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

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

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

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).

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

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

THE ABSORPTION OF LIGHT AND COLOR OF SOLUTIONS WAVELENGTH OF LIGHT ABSORBED COLOR OF LIGHT ABSORBED COLOR OF SOLUTION 380-430 Violet Yellow 430-475 Blue Orange 475-495 Greenish Blue Red-Orange 495-505 Bluish Green Orange-Red 505-555 Green Red 555-575 Yellowish Green Violet-Red 575-600 600-650 650-780

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.

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

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

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

How does a spec work Using a Colorimeter