Ion Exchange Chromatography

1
Ion Exchange Chromatography

• Ion exchange chromatography is the purification
  technique, which involves the separation of the
  proteins based on the ions exchange property
  between the proteins and the column

• Related LOs Column packing material, Retention
  and Elution time
• gt Prior Viewing IDD-6. Extraction of serum
  protein, IDD-41. Liquid chromatography - Gel
  filtration, IDD-42. Liquid chromatography -
  affinity chromatography
• gt Future Viewing IDD-36. Isobaric tag for
  relative and absolute quantitation (iTRAQ),
  IDD-37. Isotope-coded affinity tags (ICAT),
  IDD-39. LC-MSMS data analysis
• Course Name Ion exchange Chromatography
  
• Level(UG/PG) UG
• Author(s) Dinesh Raghu, Vinayak Pachapur
• Mentor Dr. Sanjeeva Srivastava

The contents in this ppt are licensed under
Creative Commons Attribution-NonCommercial-ShareAl
ike 2.5 India license
2
Learning objectives
1

• After interacting with this learning object, the
  learner will be able to
• Define buffer preparation
• Prepare ion exchange column
• Analyze the elution/purification of the sample
• Assess the troubleshooting steps involved in the
  experiments.

2
3
4
5
3
Master Layout
1
Sample preparation (Slide 5-11)
2
Resin/buffer preparation (Slide12-14)
3
Sample addition (Slide 15-17)
Elution (Slide 18-19)
4
UV-Visible spectrometry (Slide 20-26)
Analysis (Slide27-31)
5
Please animate the layout, by taking figures from
each of the steps
4
Definitions and Keywords
1
1. Stationary Phase The resins inside the column
which interacts with the protein of different
charge densities and enable it to separate . 2.
Mobile phase The buffers/ salt solution that is
used to elute the protein by interacting with the
resin and thereby free the proteins from the
column. 3. DEAE-Sepharose Diethyl amino ethyl
sepharose is an anion exchanger which interacts
more with cationic proteins. 4. CM-Cellulose
Carboxy methyl cellulose is an cation exchanger
which interacts more with anionic proteins.
2
3
4
5
5
Step 1
1
T1Reagents for Ion exchange chromatography
2
1000
500
250
3
100
The animator should draw graduated measuring
cylinder as shown in slide with graduation
100ml, 250 ml,500ml,1000ml. The user should
click on the appropriate cylinder for usage
Measuring cylinder are used to make up the
required final volume.
4
5
6
Step 1
1
T1Reagents for Ion exchange chromatography
2
3
Audio Narration (if any)?
Description of the action
Show a measuring balance, with display, ON, OFF
and TARE/0 buttons on it. let user ON it, display
reading as 0.000g, let user picks up the paper
from the rack, makes 1/10 of folding on the sides
and places it on the balance. Now the display
reading changes to 0.003g. Instruct user to TARE
the reading. And animate to click the tare
button. Once user clicks it, reading must show
0
When measuring with paper, the weight of the
paper need to be tarred from actual reading.
4
5
Video file Balancing
7
Step 1
1
T1Reagents for Ion exchange chromatography
Deionized Water
DEAE
2
CM-cellulose
Audio Narration
Description of the action
3
Show the bottle labeled as DEAE sepharose and CM
cellulose, deionized water. Let user pick up
the bottle labeled as DEAE-sepharose. Instruct
user to click on the bottle to open the lid of
the bottle and click on the spoon to take in hand
and add the DEAE from the bottle to the paper
show like gradual increase in the reading till it
weighs about 1gm. Follow the same procedure for
weighing CM Cellulose. Instruct the user to set
the pipette to 1000ul and open the lid of
deionized water bottle to pipette out 1000ul and
add to a empty DEAE containing tube. Repeat it
for one more time to make 2ml and repeat the same
for CM cellulose in another tube. Let user label
the tube accordingly. User must click on the
hands for the events to happen.
Prepare DEAE-sepharose and CM-cellulose resin.
DEAE is used to prepare the anion exchange column
while CM cellulose for cation exchange column.
4
5
8
Step 2
1
T1Reagents for Ion exchange chromatography
Monosodium phosphate
2
Disodium phosphate
Audio Narration
Description of the action
3
Instruct user to prepare 10mM phosphate buffer.
Let user pick up bottle Monosodium phosphate
and disodium phosphate, spatula, measuring
cylinder from the rack and keeps it on the table
next to balance. let user tare the balance, user
should click on the monosodium bottle, uncap it,
with help of spatula weigh the required amount on
a paper over the balance. Instruct user to weigh
0.04g of Monosodium phosphate and 0.18 g disodium
phosphate. Display a gradual increase in reading
with quantity addition. if the gram exceeds user
should remove some quantity or if it less add the
quantity to get the exact required amount.
Prepare 10mM phosphate buffer of pH7.2
4
5
9
Step 2
1
T1Reagents for Ion exchange chromatography
2
Audio Narration
Description of the action
3
Prepare 10mM phosphate buffer of pH7.2 , the
prepared buffer need to checked for pH to set it
at 7.2.
Instruct the user to click on the water bottle to
pour in the measuring cylinder till it reaches
80ml. add to the weighed phosphate bottle from
previous slide and then ask the user to take the
bottle for mixing on the magnetic stirrer (see
slide10 for proper animation instruction). Show
the clear solution, now instruct user to pour the
solution from bottle to the beaker. Let user
label the beaker accordingly as 10mM phosphate
buffer.
4
5
10
1
2
Beaker
Magnetic bead
3
Description of the action
Audio Narration (if any)?
Show magnetic stirrer instrument. Let user place
the beaker on it. Display the beaker containing
powder at bottom, liquid layer on top and a
magnetic bead at the bottom. Instruct user to ON
the instrument, let user control the speed knob
and regulate it accordingly to control the mixing
speed in the beaker. Animate powder getting into
the solution. Show a turbid solution turning
colorless
4
The magnetic stirrer is used for evenly
distribution of solute into the solvent.
5
Video file Magnetic stirrer
11
Step 3
1
T1Reagents for Ion exchange chromatography
Deionized Water
NACL
2
Audio Narration
Description of the action
Show the bottles labeled as Sodium Chloride,
deionized water. Let user pick up the bottle
labeled as Sodium chloride. Instruct the user to
click on the bottle to open the lid of the bottle
and click on the spoon to take in hand to add
sodium chloride from the bottle on to the paper.
show like gradual increase in the reading till it
weighs about 0.292gm. Follow the same procedure
for weighing1.421gm, 2.922gm of sodium chloride
and animate like putting them in separate beaker.
Instruct user to click on the phosphate buffer
(earlier prepared) to pour in the measuring
cylinder till it reaches 80ml and then ask the
user to place the bottle for magnetic stirrer
(see slide10 for proper animation instruction).
Show the clear solution after sometimes, now
instruct the user to pour the solution to the
measuring cylinder and show the volume till 90ml
and instruct user to add 10ml of phosphate buffer
to make the final volume to 100ml.
3
Prepare 50mM,250mM, 500mM of High salt buffer
solution.
4
5
12
Step 4
1
T2 pH meter standarization
2
STD 1
STD 2
3
Audio Narration
Description of the action
Display standard pH bottles and pH instrument and
deionized water, discard placed on a table.
Instruct user to calibrate the instrument. Let
user ON the instrument. Initially for the pH rod
is dipped in water, when user clicks on read
button, display must show a reading 7. Now show
like taking out the rod and washing it with
deionized-water, let user cleans the rod with
tissue. Now pick the STD-1, uncap it, dip the
cleaned rod into the solution, user must click
read button with display showing 4. now clean
the rod and repeat the step to note down the
reading for STD-2 and now the display should show
9
Before the pH reading, pH instrument need to be
calibrated with standards. Once with STD-1 at pH
4 and with STD-2 at pH 9.

4
5
Video file pH meter
13
Step 5
1
T2 pH measurement
2
NaOH
HCl
3
Audio Narration
Description of the action
Instruct user to set the pH for phosphate buffer
pH at 7.2. Now take the bottle labeled as
Phosphate buffer, uncap it, dip the cleaned pH
rod into the solution. User need to click on read
button. Initially display must show a reading 6.
now instruct user to add NaOH to adjust the pH.
Now allow the user to click on NaOH bottle so
that drops of NaOH should be added with filler,
user need to mix the solution with glass rod,
click on read button and the reading should show
anywhere near 6.1- 6.3. let user keeps adding the
NaOH drop till the pH display shows 7.2.
Prepare Phosphate buffer of pH 7.2 , buffers play
a very important role during equilibration step.
4
5
14
Step 6
1
T2 pH measurement
2
NaOH
HCl
3
Audio Narration
Description of the action

• Animate like the user transferring the beaker
  solution to 100ml measuring cylinder and reading
  should show 92ml.
• Instruct the user to click on the water and
  animate like the user pouring it to makeup the
  volume to 100ml. All action should happen when
  the user clicks the hand image.

Prepare Phosphate buffer of pH 7.2
4
5
15
Step 7
T3 Column Preparation?
1
CM
Column 2
DEAE
Column 1
2
3
Animate like the user tightening the knob at the
bottom in the column. The user must click on the
beaker labeled as DEAE and animate like the user
pouring the solution inside the tube. Show like
the particles are settling in the tube with water
at the top. Repeat the same for CM cellulose.
Animate as shown in the figure Let user click on
the hand for the things to happen Show the blue
ring with positive charge in column 1 and
negative charge in column 2
Pack the columns by DEAE and CM cellulose resins.
The packing is the main step which determines the
effective separation for the samples.
4
5
16
Step 8
1
T3 Column Preparation?
Buffer
Buffer
Buffer
2
Column
3
stopper
Instruct user to Equilibrate the column.
Animate like the user tightening the knob at the
bottom in the column. The user must click on the
beaker labeled as phosphate buffer and animate
like the user pouring the solution inside the
tube. Now instruct the user to click on hands to
place a beakers at the bottom of the columns and
open the stopper. Animate like the liquid comes
out of the tube in drop to the beaker and show
like closing the stopper and show a liquid layer
at the top of the column
Equilibrate the column using Phosphate buffer of
pH 7.2, which helps to attain a same condition
through out the column.
4
5
17
Step 9
1
T3 Sample Loading?
Buffer
2
Column
sample
3
stopper
Instruct user to take out sample stored at -80C.
animate user taking out the sample, thawing the
tube, animate frozen state to liquid state in
tube, now the user should take the pipette set to
200ul, pipette out the sample and add to the
column as shown in figure. Events must happen as
and when the user clicks on the pipette animate a
clock for 10 minutes
Load the sample to both anion exchange and cation
exchange columns to carry out separation.
4
5
18
Step 10
T4 Mechanism of separation
1
2
3
4
Column 2
Column 1
5
19
Step 10
T4 Mechanism of separation
1
2
Animate rings of different color with each ring
possesing positive or negative charges passing
through the column and bind to the
resin. Negative charge rings in the sample
should bind to resin in column1 and positive
charge to resin in column 2. Show like positive
charge ring unbinding to column 1 similarly
negative charge ring unbinding to column 2.
Cationic protein bind in CM-cellulose resin
while anionic protein bind to DEAE resin based on
the strength of charge interaction
3
4
5
20
Step 11
1
T5 Elution
50mM Nacl
MOBILE PHASE FUNNEL
2
3
4
5
21
Step 12
1
T5 Elution
Draw a mobile phase funnel as shown in previous
slide. Instruct the user to take the funnel and
place on the top of the column. Now show three
beakers labeled as 50mM NaCl, 250mM Nacl and
500mM NaCl. Now instruct the user to click on 50
mM NaCl solution beaker to pour into the funnel.
Pour the low ionic strength mobile phase through
the column for elution step.
2
3
4
5
22
Step 13
1
T5 Elution
2
3
4
5
23
Step 13
1
T5 Elution
The elution step helps for the collection of the
sample at different fractions.
Animate like the rings with the charges moving
down as in previous slide figure . Animate like
negative rings binding to the resin in column 1
while the positive rings flow through the
column. Similarly Animate like positive rings
binding to the resin in column 2 while the
negative ions rings flow through the column.
2
3
4
5
24
Step 14
1
T5 Elution
2
3
4
5
25
Step 15
)?
1
T5 Elution
2
3
4
collection tube
5
26
Step 14,15
?
1
T5 Elution
Now instruct the user to click on the knob at
side to open and show like the sample flowing out
into collection tube as shown in previous slide
Animate similarly from 20-26 with beaker
labeled as 250mM (in slide 20) Once this is done
again show the same animation now with the beaker
should be labeled as 500mM NaCl (in slide 20). So
the animation must be for 3 times starting with
500mM, 250mM and last with 500mM NaCl. When
adding the 250mM and 500mM solution show like the
movement of bound ion as in 22. Animate both
column 1 and column 2 separately as per the
instruction from 20-26 and follow the instruction
carefully in slide 23 and animate the ions
flowing accordingly
Collect the eluted sample at different fractions.
Proteins with low ionic interaction will be
eluted first and when the ionic strength is
increased proteins with high ionic interaction
will be eluted out later.
2
3
4
5
27
Step 16
1
T6 UV-visible spectrometry analysis
2
Cuvette
3
4
5
Video file UV spectrometry
28
Step 16
1
T6 UV-visible spectrometry analysis
Show a instrument labeled as UV visible
spectrometry and the samples in the stand as
shown in figure. Animate buttons like start,
auto zero, absorbance, stop on the
instrument Now instruct the user to switch on
the instrument, set the wavelength to 595nm by
pressing on numbers-open the lid of the
instrument and take a cuvette as in figure and
click on phosphate buffer to take it into cuvette
and animate like keeping it inside the instrument
to press auto zero. Display a value on the
system as 0.000 animate like the user opening
the lid to take the cuvette and discarding the
solution, now animate like the user taking the
sample-1 (collection tube-1) and adding it to the
cuvette, keeping it inside, closing the lid and
press absorbance. show the values as in next
slide for each sample follow the same for
(collection tube 2-15)
Detect the presence of protein of using the UV
visible spectrometry. The high absorbance
reading indicate the presence of protein.
2
3
4
5
29
Step 16
1

T6 UV-visible spectrometry analysis
Sample Volume of sample Absorbance
1 2ml 0.12
2 2ml 0.229
3 2ml 0.303
4 2ml 0.457
5 2ml 0.533
6 2ml 0.681
7 2ml 0.71
8 2ml 0.62
9 2ml 0.65
10 2ml 0.8
11 2ml 0.98
12 2ml 0.6
13 2ml 0.44
14 2ml 0.23
15 2ml 0.1
2
3
4
5
30
Step 17
1
T6 UV-visible spectrometry analysis
2
3
4
5
31
Step 17
1
T6 UV-visible spectrometry analysis
Draw the chromatrogram graph with absorbance at Y
axis and volume of eluent in x axis to determine
the protein using the literature or to know the
presence of protein. The sample collected can be
directly injected into the LC-MS analysis. For
further information please go through future
viewing IDD.
Animate like the user drawing the graph as shown
in previous slide with absorbance at Y axis and
volume of eluent in x axis
2
3
4
5
32
Slide 20-26
Slide 15-17
Slide 27-31
Slide 5-11
Slide 12-14
Slide 18,19
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage

• Animation area
• Interaction 1 slide-19 provide user sample
  mixture of both cation and anion proteins, ask
  for user input.
• Instruction user can start with any one column
  at first place, later by going through the
  absorbance reading user must make out the with
  high protein concentration the sample must
  undergo another step of column separation.

Interactivity area
Instructions/ Working area
Credits
33
Questionnaire
APPENDIX 1

• Question 1
• The cation exchange resin is
• CM cellulose
• DEAE
• Sepharose
• Cellulose
• Question 2
• The anion exchange resin is
• DEAE-sepharose
• CM-cellulose
• Cellulose
• Sepharose
• Question 3
• The stationary phase of the ion exchange
  chromatography is
• Nacl

34
Questionnaire
APPENDIX 1

• Question 4
• The mobile Phase in ion exchange chromatography
  is
• High salt phosphate buffer containing Nacl
• Phosphate buffer
• Resin
• Proteins
• Question 5
• What is the absorption wavelength of the proteins
  used
• 595nm
• 500nm
• 600nm
• 800nm

35
APPENDIX 2
Links for further reading

• Reference websites
• http//oscar.iitb.ac.in
• http//www.mnstate.edu/provost/ionexchangeprotocol
  .pdf
• http//sbio.uct.ac.za/Sbio/documentation/Ion_excha
  nge_chromatography.pdf

36
APPENDIX 3
Summary
The experiment mostly involves the sample
preparation, column preparation, buffer
preparation, elution buffer, followed by sample
addition to the column, separation and mobile
phase addition to the column in increasing
concentration followed by elution, collection of
sample for analysis and spectrometric analysis
and interpretation.