Use of spectrophotometers                                    

Essentially, a spectrophotometer sends a  beam of light through your sample, and measures how much of it gets through (is transmitted).  This is a function of how much light is absorbed by the specific chemicals contained in the sample.  Spectrophotometers can read either "%T" (=percentage of light that is transmitted through the sample) or "Absorbance" (=the amount of light absorbed, expressed in arbitrary units called absorbance units, or optical density).  Several types of spectrophotometers are routinely used in biological laboratories.  This first is a relatively simple one also called a colorimeter, that measures only the visible range of light waves.  For many years the standard colorimeter used in teaching laboratories has been the Spectronic 20.  A newer version is the Spec 20 Genesys, described below; its improvements include digital instead of analog readout. 

Spectronic 20 Genesys Spectrophotometer™

When you turn on the Spectronic 20 Genesys instrument, it performs its automatic power-on sequence (check to be sure the cell holder is empty and its cover closed before turning on the instrument).  This includes a self-check of the software, and initializing the wavelength filter mechanism.  The sequence takes about 2 minutes to complete; do not interrupt during this sequence.  Allow the instrument to warm up for about 15-20 minutes before you are ready to use it.  When your samples and blanks (or calibration tubes) are ready, follow the steps below to operate the instrument.  

1.  Press 'A/T/C' to select the absorbance or % transmittance mode.  The selected mode will appear on the display.

2.  Press 'nm^' or 'nm" to select the wavelength specified in your exercise.  Note:  holding either key will cause the wavelength to change more rapidly than pressing many times.

3.  Insert your blank into the cell holder and close the sample door.  Be sure to position the cuvette so that the light passes through the clear walls from front to back.

4.  Press '0 ABS/100%T' to set the blank to 0 absorbance, or to 100% transmittance, depending upon which mode was selected in #1 above.

5.  Remove your blank and insert your sample into the cell holder.  The sample measurement appears on the LCD display.  Repeat as often as necessary to read the values for all of your samples, periodically returning to the blank (steps 3-4) to check that the calibration of the instrument remains stable.

6.  The Spectronic 20 Genesys may also be used to directly determine concentration using either a known factor or a freshly prepared standard curve.  See your instructor or the manufacturer's Operator's Manual for directions.  The "Print" option is only functional when the instrument is connected directly to an external printer.  Do not press buttons to access other functions of the spectrophotometer unless instructed to do so, including the Utilities option.
Note:  there are two arrow keys and two unlabeled keys below the LCD display.  These keys are used to select or access alternate functions available on certain menus.  Do  not select these keys unless instructed to do so.

7.  When you have completed all of your sample measurements, remove all samples from the spectrophotometer.  Rinse your cuvettes and leave to dry.  Always turn off and cover the instrument before leaving the laboratory.