Topic 1
Tools of the Biology Laboratory
Exercise I. The spectrophotometer.
Exercise II. Liquid Measurements.
Exercise III. Sedimentation and Centrifugation.
Bio 185 Labs: goal
When:
Self-paced:
Lab-Report:
Bio 185 Labs: set-up.
Exercise I.
The Spectrophotometer
Experiment 2: Absorption Spectra of Dyes.
Experiment 1: Concentration of an unknown Dye.
Exercise I. The Spectrophotometer
Spectrophotometry:
Spectrophotometry:
Exercise I. The Spectrophotometer
Exercise I. The Spectrophotometer
Exercise I. The Spectrophotometer
Exercise I. The Spectrophotometer
Exercise I. The Spectrophotometer
Spectrophotometry:
You need to “Blanc” the spectrophotometer for each solvent and
change of wavelength.
Exercise I: Experiment 2: Absorbance spectrum:
Absorbance spectrum:
Absorbance spectrum:
Exercise I. The Spectrophotometer: Absorption
spectrum.
1. Change wavelength.
2. Blanc.
3. Measure.
Exercise I. The Spectrophotometer: Absorption
spectrum.
What wavelength to determine the concentration of a solution?
Exercise I. The Spectrophotometer: Concentration
of an unknown dye.
Standards of known protein concentration
Optical density of
solutions with
unknown
concentration.
LAB REPORT FORMAT: (1 report per group!)
LAB TITLE: Concentration of a dye.
Experiment 1.
Goal: one sentence.
Method: Relevant.
Results: (based on class data). A (mean
and SEM) is significantly different
(P<0.05) from B (mean and SEM).
Conclusion: A is different from B.
Explanation.
Standard Curve Yellow Dye
0.9
0.8
Optical Density at 420 nm
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
1.5
2
2.5
3
Concentration (drops)
3.5
4
Figure 1. Standard curve of yellow dye at 420 nm. We measured
the optical density at 420 nm of four concentrations of yellow dye
(1, 2, 3, and 4 drops per sample) using a Spectronix 21
spectrophotometer. The OD of Standard 1 ((0.217 ± 0.0070) was
significantly smaller (P<0.05, Student’s t-test) than that of Standard
2 (0.410 ± 0.0057), the OD of Standard 2 was significantly smaller
(P<0.05) than that of Standard 3 (0.619 ± 0.0045) and the OD of
Standard 3 was significantly smaller (P<0.05) than that of Standard
4 (0.806 ± 0.0087). We calculated the standard curve (linear
regression y = 0.1977x + 0.0186, R2 = 0.9996).We measured the
optical density at 420 nm of an unknown concentration of yellow
dye (OD equals 0.699 ± 0.0054) and used the standard curve to
determine its concentration (3.44 drops per sample).
Exercise II.
Liquid Measurements
Accuracy of (1) 100 ml Graduated cylinder, (2) 10 ml
serological pipette, and (3) 1.0 ml micropipette.
Percent error when device is used at maximal and
1/10th maximal nominal volume.
Exercise II. Liquid Measurements
Measure by weight (mass).
Read mass in g.
Tare or zero the balance.
Add volume of water.
Measure.
What mass (g) do you
expect?
Exercise II. Liquid Measurements: 50 ml cylinder.
Exercise II. Liquid Measurements: 10 ml pipette.
Exercise II. Liquid Measurements
Exercise II. Liquid Measurements: 1.0 ml
micropipettor.
Exercise II. Liquid Measurements
Exercise II. Liquid Measurements
Accuracy of Volumetric Devices.
Percent Error
8
4
0
100 ml
10 ml
cylinder
10 ml
1 ml
serological pipet
Volumetric Devices
1 ml
0.1 ml
micropipetor
Exercise III. Sedimentation and Centrifugation.
Exercise III. Sedimentation and Centrifugation.
Exercise III. Sedimentation and Centrifugation.
Sedimentation at 1 g
Sedimentation in 50
ml conical tube at
slow centrifugation
Sedimentation in
microfuge tubes at
high speed
centrifugation
Exercise III. Sedimentation and Centrifugation.
Exercise III. Sedimentation and Centrifugation.
Exercise III. Sedimentation and Centrifugation.
Exercise II. Liquid Measurements
Exercise II. Liquid Measurements
Exercise II. Liquid Measurements
Exercise III. Sedimentation and Centrifugation.
Exercise III. Sedimentation and Centrifugation.
Microscope:
Exercise III. Sedimentation and Centrifugation.
Sedimentation at 1 g
Sedimentation in 50
ml conical tube at
slow centrifugation
Sedimentation in
microfuge tubes at
high speed
centrifugation