an introduction to the spectrophotometer. meet your spectrophotometer meet your spectrophotometer
TRANSCRIPT
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An Introduction to the Spectrophotometer
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Meet your SpectrophotometerMeet your spectrophotometer
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You willPractice Calibration
Calibration= assure equipment is accurate
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1. Set wavelength at 580nm
2. 0% T
3. 100% T
Calibration wavelength
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3. Pipet 4 ml of water into cuvette
4. Place cuvette into spectophotometer
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5. Close lid
6. Set transmittance to 100%
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• Step 1: Make a standard curve of known concentrations
Standard Curve
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A graph that allows the correlation between a qualitative measurement such as absorbance and known concentrations.
What is a standard curve?
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HOW TO PREPARE YOUR PRACTICE STANDARD CURVE
• How to use the “pipetman”
• How to make dilutions
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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
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HOW TO PREPARE YOUR PRACTICE STANDARD CURVE
Abs.
Concentration (independent)
(dependent)
What you manipulate
What you measure
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How to use your standard curve:
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(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
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Example of data & standard curve
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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
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(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
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ENZYME LAB
Next class:
Apply principles to
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Preview of things to come:Important Terms
• Enzyme
• Substrate
• Product
• Standard Curve
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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
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Substrate
Products
Enzyme Active Site
Enzyme
Binding of Substrate to Catalyst
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