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An Introduction to the Spectrophotometer
Meet your SpectrophotometerMeet your spectrophotometer
You willPractice Calibration
Calibration= assure equipment is accurate
1. Set wavelength at 580nm
2. 0% T
3. 100% T
Calibration wavelength
3. Pipet 4 ml of water into cuvette
4. Place cuvette into spectophotometer
5. Close lid
6. Set transmittance to 100%
• Step 1: Make a standard curve of known concentrations
Standard Curve
A graph that allows the correlation between a qualitative measurement such as absorbance and known concentrations.
What is a standard curve?
HOW TO PREPARE YOUR PRACTICE STANDARD CURVE
• How to use the “pipetman”
• How to make dilutions
Push plunger to “first stop”Push plunger to “first stop”
Place tip in solutionPlace tip in solution
Aspirate sample by releasing plungerAspirate sample by releasing plunger
HOW TO PREPARE YOUR PRACTICE STANDARD CURVE
Abs.
Concentration (independent)
(dependent)
What you manipulate
What you measure
How to use your standard curve:
(independent variable)
(dep
end
ent variab
le)
Ab
sorban
ce
concentration
0
.2
.4
.6
.8
1.0
.2 .4 .6 .8 1.00
Unknowns
Abs. 0.77
Abs. 0.40
XX
X
Sample graph to calculate unknown concentrations
Example of data & standard curve
DNA ug/ml
0.00.20.40.60.81.0
Absorbance 260 nm
0.180.350.600.700.95
0.0
Sample DATA (example only!)
Concentration
(independent variable)
(dep
end
ent variab
le)
Ab
sorban
ce (260nm
)
DNA (ug/ml)
0
.2
.4
.6
.8
1.0
.2 .4 .6 .8 1.00
Sample graph from sample data
Low sensitivityValues>1.0Abs
ENZYME LAB
Next class:
Apply principles to
Preview of things to come:Important Terms
• Enzyme
• Substrate
• Product
• Standard Curve
Next Week Experiment: To study the affect of various parameters on enzyme activity by measuring product
formation
– A. p-nitroaniline Standard curve
– B. of substrate on product formation over time
– C. of temperature on product formation over time
– D. of pH on product formation over time
Substrate
Products
Enzyme Active Site
Enzyme
Binding of Substrate to Catalyst