Appendix B

APPENDIX B: Use of the Spectronic20+ Spectrophotometer

An animation of how to use a Spec 29 can be found at: [| http://www.wellesley.edu/Biology/Concepts/Html/analogspec20instructions.html]

A diagram and general instructions for use of the Spectronic20+® (Spec20, informally) are provided. Please read the instructions before beginning the laboratory exercises. The meter on the Spec20 has 2 scales: a logarithmic scale for absorbance (A) and a linear one for percent transmittance (%T). We will use the %T scale to zero and to blank the instrument. We will use the A scale to take absorbance readings. Your instructor is available to answer any questions regarding the use of the spectrophotometer.

Figure 4. A Diagram of the Spectronic20+ Spectrophotometer.

1. Turn on instrument at least 30min before use with On-Off Knob (A).

2. Set the desired wavelength using Wavelength Selector (D).

3. Insure that the filter lever located at bottom front of instrument is in correct position for the wavelength that you have selected (to the left for wavelengths 340-599nm; to the right for wavelengths 600-950nm).

4. Use Zero-Adjust Knob (A) to set meter to 0%T.

5. Wipe outside of test tube that contains the blank solution with a Kimwipe, insert it into the Tube Holder (C), and close the lid.

6. Use 100% Adjust Knob (B) to set meter to 100%T, which is equivalent to zero absorbance on the absorbance scale. Remove the blank tube.

7. Wipe the outside of the test tube that contains the sample, insert it into the Tube Holder (C), and close the lid.

8. Record the absorbance reading from the absorbance scale, and remove the sample tube.

9. Repeat steps 7-8 for other samples to be read at the same wavelength (e.g., standard curve data). Repeat steps 2-8 for samples to be read at different wavelengths (e.g., absorption spectrum data).