction coefficient units are L/mole cm
length of light path measured in cm
molar concentration (moles/L)
* n.b. The molar extinction coefficient indicates the ability of the molecule to absorb light at a certain wavelength.
One looks this number up.
**n.b. The length of the light path has a defined and measurable length measured in cm
What you need to pay attention to is that when
A
A
A increases c will increase the
same amount. If doubles so does C. If
increases 1.25 times, so does C, etc.
Bottom line, one can use a spec to measure the absorbance of a sample at 280
nm and 260 nm to estimate how much protein and nucleic acid is in a sample.
To do this for your mosquito DNA samples you will use a machine called a nanodrop. This very expensive
and delicate technology is a spec that can measure nucleic acid concentrations in very small volumes. In
fact only 0.5 to 1 ml is needed! That means you only need 0.0000005 to 0.000001 L! As you will find-out
your samples are precious. Sometimes one can only get a few ml of samples and they may even be one
of a kind! So they can be precious!
Arm
A
B
Figure 6- Pictures of a nanodrop- Panel A shows the arm (note the arrow). Panel B shows a sample
being pipette on the pedestal. (http://www.nanodrop.com/Productnd2000coverview.aspx)
Procedure to use the nanodrop
1. Blank the instrument.
 Even the liquid your sample is in (the solvent) absorbs light. One must tell the machine
not to detect those molecules
 If the DNA is dissolved in ultrapure water, then use that to blank the nanodrop.
 Gently lift the arm, place 1 l of solvent on the pedestal with a micropipetor and very
gently lower the arm.
 Click on the blank button on the computer.
 Click on the run button on the computer to ensure the result will read and absorbance
of zero.
2. Read the sample
 Gently lift the arm, place 1 l of the sample on the pedestal and very gently lower the
arm
 Click on the run button on the computer
3) The computer will show a value for concentration that is in ng/l.
4) It will also show a 260/280 ratio, which is the ratio of absorbances at those wavelengths.
Clean DNA will have a 260/280 ratio from 1.5 to 1.8.
5) The absorbance spectra will probably show at least 3 peaks. The one around 230 is for
contaminants from the DNA isolation or carbohydrates in the sample. Roughly speaking, the
peak at 260 nm (DNA) should be as large or at least ½ the size of the peak at 230 nm for the next
experiments to work.
It is important to realize that the Beer Lambert Law is used by the computer to calculate the amount
of DNA in the sample. The computer program has already been set with the path length for the
nanodrop and molar extinction coefficient for DNA.
Activity
Your instructor will introduce you to using the p20 micropipetor.
Practice taking a nanodrop reading for the 3 DNA samples provided.
Explain whether or not you think the samples will be good ones for our future work.
Data Table
Sample #
1
2
3
A 260
A 280
260/280
230/260
Explain why you think it will or will not
work?