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