Ion Exchange Laboratory

Today’s Schedule Pre-lab discussion Ion Exchange and Spectrophotometer Ion exchange experiment

Examples of Column Chromatography Ion Exchange Gel Filtration Affinity

Basis for molecular separation Ion Exchange Gel Filtration Affinity Charge Size Conformation

Components of ion exchange A charge solid phase or matrix Liquid phase contains molecules of different charges Solutions (eluant) of different charges to influence interactions between liquid and solid phases

Solid matrix exchangers 1. A cation exchanger: –Matrix negative charge –Exchanges cations 2. An anion exchanger: –Matrix positive charge –Exchanges anions

You will use ion exchange to separate a sample mixture : Separate dextran blue from cytochrome c

Dextran Blue: large non-ionic polysaccharide (500,000 MW) Cytochrome c: positively charge protein (12,400 MW) Which type of solid phase exchangers should you use to separate these two compounds from a mixture?

Cation Exchanger: Solid Matrix has a negative charge Cytochrome c is a cation and

Cytochrome c is bound to column Beads have a negative charge Cytochrome c: positive charge What happens to the dextran blue? How do I remove the cytochrome c from the bead?

Remove cytochrome c with liquid phase of increased ionic strength. Add a potassium acetate (0.5M) Potassium acetate is a positively charged “eluant” Dextran blue eluted earlier

Steps to separate the mixture : Separate dextran blue from cytochrome c –Add sample mixture –Add 0.01 M KOAc first –Remove (elute) dextran blue –Add 0.5M KOAc next –Remove (elute) cytochrome c

Preparing the column

Add the mixture and elute with potassium acetate

Analyze Fraction Spectrophotometer

Calibration Empty Pure water Zero transmission 100% transmittance

Calibration Spectrophotometer plug TATA Cuvette (requires 4 ml) Set: 100% transmission with cuvette + water Set: 0% transmission without cuvette

Cytochrome c standard curve: Dilute Standard curve –You must know how to make serial dilutions for Bio 22. Determine unknown concentration of cytochrome c

Standard Curve Obtain tube of 10mg/ml of pure cytchrome c Dilute (as outlined in your lab manual) Known cytochrome c concentrations Measure absorbance for known standards Measure absorbance of unknowns

Each group must have Sample mixture –(contains 4 mg/ml cytochrome c) Pure cytochrome c (10 mg/ml)

At bench Ion exchange column 0.01 M Potassium acetate 0.05 M Potassium acetate Spectrophotometer Water Pipets (I’ll review how to use them) Test tubes and racks should be at your lab bench

Ion exchange Hand in: due date 9/25: beginning of class Please hand in a neatly presented: 1. Table with absorbance and corresponding concentration values (construct your OWN table.) 2. A graph of your cytochrome c standard curve, be sure to label the axis 3. The concentration of your purified cytochrome c (mg/ml) 4. The volume (ml) of your purified cytochrome c 5. The total amount of cytochrome c purified (mg) (% recovery) Note: If you diluted your purified cytochrome c remember to use the dilution factor in your calculations

Let’s start (I have included some review slides about the spec. and standard curve)

FYI: Instrumentation: Review Spectrophotometer plug TATA 0 100

Calibration Spectrophotometer plug TATA Cuvette (requires 4 ml) Set: 100% transmission with cuvette + water Set: 0% transmission without cuvette

 A graph that allows a quantitative determination known concentration. What is a standard curve?

Why do we use standard curves? To obtain quantitative measurements In clinical settings –Measurement of blood hormones –Measurement of environmental carcinogens –Measurement of drugs –Measurement of antibodies (such as anti-HIV)

DNA ug/ml Absorbance 260 nm Example of Standard Curve Step 1: Data Collection of Known Values

(independent variable) (dependent variable) Absorbance (260nm) DNA (ug/ml) Step 2. Standard Curve

(independent variable) (dependent variable) Absorbance (280nm) DNA (ug/ml) Unknowns Prostate Tumor DNA Abs Normal Prostate DNA Abs X X X = unknown values Step 3. Use the curve to calculate unknowns

FYI: Ion Exchange Chromatography Generally speaking, a protein will bind to a cation exchange resin if the buffer pH is lower than the isoelectric point (pI) of the protein, and will bind to an anion exchange resin if the pH is higher than the pI.