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Analytical Techniques > Separation Science > Flashcards

Separation Science Flashcards

1
Q

Properties of proteins

A

Mass Charge – pH Hydrophobic/Hydrophilic Properties Differential Solubility Mobility in Applied Fields

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2
Q

Sep science

A
  • Centrifugation
  • Mass – acceleration
  • Density – mass to volume ratio
  • Dialysis
  • Mass transport
  • Size exclusion
  • Lyophilisation
  • Freeze-drying
  • Precipitation
  • Differential solubility
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3
Q

Centrifugation

A
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4
Q

Centrifuge g-value – Relative Centrifugal Force (RCF)

A

•The ‘g-force’ of a centrifuge is calculated from the following formula:

g=11.18 ×r × (n/1000)2

  • where g is the equivalent gravitational force (1 ×g = 9.8 ms-2)
  • ris the length of the rotor, cm
  • nis the number of revolutions per minute, RPM
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5
Q

Centrifugation field

A
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6
Q

Principles of centrifugation

A
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7
Q

Centrifuge classes

A
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8
Q

Differential centrifugation

A

•Differential Sedimentation (standard pelleting)

•

•Change the properties of the centrifugation medium to provide differential mobility of target proteins or organelles.

•

  • DensityGradientCentrifugation
  • RateZonal Centrifugation –eg. Sucrose
  • IsopycnicCentrifugation –eg. CsCl
  • Isopycnic– fluid of the same density
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9
Q

Preparation of the Density Gradient

A
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10
Q

Sucose density gradient

A
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11
Q

Isopycnic centrifugation

A

Isopycniccentrifugation of 15N

labelled community DNA in

CsCl/ethidiumbromide densitygradients.

A mixture of labelled(15N)

and unlabelled (14N) DNA,

extracted from Nitrosomonaseuropaea.

L, M, H in (A) indicate the positions of light,

medium, and heavy DNA bands, respectively

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12
Q

Gradient centrifugation

A
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13
Q

Sedimentation Coefficients

A
  • Depends on the Mass and shape
  • Transport property
  • Svedberg Unit (non-SI unit)
  • Molecule of 26 S will travel
  • 26 µm per second in 106g
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14
Q

Ribosome Subunits

A
  • Nobel Prize in Chemistry 2009
  • “for studies of the structure and function of the ribosome
  • The two eukaryotic ribosomal subunits have sedimentation coefficients of 40 x 10-13 and 60 x 10-13. As one Svedberg (S) unit is 10-13, the two ribosomal subunits are referred to as the 40S and the 60S ribosomal subunits.
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15
Q

Dialysis Membranes

A
  • Pore sizes in the membrane
  • Size separation
  • Porous to small molecules
  • Size cut-off
  • Semi-permeable membranes
  • Desalination
  • Chemical Potential
  • Partial Molar Free Energy
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16
Q

Dialysis

A
17
Q

Dialysis examples

A
  • Membranes–collodion, cellophane and cellulose. ‘Viskingtubing’. Needs to be boiled and treated with EDTA to remove contaminating materials eg. Heavy metals before use. Usual tubing 10-14,000 MW cut-off.
  • Ultrafiltrationdevicesuse a membrane (polycarbonate or cellulose esters) and rely on pressure or centrifugation to force liquid through.

These are used toconcentrate protein to smaller volume remove turbidity or insoluble material

18
Q

Lyophilisation - phase diagram

A
  • Freeze-Drying- removal of solvent from frozen sample.
  • Often used for storage of material.
  • Can cause protein denaturationand aggregation.
  • Good for temperature sensitive material.
  • Freeze sample and then put under a vacuum to remove water.
  • Need to control since if you remove all of the water from a protein it will denature.
  • Protein protectants– sugars polyols, glocols
19
Q

Separation by precipitation

A

•Precipitation with organic solvent:

  • Not used much today since it can result in protein denaturation.
  • Solvents used ethanol or acetone.
  • Principal effect is reduction of water activity.

•DNA Extraction:

•Base sequence is preserved

20
Q

Salting out

A
  • Salts compete for the solvation shell of the protein and destabilise solution
  • Multivalent cations
  • phosphate and sulfate
  • Usually potassium, sodium or ammonium.
  • Salt used most commonly is ammonium sulfate.
  • Needs to be ‘enzyme grade’ as often contaminated with heavy metals which interact with cysteine residues
21
Q

Solubility Stability

A

•Proteins are usually least soluble around their isoelectricpoints (electrically neutral)

•Kosmotropes and Chaotropes

  • Kosmotropes– order maker
  • Chaotropes– disorder maker
  • Entropy – fundamental measure of order and disorder
  • Small ionic species that cover the surface of a charged protein
22
Q

Kosmotropes and Chaotropes

A

Kosmotropes increase the interactions order within the water by increasing or making order

Chaotropesdecrease the interactions within the water decreasing the order

23
Q

Ammonium Sulfate Fractionation

A
  • Ammonium Sulphate Saturation Table
  • Quantities of ammonium sulphate required to reach given degrees of saturation at 20oC
  • Work out how much ammonium sulphate you need to add depending on your volume of extract. Add slowly while stirring on ice.
  • Centrifuge, separate pellet and supernatant and add further ammonium sulphate to supernatant to reach next required concentration and repeat above.
24
Q

Ammonium Sulphate Precipitation
Procedure

A
  • Take sample in a beaker containing a stir bar and place on magnetic stirrer
  • While sample is stirring, slowly add ammonium sulfateof a desired saturation level (Can refer ammonium sulphate precipitation chart)
  • Add ammonium sulfatevery slowly to ensure that local concentration around the site of addition does not exceed the desired salt concentration.
  • Once total volume of ammonium sulfateis added, move beaker to 4°C for 6 hours or overnight.
  • Collect the precipitate from the beaker and centrifuge the precipitate at 5000g for 20 minutes.
  • Carefully remove and discard supernatant. Invert the tube and drain well
  • Give two or three wash with distilled water
  • Dissolve the precipitate in phosphate buffer saline and dialyze protein solution at low temperature overnight to get removed salt
  • Determine the concentration and store at -10°C for long term storage.
25
Q

Ammonium SulfateFractionation

A

Number of g of Ammonium Sulphate

to be added to 1 L to produce a

change in % saturation

Top row – 0 – 65% requires 430g

33 – 70% requires 250 g

Analytical Techniques (6 decks)

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