Upload
bonnie-golden
View
215
Download
0
Tags:
Embed Size (px)
Citation preview
Biotechnology Explorer™ | explorer.bio-rad.com2
Instructors - Bio-Rad Curriculum and Training Specialists
Sherri Andrews, Ph.D., Eastern US
Damon Tighe, Western US
Leigh Brown, M.A., Central US
Biotechnology Explorer™ | explorer.bio-rad.com3
Separation technology: How do you separate two or more compounds from each other?
Materials to be Separated Property Used to Separate Components
Explanation
Iron filings from other metals Magnetism Iron filings will be attracted to a magnet while other metals will not
Salt from sand Solubility Salt will dissolve in water and can be separated from the sand
Caffeine from coffee beans Solubility A solvent is used that selectively dissolves the caffeine (such as dichloromethane or ethyl acetate) but not most of the remaining coffee oils
Ethanol from fermented grain Boiling point Ethanol will boil off at a lower temperature than the other components of the fermented grain
Wheat from chaff Density Wheat kernels are more dense and will fall to the ground while chaff can be blow away by the wind
Tea from tea leaves Size Filtration can be used to separate the liquid extract from the large tea leaves
Biotechnology Explorer™ | explorer.bio-rad.com4
Combining Science, Engineering and Math to Develop a Separation Technology
Engineering – Design parameter based; no single “correct answer”
Science – Hypothesis driven; try to find specific cause/effect
Inexpensive separates well
Understanding the chemistry of mobile phase- Salt? Polar? Non-polar?
Best separation material?
Mea
sure
men
ts
An
alys
is
Biotechnology Explorer™ | explorer.bio-rad.com5
Chromatography
• Used to separate biomolecules based in their physical characteristics may include:• size• charge• hydrophobicity• interaction with other molecules
Biotechnology Explorer™ | explorer.bio-rad.com6
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)
Biotechnology Explorer™ | explorer.bio-rad.com7
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
Biotechnology Explorer™ | explorer.bio-rad.com8
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
Biotechnology Explorer™ | explorer.bio-rad.com9
What are some of the design factors we want to think about?
Type Mobile Phase
Stationary Phase
Column Type
Paper Water Paper type
Column 1X PBS HIC Media Syringe
Alcohol Cotton Commercial Column
Biotechnology Explorer™ | explorer.bio-rad.com10
Dye separation from Kool Aid Using Paper Chromatography
1. Choose your paper type (stationary phase)2. Cut it into .75 x 4 inch strips3. Place 50ul of Kool-Aid at one end using a
DPTP
Biotechnology Explorer™ | explorer.bio-rad.com11
4. Place 1 ml of your mobile phase into a medicine cup
5. Place the strip Kool-Aid side down into the mobile phase
Biotechnology Explorer™ | explorer.bio-rad.com12
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
Biotechnology Explorer™ | explorer.bio-rad.com13
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
Biotechnology Explorer™ | explorer.bio-rad.com14
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
Building and Using A Column to Separate Your Kool-Aid