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Column Choices For Proteins - Agilent
Column Choices For Proteins - Agilent
Page 2
Chromatographic Methods for Peptide/Protein
Separations
• Ion-Exchange • Charge
Chromatography
• Reversed-Phase • Hydrophobic Interaction
Chromatography
• Gel Filtration (SEC) • Molecular Size
Page 3
IMAC: Immobilized Metal Affinity Trp
Chromatography H2N O
NH2
O
HS OH HO
N
Cys H
O
2. Loading/binding of
peptides to free OH
N
coordination spaces Selection for His
H N
of metal ion N containing peptides H
3. Elution with N
Peptide sample
Column material
Unbound
carries spacer
peptides are
with covalently
washed off
attached ligand
Specifically
bound molecules
Elution by competitive
Ligands can be antibodies, replacement, pH, or salt
hormones, dyes, avidin, lectins...
Eluted
peptides
http://www.chem.agilent.com/Scripts/PDS.asp?lPage=10784
Page 5
Hydrophobic Interaction Chromatography (HIC)
CH3
A mild method of protein separation
Si - 0 - Si - (CH ) - CH 3
23
without denaturation
2
CH 3
2
CH
CH
2
2
NH
2
CH
CH
CH3
Si - 0 - Si - (CH ) - CH 3
23
Columns
-H
Reversed-phase columns with very
H
CH 3
-C
-
N
C
CH3
=
O
OH
low carbon loading of short chain
Si - 0 - Si - (CH2) - CH 3
bonded phases
O
=
3
C
CH
3
2
CH
H
CH
C
3
O Mobile Phase
N-
H
N
-
=
Si - 0 - Si - (CH ) - CH 3 OH H
23
C
CH 3 Pure aqueous mobile phase with
salt (ammonium sulfate, lithium
sulfate, sodium perchlorate) gradient.
Page 6
Separation of Protein Mixture Using
Hydrophobic Interaction Chromatography (HIC)
Agilent no longer sells this column
but does sell TSK HIC columns
TSKgel Ether-5PW
TSKgel Phenyl-5PW
Page 7
Ion Exchange Chromatography of Proteins
• Ion-exchange chromatography (IEC) discriminates between
proteins on the basis of accessible surface charges and their
corresponding electrostatic interaction with the column’s
stationary phase.
• The degree of protein retention is dependent on the strength and
number of interactions.
• The 3-D structure of the protein determines which surface
residues will be available to contact the column’s stationary
phase.
• The net charge determines the form of IEC (anion exchange or
cation exchange) to be applied.
• Cation exchange is used at pHs below a protein’s pI, while anion
exchange is used at pHs above a protein’s pI.
• Sample pI is a guideline and not absolute. Protein interaction
with a column’s stationary phase is dependent on the
microenvironment of the interaction site.
Page 8
000855P1.PPT
Cation Exchange Chromatography
+ +
Principle: +
+ +
competitive interaction of ions: + + + Positively
charged sample molecule competes charged sample
with salt ion about fixed charges of is loaded and
stationary phase - -- - -- bound to
-- - - column
Cation exchange: - - ---
stationary phase carries negative - Na +
charge, analyzed peptide molecules
Cl-
are positively charged (at acidic pH) Na+
Na+ Na+
Functional groups of column are: Positively - -- - -- Elution with salt or
Sulfonic acid, sulfomethyl, sulfoethyl, charged salt ions - - - - pH
sulfopropyl replace bound - - ---
peptide - Peptides are
separated
molecules
Elution: according to
by increasing salt concentration or Fraction difference in their
pH change collection net charge
Page 9
Use of Cation Exchange to Separate
Basic Proteins
Page 10
2-D HPLC: Cation Exchange and
Reversed Phase Chromatography
Digest pH < 3 SCX (SEC)
Waste
ICAT
1. big molecules
Protein/peptide sample cannot enter pores
contains molecules of
different size fast elution
Column contains
porous particles
Separation according to
size
Page 12
Mechanism of SEC Separation – Pore Size
Determines Linear Separation Range
FLOW
• Molecules are separated by size based on their ability to penetrate the pores of
the column support. Multiple columns can be put together with different pore
sizes to extend the separation range.
• Which molecules separate in the linear range depends on the pore size of the
packing
Page 13
SEC Applications with Proteins
Page 14
SEC of Proteins on ZORBAX GF-250
Separation of Albumin Monomer, Dimer and Aggregate
3
Column: ZORBAX GF-250, 9.4 x 250 mm
Mobile Phase: 0.2M Sodium Phosphate, pH 7.0,
0.1% Sodium Azide,
Detection: UV 280 nm
Sample: 1. Aggregate
2. Albumin dimer
3. Albumin
mAU Sample:
1. Thyroglobulin – 660K
2. Gamma-Globulin – 150K
400
3. BSA – 66K
4. Ovalbumin – 45K
5. Carbonic anhydrase – 29K
300
6. Cytochrome C – 12.4K
7. Insulin – 5.8K
20
8. Lysozyme – 14.3K
0 9. Tyrosine – 180
10. Leucine-Enkephalin - 555
100
0
0 5 10 15 20 25 30 min
70 mM
• Chromatograms were
generated at each of the 80 mM
sodium phosphate
concentrations indicated. 100 mM
Column: ZORBAX GF-250, 9.4 x 250 mm
• Lysozyme (pI=10) increases Mobile Phase: Sodium Phosphate, pH 7.0
in retention volume when the concentration as indicated
sodium phosphate Sample: Lysozyme
Flow: 1 mL/min
concentration is very high or Temperature: Ambient
very low. 200 mM
• Protein exhibits both basic
and hydrophobic properties.
600 mM
1000 mM
Page 17
Break Number 1
Page 18
Reversed-Phase Chromatography
Page 20
Reversed Phase Columns for Separations of
Proteins and Peptides
• Requirements • Columns available
• Wide pore - 300Å for • 300StableBond:
unrestricted access to C18, C8, C3, CN
bonded phase • 80A StableBond:
• Multiple bonded phases for C18, C8, Phenyl, CN, C3, AQ for
selectivity optimization peptides (< 4000-6000 Da)
Page 21
Wide Pore (300Å) Columns are Needed for
Proteins and Polypeptides
Effect of Pore Size and Molecular Size on Peak Width, Gradient Separations
0.2
0.12
0.1
0.08
0.06
0.04
0.02
0
6 n
46 II
1 3 me
3, B
55 ali
27
84
00
6
1 0 in
13 e
49
. = lin
.= tC
. = eph
. = ns
. = as
. = zy
,3
,6
,9
.W u
12
.W Cy
.W te
.W so
.W RN
M Ins
.W k
M g io
M En
M Ly
n
u
A
Le
Page 23
Initial Conditions for Separations of
Peptides and Proteins on 300SB Columns
Page 24
Demonstration of ZORBAX StableBond Column
Lifetime at Low pH
Column: ZORBAX 300SB-C8, 4.6 x 150mm Mobile Phase Gradient 0 - 60% in 120 min.A= 0.1% TFA in Water,
B= 0.086% TFA in ACN Temp.: 40°C Flow Rate: 1mL/min. Det. UV 210nm Sample: 50 µg of rhGH Tryptic Digest
After 495 mL
After 13680 mL
Page 25
100% B Optimize Resolution:
Gradient Steepness and Retention
tG = 5 ( k*)
0% B
100% B
tG = 10
0% B
100% B
tG = 40
0% B
0 10 20 30 min. 40
Page 26
Optimize Resolution - Separation of
Polypeptides on 300SB Bonded Phases
2,3
300SB-C18 1
4 5 7 8 Columns: ZORBAX StableBond 300SB
6 9
10 4.6 x 150 mm, 5 µm
Mobile Phase: Linear Gradient, 25- 70% B in 40 min
A: 0.1% TFA in Water
6,7 B: 0.09% TFA in 80% ACN/20% water
2
300SB-C8 1
4 5 8 Flow Rate: 1.0 mL/min
3 9
10 Temperature:60°C
Sample: 3 µg each protein
1. RNase 6. CDR
2. Insulin 7. Myoglobin
300SB-C3 1 2
4 5 7
8
3. Cytochrome C 8. Carbonic Anhydrase
3 6 4. Lysozyme 9. S-100β
9
10 5. Parvalbumin 10. S-100α
1 2 7 8
4 5
300SB-CN 3 6 9
10
0 5 10 15 20 25 30 35 40
Retention Time (min)
300SB-C18
Conditions:
Columns: ZORBAX 300SB, 4.6 x 150 mm, 5 µm
Mobile Phase: Gradient, 0 - 26% B in 30min.
A = 0.1% TFA in Water
300SB-C8 B = 0.1% TFA in Acetonitrile
Temperature: 40°C
Sample: 2 µg of each peptide
Flow Rate: 1.0 mL / min.
Detection: UV-210nm
300SB-C3
300SB-CN
Page 28
Optimize Resolution: Change Temperature to
Improve Resolution
8. Myoglobin
9. Calmodulin
10. Carbonic
Anhydrase
Page 29
Optimize Resolution: Column Length, Particle
Size and Gradient Time
1. Met-enkephalin
ZORBAX 300SB-C8 4.6 x 250 mm, 5 µm 2. Leu-enkephalin
10-60% B in 50 min.
3. Angiotensin II
1 2 6 7 40 min. 4. Neurotensin
5
3 4 9 10 5. RNase
8
6. Insulin (Bov)
0 5 10 15 20 25 30 35 40 45 7. Lysozyme
50
8. Calmodulin
Rapid Resolution 4.6 x 150 mm, 3.5 µm 9. Myoglobin
10-60% B in 30 min. 10. Carbonic
24 min. Anhydrase
Conditions:
Mobile Phase:
A: 95:5, Water : ACN with 0.1% TFA
0 2 4 6 8 10 12 14 16 18 20 22 24 26 B: 5:95,
28 Water30 : ACN with 0.085% TFA
. 6
1 2 1
9 min.
0 1 2 3 4 5 6 7 8 9 10
Page 30
Optimize Recovery
Page 31
Recovery of Polypeptides from
ZORBAX 300SB Columns
110%
Parvalbumin
Relative Recovery to 300SB-C18
Myoglobin
100%
RNase A
Insulin
90% Lysozyme
Carbonic Anhydrase
80% Calmodulin
Columns: 4.6 x 150 mm
70% Mobile Phase: 5 - 40% B in 20 min.
A: 0.1% TFA / Water
B: 0.1% TFA / ACN
60% Flow Rate: 1 mL / min.
Temperature: 60°C
Sample: 4 µg each protein
50% 25 µL injection
300SB-C8 300SB-C3 300SB-CN
ßAP(1-38) 25°C
ßAP(1-43)* Recovery <10%
Absorbance (210 nm)
40°C
60°C
80°C
Recovery >70%
0 5 10 15 20 25 30 35 40
Time (min.)
Page 34
Optimize Solubility
Selected Sample Solvents and Application for
Proteins and Peptides
Page 35
Break Number 2
Page 36
Evaluate High pH for Improved Selectivity
and Resolution
Page 37
Conditions for Separations of Proteins on ZORBAX
300Extend-C18 Columns at High pH
Si Si
O O
Silica Support
Page 38
Angiotensins Separation at High and
Low pH on Extend-C18 Angiotensin I: Asp Arg Val Tyr Val His Pro Phe His
Leu: Calc pI = 6.92
12.050
Angiotensin III: Arg Val Tyr Val His Pro Phe
4.0E6 Calc pI = 8.75
Acidic Conditions
AII + AIII
A- 0.1% TFA in water
3.0E6
13.261
B- 0.085% TFA in 80%ACN
2.0E6
AI
Column: ZORBAX Extend-C18,
1.0E6 2.1 x 150 mm, 5 µm
Agilent 1100
0 MSD: Pos. Ion ESI
0 2.5 5 7.5 10 12.5 min Vf 70V, Vcap 4.5 Kv
5.0E7
N2=35psi, 12L/min.
325°C
4.0E7
Gradient: 15-50%B / 15 min.
9.621
Basic Conditions 0.2 mL/min
A- 10 mM NH4OH
3.0E7in water Temp: 35°C
AIII
5.499
0
0 2.5 5 7.5 10 12.5 min
Page 39
High pH Can be Used for Separating Hydrophobic
or Other Low-Solubility Peptides
Comparison of Aß Peptide RP-HPLC Separations at Low and High pH
TFA Conditions, 25°C Column: ZORBAX 300Extend-C18
A- 0.1% TFA in water β (1-38)
Aβ 2.1 x 150 mm, 5 µm
B- 0.085% TFA in 80%ACN β (1-40)
Aβ Flow Rate: 0.25 mL/min
33-45%B in 30 min. β (1-42/3)
Aβ Sample: 5 µL sample
(100 pmol each)
Absorbance (210 nm)
β (1-43)
Aβ
• High pH and room temperature improve peak shape, recovery and change
selectivity.
Page 40
ZORBAX Extend-C18 is Very Stable at High pH
Aging of Extend-C18 column in NH4OH at pH 10.5
10
9 1-Chloro-1-nitrobenzene
Trimipramine
8
Column: ZORBAX Extend-C18
7 4.6 x 150 mm, 5 µm
6 Mobile phase:80% Methanol
k Values
0
0.005
1-Chloro-1-nitrobenzene
Trimipramine • Consistent retention and
0.004 plate heights over time
Plate Heights, cm
0.001
0 10000 20000 30000 40000 50000
Page 41
High Sensitivity, High Resolution and High Speed
LC and LC/MS of Proteins and Peptides
Page 42
Poroshell Particles Provide More Efficient Peaks
for Peptides and Proteins
Page 43
Comparison of Totally Porous and Poroshell
300SB-C18 at High and Low Flow Rates
mAU
800
Totally Porous Flow = 0.5 mL/min
600 Particle
400 (5 - 100 %B / 4 min)
200 309 bar
0
0 1 2 3 4 5 min
mAU
800
Superficially
600 Porous Particle
400
200
222 bar
0
0 1 2 3 4 5 min
mAU
Totally Porous
800
Particle Flow = 3.0 mL/min Agilent 1100 WPS with AutoBypass
600
400
309 bar
(5 - 100 %B / 0.67 min) Column: Poroshell 300SB-C18,
200 2.1 x 75 mm, 5 µm
0 Mobile Phase:
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 min A= 95% H2O, 5% AcN with 0.1%TFA
mAU
800
Superficially B= 5% H2O, 95% AcN, with
0.07%TFA
600
Porous Particle
Gradient: as shown
400
200
222 bar Piston Stroke: 20µL
0
Temp.: 70°C,
Det.: UV 215 nm
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 min
Page 44
Poroshell for Fast Analysis of High MW Proteins
- ß-amylase (200kdal)
Agilent 1100 WPS with AutoBypass; Piston Stroke: 20µL, Column: as shown Gradient: as shown Mobile Phase: A= 95% H2O, 5% ACN with
0.1%TFA; B= 5% H2O, 95% ACN, with 0.07%TFA Flow Rate: as shown Temp.: 70°C, Detection.: UV 215 nm
mAU
500 F =2 mL/min, 5-100 %B / 1 min
400
ß-amylase
300
200
100
0 12 min Poroshell 300SB-C18
-100
0 2 4 6 8 10 12 14 2.1x75
16 18, 5 µmmin
mm
mAU
500
Poroshell chromatogram
400 expanded
(Poroshell chromatogram - expanded)
300
ß-amylase
200
100
2 min
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
-100
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 min*
-100 12 min
0 2 4 6 8 10 12 14 4.6x150
16 18 , 5 µm
mm min*
Page 45
LC/MS of Proteins with Poroshell
Formic acid Provides High Sensitivity
Column: Poroshell 300SB-C18, 2.1 x 75 mm, 5 µm Mobile Phase Gradient: 20-100% B in 5.5 min. A: water + 0.1% TFA or FA B: ACN + 0.1% FA
or TFA Flow Rate: 500 µL/min Temperature: 60°C Injector: 1 µL Sample: 10 pmol of BSA Electrospray ionization: positive ion
Vcap:6000V Drying Gas: 12L/min 350ºC Nebulizer: 45 psi Scan: 600-2500 amu Step size:0.15 amu Peak width:0.06 min
1254.4
1187.3
1231.2
1146.4
1303.6
Abundance
Formic Acid
1278.7
1166.5
1356.7
1208.8
TIC
1108.2
60000
1128.7 1126.9
1331.8 1329.7
25000000 50000
1189.6
1090.0
1211.9
1445.2
40000
1233.2
1257.3
1385.5
1149.1
1414.4
1306.8
1168.4
1110.3
1360.0
1510.9
1545.8
1477.2
1448.7
1055.5
1282.0
30000
1621.3
1075.2
20000000
1582.6
1661.8
1704.1
1749.1
1023.3
1796.7
20000
10000
15000000 0
1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 m/z
1385.2
TFA
1796.5
4000
1513.7
1704.2
10000000
3500
1955.1
1621.8 1624.3
1749.1
1303.6
1546.2
3000
1414.4
1665.6
1480.6
2500
1586.3
1388.2
1359.6
1705.9
2004.7
1510.7
1801.0
1904.5
5000000
2144.6
2171.8
1848.8
2076.9
2000
1734.8
1254.4
1500
1000
0 500
0
0.5 1 1.5 2 2.5 3 3.5 min 1000 1200 1400 1600 1800 2000 m/z
Page 46
Conclusions
Page 47
Wrap-up e-Seminar Questions