An Introduction to the Spectrophotometer

Meet your Spectrophotometer Meet your spectrophotometer

You will Practice 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” Place tip in solution Aspirate 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) (dependent variable) Absorbance concentration Unknowns Abs Abs X X X Sample graph to calculate unknown concentrations

Example of data & standard curve

DNA ug/ml Absorbance 260 nm Sample DATA (example only!) Concentration

(independent variable) (dependent variable) Absorbance (260nm) DNA (ug/ml) Sample graph from sample data Low sensitivity Values>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