Gel Permeation Chromatography

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Gel Permeation Chromatography

• GPC 1 Pre-lab lecture
• Lead TA Tom Gisel
• Gisel_at_wisc.edu

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• How does the column work
• Hydrodynamic radius (Stokes radius)
• Hydration
• Shape

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Definitions of Volumes
Vt
Vo
Vi
VG
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Distribution Coefficient

• Kd (Ve - Vo)/Vi
• Or since Vt Vi Vo Vgel
• Kd (Ve - Vo)/(Vt - Vo - Vgel)
• And if we assume that Vgel 0 we get
• Kav (Ve - Vo)/(Vt - Vo)
• (This is an approximation)

• Both are normally values between 0 and 1
• Molecules that are too big to penetrate any of
  the pores elute in Vo and give Kd 0
• Molecules that are small enough to penetrate all
  of the pores elute in Vi Vo and give Kd 1
• Between 0 and 1 larger molecules give smaller Kd
  values.
• If a molecule gives a Kd larger than 1 it is
  likely interacting with the gel beads themselves
  (adsorption).

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Practical Stuff

• Column packing
• Sephadex 75
• fractionation range of 3 to 80 kDa
• Column buffer Tris buffer at pH 8.2
• What we will be loading into the column
• Blue dextran
• Egg white
• Inorganic phosphate
• Phenol red

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Setting up the column

• Add 10 ml column buffer to column to check
  tubing and leakage
• gt what happens when liquid is inside an
  open-ended column

DO NOT RUN DRY!!
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Pouring the Column

• Get resin slurry from TAs and IMMEDIATELY pour
  down glass rod gently to prevent air bubbles

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Packing the Column

• Let resin form 1-2 cm bed and then start buffer
  flow. Adjust to 0.5-1 ml/min (no faster!).
• Collect buffer in a clean beaker to be reused
  later.

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Packing the Column

• The column height should be at least 25 cm at
  this point.
• Watch for any bubbles in resin before it settles
  all the way

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Adding Additional Resin if Needed

• - Gently make a vortex in buffer above resin to
  create a slurry
• Add extra resin down glass rod and let it
  compact
• Dont let the column run dry. EVER!

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Fraction Collector Calibration

• Measure volume of 150 drops to determine number
  of drops that will give 1.5 ml fractions

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Loading Sample Onto the Column

• Ooze sample on gently with pipet to avoid
  disturbing the resin
• Start collecting buffer in an empty beaker
  immediately

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Adding Buffer Head and Mariotte Flask

• Add 5 cm buffer head gently and then connect
  Mariotte flask to maintain buffer head
• Keep collecting buffer eluate in the beaker

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Determining the Void Volume with Blue Dextran

• Band will broaden as it filters down column
• Keep collecting buffer eluate in beaker until
  band nears the bottom

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Collecting Blue Dextran Fractions

• Start collecting fractions as Blue Dextran nears
  bottom of column
• Save eluant collected before first fraction and
  measure volume in graduated cylinder.
• Stop column flow once all Blue Dextran and TWO
  clear tubes have been collected

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While collecting Blue Dextran..

• Take 1 ml of egg white you got from buffet and
  save it in an Eppendorf tube.
• With remaining egg white prepare your loading
  sample.

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Before loading egg white sample..

• Reset the fraction collector
• Decide who is going to do egg white while the
  other person measures the absorbance of the Blue
  Dextran fractions
• Number ALL tubes to avoid mix-ups during analysis
  next week

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Clean-up

• Save all column fractions TAs will freeze them
  until next week.
• Dont forget to save 1 ml of your original egg
  white sample for assays next week! Keep this
  with your column fractions in an eppendorf tube.
• Transfer resin to large collection beaker with
  column buffer this will be used by other lab
  sections this week.
• Rinse gel resin off of ALL equipment with water
  dried Sephadex is hard to remove.

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Pre-Lab Question 1

• What we will be loading into the column
• Egg white (14.3 kDa)
• Blue dextran (2000 kDa)
• Inorganic phosphate (95 g/mol)
• Phenol red (354 g/mol)
• Given the above information and based on what you
  know about how size exclusion chromatography
  works what is the order of elution you expect

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Pre-Lab Question 2 3

• As I mentioned earlier the column can only
  resolve a molecular weight range of 3 to 80 kDa.
  Then why are we loading 2000 kDa Blue Dextran
  into the column
• What is the reason that we cannot mix Blue
  Dextran and lysozyme together to run in one
  sample

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References

• Jenna Eun GPC 1 Powerpoint presentation Fall
  2007 UW-Madison
• http//ecx.images-amazon.com/images/I/31DTpD59byL.
  _SL500_AA280_.jpg

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Appendix I

• Partial structure of Sephadex