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Chromatography Flashcards

1
Q

How does the salting in/out technique separate proteins?

A

-> Salting in: As salt is added, solubility increases at low [ion]

-> Salting Out: in (aq) conditions at high [ion] , reduce solubility
= causes certain proteins to ppt.

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2
Q

How can we identify the correct technique to separate a specific protein?

A

Combining two protein properties makes it possible to derive a purification process

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3
Q

Outline the process of column chromatography

A
  1. Sample mixture dissolved in solvent poured in top
  2. Component with greatest affinity to stationary phase
    takes longest to flow through
  3. Each component collected as effluent reaches bottom
    of column
    (fractionated molecules)
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4
Q

What is chromatography?

A

Laboratory technique for separating mixtures in which components partition between a moving phase and a stationary phase

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5
Q

Why is ion exchnage often carried out as the first separation technique?

A

IEX separates larger amounts of materials than gel filtration

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6
Q

How are proteins eluted off the cation exchanger to ensure fractions are separated?

A

Column is washed with buffer to elute low affinity proteins

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7
Q

Why do we ensure the first wash in affinity chromatography isn’t too extreme?

A

Can cause the isolate to dissociate from the ligand, contaminating effluent

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8
Q

What are the disadvantages of HPLC?

A
  • costly
  • complex
  • coelution
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9
Q

What are cation exchangers?

A

Acidic groups of resin that interact with positively (basic) charged proteins

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10
Q

How can elution be determined?

A

By [salt] of buffer or pH (stepwise/gradient elution)

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11
Q

In a chromatogram what does the area under the curve represent?

A

Area under curve = measure of [compound]

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12
Q

What stationary phase is used in IEX?

A

Stationary phase = polymer (matrix/resin) attached with charged groups

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13
Q

What is an elution volume (Vₑ)?

A

The volume of solvent required to elute a given solute from the column

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14
Q

What is the charge on the polymer beads in a cation exchanger?

A

Polymer beads have negatively charged functional groups

- (beads can be positively charged)

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15
Q

Which part of the purification process is GFC used?

A

GFC generally used near the end of the purification process

e.g. to separate correctly folded native protein from denatured protein

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16
Q

What biological molecules is affinity chromatography used to separate?

A

Antibodies
Antigens
Hormones
Other proteins

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17
Q

Why is chromatography significant?

A

It is important to isolate proteins in order to study their individual properties

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18
Q

What are the advantages of HPLC?

A
  • speed
  • high resolution
  • sensitivity
  • reproducible
  • accuracy
  • automation
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19
Q

How does GFC work?

A

Proteins separated based on size and shape
using a porous matrix to which molecules have different degrees of access
Larger molecules excluded form matrix first (elute faster)
Smaller molecules able to enter pores - take longer to elute

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20
Q

Which separation techniques are used to isolate proteins?

A

A number of separation methods are used in succession

- methods capable of using large quantities used first
- smaller quantity methods used last
e. g. mg

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21
Q

How is stepwise elution carried out?

A
  1. Protein mixture bound to upper most portion of ion
    exchanger in coulmn
  2. As elution progresses, various proteins separate into
    discrete bands due to their different affinities for the
    exchanger under the conditions
  3. [salt] in the elution is increased to increase the mobility
    • elute remaining bands
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22
Q

Why is it hard to observe the bands formed during HPLC?

A

Most compounds have no colour so can’t be seen by our eyes
The injected sample appears black to start off with and separates into yellow, red and
blue bands

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23
Q

How does ion exchange chromatography (IEX) separate proteins?

A

Proteins bind to an ion exchanger with different affinities
- the greater the binding affinity of a protein for the ion
exchanger column, the longer it takes to elute off the
column

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24
Q

How does HPLC enable a much better sample separation?

A
  1. solvent forced under 400 atm pressure through column

2. Allows use of smaller particles for column material (increases SA)

25
How does normal phase chromatography work?
Polar, solid stationary phase (e.g. Sillica) Less polar mobile phase - the more polar the analyte, the greater the retention time
26
How are molecules separated in affinity chromatography?
Molecules separated by taking advantage of their binding affinities for their respective ligands
27
What does a gel filtartion column look like?
Column consists of porous beads made of dextran or agarose
28
Outline the stages of HPLC
1. Reservoir hold solvent (mobile phase) 2. High pressure pump generates and monitors the specified flow rate of the mobile phase (mm/min) 3. Injector injects sample into the continuously flowing mobile phase stream 4. The mobile phase stream carries the sample into the HPLC column 5. Detector detects the separated compound bands as they elute from the column
29
How do we ensure all the unbound impurities are washed off in the first wash?
The first wash should have a sufficient [salt], pH or temperature in order to elute all the unbound impurities from the solution - need to be careful it's not too extreme
30
What are the various components of HPLC?
- reservoir - pump - mixer - injector - column - detector - fraction collector
31
What are the different separating techniques used to differentiate between proteins?
- Salting in/out - Chromatography (Column, Ion Exchange, Elution, Gel Filtration, Affinity, HPLC) - Electrophoresis (IEF) - Isoelectric Focusing (electrophoresis)
32
How is the elution of the hydrophobic bound proteins achieved?
By increasing [organic solvent], increases the retention time
33
How does the 2nd AC wash differ from the first?
Second wash needs to elute the isolate so must be significantly extreme in [ ], pH or temperature
34
Give an example of a cation exchanger
CM (carboxymethyl) cellulose formed via a reaction with alkali and chloroacetic acid
35
How do proteins interact with the reverse phase stationary phase?
All proteins contain hydrophobic and hydrophilic amino acids - those with high net hydrophobicity participate in hydrophobic interactions with the stationary phase
36
How does chromatography work?
Samples are dissolved in a mobile phase (gas/liquid) then forced through a stationary phase
37
How are proteins separated using 2DE and SDS?
1. Isoelectric focusing occurs 2. Isoelectric focusing gel placed on SDS polyacrylamide gel 3. SDS PAGE electrophoresis - protein subjected to electric current perpendicular to IEF
38
Why are proteins able to be separated using affinity chromatography?
Proteins can bind specific molecules very tightly but not covalently
39
What is HPLC?
High performance liquid chromatography
40
How does each technique differentiate between samples?
``` IEX - based on charge distribution GFC - separates on molecular size AC - binding properties IEF - based on pI HPLC - hydrophobicity and high pressure ```
41
How does Reverse phase chromatography separate proteins?
Separates molecules based on their hydrophobicity using a non polar stationary phase and a polar mobile phase Polar proteins elute first Non polar proteins bind to column - longer retention time
42
Outline the stages of affinity chromatography
1. Protein mixture added to column containing polymer bound ligand specific for protein of interest 2. Unwanted proteins are washed through column 3. Ligand solution poured in 4. Proteins of interest eluted by ligand solution
43
How is gradient elution used to increase the quality of separation?
The mobile phase is progressively changed This increases the [organic solvent] Causing a decreased retention time - hydrophobic proteins bind to hydrophobic stationary phase - hydrophillic proteins don't bind and wash out
44
How are proteins separated using a cation exchanger?
1. Protein mixture added to column containing cation exchangers 2. Proteins move through column - rate depends on net protein charges at the pH being used 3. Proteins with -ve charges move faster and elute earlier with cation exchangers
45
What is the reverse phase mobile and stationary phases composed of?
Stationary Phase packed with silica modified with silyl esthers containing non polar alkyl groups (c18 or c8) - creating a hydrophobic end Mobile phase contains organic solvents (e.g. methanol or acetonitrile)
46
What is a common use for gel filtration chromatography (GFC)?
Separating complex samples of biological molecules e.g. blood Can use GFC to determine molecular weight of molecules
47
How do we remove the protein of interest from the column?
Use a higher affinity for ligand (competitive) solution | Can alter pH, ionic strength and/or temperature to destabilise the protein-ligand complex
48
What are anion exchangers?
Basic groups of resin that interact with negatively charged (acidic) proteins
49
How does IEF + SDS-PAGE electrophoresis separate proteins?
2D gel electrophoresis (IEF) separation is based on osmotic potential Followed by SDS-PAGE which is based on protein size
50
What basis are samples separated on in GFC?
Based solely on molecular size
51
What components are required for affinity chromatography?
- beads matrix (agarose in column) - solution containing substance to be isolated - ligand (binds specifically to protein) - wash to elute non bound substances - final wash containing competitive ligand
52
What factors affect a proteins affinity for the stationary phase?
Proteins contain multiple charged groups which alter affinity Solubility effects affinity
53
What factor does Reverse Phase Chromatography use to separate molecules?
Reverse Phase Chromatography is separation based on protein solubility
54
Why is sillica a popular choice of material for the stationary phase?
Readily available Low cost OH- group on surface increases polarity
55
What is Reverse Phase chromatography?
The most widely used mode of high performace liquid chromatography (HPLC)
56
What does a proteins solubility depend on?
Solubility depends on: - [dissolved salts] - solvent polarity - pH - temperature
57
How is the affinity material prepared?
Inert support + spacer arms + ligand --> affinity material
58
How can proteins be selectively precipitated using salting in/out?
Can alter the type of salt and [salt] to precipitate certain molecules
59
Name an anion exchanger used in IEX
Diethylaminoethyl (DEAE) cellulose

FLC (34 decks)

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