1. Purpose of the Lab 3E, 3F, 3H
Learn how to use a spec to figure out concentration of solutions
Learn how to use a GLX
Practice application of calculations
Practice graphing 3E 3F 3H
Practice correct waste disposal

2. SPECTROPHOTOMETERS
Are instruments that measure the interaction of light with materials in solution

3. INTERACTION OF LIGHT WITH MATERIALS IN SOLUTION
When light shines on a solution, it may pass through – be transmitted OR
Some or all of the light energy may be absorbed

4. Colorimeter Are a type of Spectrophotometer
Works on the basis of Beer-Lambert's law
Beer-lambert law states that the absorption of light transmitted through a medium is directly proportional to the concentration of the medium.
The % transmission or the % absorbance is recorded (you can use either).

5. Colorimeter
A colorimeter is an instrument which compares the amount of light getting through a solution with the amount which can get through a sample of pure solvent.
Pigments absorb light at different wavelengths.
A cloudy solution will simply scatter/block the passage of light such as monitoring the growth of a bacterial or yeast culture

6. BIOLOGICAL SOLUTIONS Usually appear clear to our eyes – have no color
DNA, RNA, most proteins do not absorb any visible light
Indicators are added to colorless solutions so that concentration can be checked without a UV spec.
But they do absorb UV light, so UV spectrophotometers are useful to biologists
Example, can use a detector that measures absorbance at 280 nm, or 254 nm to detect proteins

7. THE ABSORPTION OF LIGHT AND COLOR OF SOLUTIONS
WAVELENGTH OF LIGHT ABSORBED
COLOR OF LIGHT ABSORBED
COLOR OF SOLUTION
Violet
Yellow
Blue
Orange
Greenish Blue
Red-Orange
Bluish Green
Orange-Red
Green
Red
Yellowish Green
Violet-Red

8. How the Colorimeter Works
Light from a LED light source passes through a Cuvette containing a solution sample,
Some of the incoming light is absorbed by the solution.
As a result, light of a lower intensity strikes a photodiode.

9. Beer-Lambert Law and Specs
Detects the transmittance of a certain wavelength and then calculates the absorbance of light using the formula:
A = 2 – log%T

10. THE BLANK
Spectrophotometers compare the light transmitted through a sample to the light transmitted through a blank.
The blank is treated just like the sample
The blank contains everything except the analyte (the material of interest)
Contains solvent
Contains whatever reagents are added to the sample

11. Lab Reminders
Remember Copper Sulfate and Heavy medals must go in the waste container in the hood
Anything the contains gelatin, agar or agarose must never go down the sink

12. How does a spec work Using a Colorimeter