CC 01 Lecture: SPECTROPHOTOMETRY

SPECTROPHOTOMETER

• Is a device that measures the concentration of an unknown solution by measuring the light absorbed and transmitted through a specific wavelength of light.

COMPONENTS OF SPECTROPHOTOMETER

1. sample compartment
2. digital read-out
3. mode indicators
4. mode selection
5. decrease
6. increase
7. print
8. wavelength control
9. transmittance/absorbance control (100%T 0A)
10. power switch Zero control
11. light source

Watch the video on how to use the spectrophotometer:

Spectrophotometer Operation
Video credit:meowmo3/YouTube


Spectrophotometer Operation
Video credit: Wendytrees/YouTube


PRINCIPLE

A source of light provides a continuous beam which when passed through a cuvette (tube containing the unknown solution), the intensity of light absorbed is detected and this absorbed light is inversely related to the amount of transmitted light.
This relationship is called Beer-Lambert's law

• the Spectronic 20 D is spectrophotometer used in this laboratory provides a direct reading of absorbance, so the concentration is proportional to the dial reading. Absorbance depends on ration, and therefore has no units. Absorbance is sometimes called OD or optical density

Procedure

1. check that the instrument is turned on. The left-hand knob should be turned clockwise. Allow 10 minutes for warming up
2. set the wavelength to the desired value using the knob on top
3. with empty, closed sample compartment, turn the left-hand knob to obtain a reading of 0%T
4. wipe the cuvette with dry tissue to remove drops of solution or finger prints
5. line up the mark on the cuvette with the line on the sample compartment
6. insert cuvette filled with the solvent in the sample compartment
7. close the cover
8. turn the right-hand knob to obtain a reading of 100%T
9. to analyze your sample, insert sample cuvette and read the absorbance value on the scale. Use the mirror behind the needle to avoid parallax error.

INTRINSIC PRECAUTIONS

• preparing the cuvette
1. never use a brush to clean the inside of the cuvette
2. fill the cuvette about ¾ full of the solution you wish o test
3. wipe the outside of the cuvette with a lint-free, soft tissue to remove any moisture or fingerprints from the outside surface

• calibrate the spectrophotometer
1. plug in and turn on the Spec. 20. It must warm up for at least 10 minutes to avoid fluctuations.

Beer Lambert's Law

Beer Lambert’s Law
Video credit: brightstorm2/YouTube

Enter the LINK below for more information.
'Spectrophotometer Simulation! link';
From: http://www.chm.davidson.edu/java/spec/spec.html