STAM Winnipeg October 25, 2013

Inspire your students – tomorrow’s researchers

STEM Electrophoresis with Food Dyes

Bio-Rad (Canada) Life Science Education – Biotechnology Explorer Consultant

Robert Malyk BSc., MEd.

Senior Biology Teacher (retired)Ridley CollegeSt. Catharines, ON Canada

biobob4@hotmail.com

Combining Science, Engineering and Math to Develop a Separation Technology

Why Use Bio-Rad?

• Real research!!

• Guaranteed to work

• Easy to prep

• Cost Effective per student group

• Easy, inexpensive first exposure to electrophoresis

• Independent inquiry opportunities for what dyes are present in food items, what is the charge of the dyes, can they be separated based on charge/size?

• Engineering inquiry with STEM kit ranging from optimizing metal composition and diameter for electrodes, gel matrix composition, buffer composition, etc.

• Extensions including paper chromatography, spectroscopy, and researching food dyes commonly used

Separation technology: How do you separate two

or more compounds from each other?

Electricity can be used to separate molecules by charge

Sieves can be used to separate molecules by size

Electrophoresis separates molecules by CHARGE and SIZE

Electrophoresis means “to carry with electricity”

Food dyes –

Think about what you ate for breakfast or lunch…

Did you eat any cheddar cheese? Was it orange?

A Starbucks Strawberry Frapuccino?

Food dyes –

What flavor do you think this Skittle would have?

Food dyes –

We have been dying our foods for hundreds of years

In the early 1800s some cheese and cayenne pepper was colored with LEAD tetroxide

Pickles with COPPER sulfate

Green tea with COPPER carbonate

Food dyes –

Canada Food Inspection Agency

There was NO control or regulations on food dyes until the early 1900s

Food dyes –

Canada Food Inspection Agency

Permitted Natural Colours in Canada and Corresponding European Names

FD&C Dyes and Natural dyes FD&C Dyes

FD&C Red 40 or FD&C Red 40 LakeFD&C Yellow 6 or FD&C Yellow 6 LakeFD&C Yellow 5 or FD&C Yellow 5 LakeFD&C Blue 1 or FD&C Blue 1 LakeFD&C Blue 2 or FD&C Blue 2 LakeFD&C Red 3 or FD&C Red 3 LakeFD&C Green 3 or FD&C Green 3 Lake

Natural DyesCarminic acid, carmine, or cochineal

(from ground up beetle abdomens)Beetroot red or betanin (from beets)Curcumin (from tumeric)Caramel coloring (from sugar)Annatto (seeds of achiote trees)Lycopene (from tomatoes, watermelons,

papayas, and red carrots)

Food dyes in the news

Food dyes have an intrinsic SIZE and CHARGE and therefore can be separated using Electrophoresis

So how do we design an electrophoresis chamber to separate food dyes?

Dye Electrophoresis Commercial versus built box comparisons

What are some of the design factors we want to think about?

Dye extraction from candies

Building and running your electrophoresis system to separate the dyes

Building and running your electrophoresis system to separate the dyes

Building and running your electrophoresis system to separate the dyes

Building and running your electrophoresis system to separate the dyes

Building and running your electrophoresis system to separate the dyes

Extensions Paper chromatographySpectroscopyFood diaryElectrochemistry study

pH changes at the cathode and anode

Effect of material choice for electrode

Optimization of STEM box systemElectrode material choice

and thicknessDifferent materials for gel

thicknessImpact of TAE Concentration

and VolumeGel percentages

Copper electrode

Gelatin matrix

Other Activities - Chromatography

• Used to separate biomolecules based in their physical characteristics may include:

• Size

• Charge

• Hydrophobicity

• interaction with other molecules

The System

• All forms of chromatography have a

–Stationary phase (remains stationary and is a solid or a liquid supported by a solid)

–Mobile phase (a liquid or a gas that travels through the stationary phase and carries the parts of the mixture)

Types of Chromatography

• Paper

–Paper serves as the stationary phase. –The paper strip is placed in a solvent (mobile)

phase which carries the mixture through the paper.–Parts of the mixture will travel at different rates

separating the parts

• Column

–The stationary phase is packed into a column–The column is equilibrated (saturated with

solvent)–Sample is added to the column–Buffers are added to separate the mixture–Samples are collected in intervals called fractions

Be an Engineer

– Design your own!

• Select materials that you will use to separate your Kool Aid into the food dyes that make it the color it appears

• You will do this by

–Paper chromatography–Column chromatography

What are some of the design factors we want to think about?

Dye separation from Kool Aid Using Paper Chromatography 1. Choose your

paper type (stationary phase)

2. Cut it into .75 x 4 inch strips

3. Place 50ul of Kool-Aid at one end using a DPTP

Dye separation from Kool Aid Using Paper Chromatography 4. Place 1 ml of

your mobile phase into a medicine cup

5. Place the strip Kool-Aid side down into the mobile phase

Building and Using A Column to Separate Your Kool-Aid 1. Choose your

Column Type (HIC, Empty Column, Syringe)

2. If empty column or syringe add stationary phase by stuffing it with a cotton ball

Building and Using A Column to Separate Your Kool-Aid 3. Choose your

mobile phase (water, alcohol, or 1xPBS)

4. Place column in collection tube

5. Add 2 ml of mobile phase to column

6. Let it flow until it no longer drips

Building and Using A Column to Separate Your Kool-Aid 7. Move column to

next collection tube

8. Using a DPTP add 1ml of Kool-Aid to the top of the column

9. Let it flow until it no longer drips (this is fraction 1)

10. Move to the next column

11. Continue steps 5 and 6 until the eluate (fluid coming off the column) is clear

Compare Results

• Which paper, mobile phase worked best for paper chromatography?

• Which column, mobile phase worked best for column chromatography?