analysis of proteins and peptides hplc column … choices... · page 2 outline of talk •...
TRANSCRIPT
HPLC Column Choices for the Analysis of Proteins and Peptides
Page 2
Outline of Talk
• Chromatographic methods for protein/peptide separations
• Ion exchange• Gel filtration• Reversed phase
• Method development for proteins• Optimizing resolution• Optimizing recovery
• Special Applications• High pH separations• High speed separations
Page 3
Chromatographic Methods for Peptide/Protein Separations
• Ion-Exchange Chromatography
• Reversed-Phase Chromatography
• Gel Filtration (SEC)
• IMAC (immobilized metal affinity chrom.)
• Affinity Chromatography
• Hydrophobic Interaction Chromatography (HIC)
• Charge
• Hydrophobic Interaction
• Molecular Size
• Non-specific affinity
• (Bio)specificity for a defined ligand
• Hydrophobic interaction under high salt conditions
Page 4
IMAC: Immobilized Metal Affinity Chromatography
Matrix with metal chelating group (e.g. iminodiacetic acid)
1. Binding of metal ions(Cu2+,Ni 2+, Zn 2+, Co 2+,Fe2+, Fe3+, Ca2+)
2. Loading/binding of peptides to free coordination spaces of metal ion
3. Elution with Imidazole gradient
Selection for His containing peptides
Application for His peptides(Cu2+) and Phosphopeptides(Fe3+)
N
NH
N
NH
O
NH2
OH
NH
O
NH2
OH
TrpONH2
OHSH
Cys
Page 5
Affinity Chromatography
Biospecific binding of immobilized ligand and binding partner
Peptide sample
Specifically bound molecules
Unbound peptides are washed off
Elution by competitive replacement, pH, or salt
Column material carries spacer with covalently attached ligand
Eluted peptides
Ligands can be antibodies, hormones, dyes, avidin, lectins...
http://www.chem.agilent.com/Scripts/PDS.asp?lPage=10784
Page 6
3
3
CH
CH3
Si - 0 - Si - (CH ) - CH2 3
Si - 0 - Si - (CH ) - CH
CH3
CH3
2 3
Si - 0 - Si - (CH ) - CH
CH3
CH3
2 3
Si - 0 - Si - (CH ) - CH
CH3
CH3
2 33
3
3
A mild method of protein separationwithout denaturation
ColumnsReversed-phase columns with verylow carbon loading of short chainbonded phases
Mobile PhasePure aqueous mobile phase withsalt (ammonium sulfate, lithiumsulfate, sodium perchlorate) gradient.
CO
=
N -
C
CO =
H
- N
CH2
H
N - C
OC=
- HH -
CH
2
CH
2
CH
2
CH 2
NH
2
H
O H
O H
Hydrophobic Interaction Chromatography (HIC)
Page 7
Separation of Protein Mixture Using Hydrophobic Interaction Chromatography (HIC)
Column: SynChropak H-PropylMobile Phase: A: 1M (NH4)2SO4, 0.02 M KH2PO4, pH 7
B: 0.01M (NH4)2SO4, 0.02 M KH2PO4, pH 7Gradient: 0 – 100% B in 30 min.Flow Rate: 1.0 mL/minSample: Proteins
1. RNA polymerase2. Ovalbumin3. Lysozyme4. α-Chymotrypsin5. Chymotrypsinogen
• HIC uses a decreasing salt gradient to release the proteins bound to the column.
Agilent no longer sells this column but does sell TSK HIC columns
TSKgel Ether-5PWTSKgel Phenyl-5PW
Page 8000855P1.PPT
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 andnumber 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 anionexchange 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 9
Cation Exchange Chromatography
-- -- -- --
----
- -- -Na+
Principle:competitive interaction of ions:charged sample molecule competes with salt ion about fixed charges of stationary phase
Cation exchange:stationary phase carries negative charge, analyzed peptide molecules are positively charged (at acidic pH)
Functional groups of column are:Sulfonic acid, sulfomethyl, sulfoethyl, sulfopropyl
Elution:by increasing salt concentration or pH change
++
+
+++
+
+
-- -- -- --
----
- -- -
Positively charged sample is loaded and bound to column
Elution with salt or pH
Fraction collection
Positively charged salt ions replace bound peptide molecules
Peptides are separated according to difference in their net charge
Na+Na+
Na +Cl-
Page 10
Use of Cation Exchange to Separate Basic Proteins
Column: SynChropak SCX, 4.6 x 100 mm, 6.5 µmMobile Phase: A: 0.02 M tris, pH 7
B: 0.02 M tris in 0.5M sodium acetate, pH 7Gradient: 0 – 100% B in 30 min.Flow Rate: 1.0 mL/minDetection: UV 254 nmSample: Basic proteins
1. RNA polymerase2. Chymotrypsinogen3. Lysozyme
Page 11
2-D HPLC: Cation Exchange and Reversed Phase Chromatography
Waste
Mass Spec
Data Analysis
SCX (SEC)
Peptides
RP
Protein mixture
Digest pH < 3
MS/MS Data
1) Load peptides on SCX at 0% salt
2) Elute w/ increments of salt (0.1 M - 1 M) using injector program
3) a. Collect fractions and re-inject on RP column (OFF-LINE approach)
orb. Inject directly on RP column
(ON-LINE approach)
2D approach results in more resolved peptides than either single dimension
ICAT
Eluted and separated peptides are directly analyzed by MS/MS
Page 12
Gel Filtration (Size Exclusion) Chromatography
1. big molecules cannot enter pores
fast elution
Separation according to size
Protein/peptide sample contains molecules of different size
2. Small molecules enter pores
later elution
Column contains porous particles
Particles act as molecular sieve
Page 13
Mechanism of SEC Separation – Pore Size Determines Linear Separation Range
• 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
FLOW
Page 14
SEC Applications with Proteins
• Impurity testing (separation of monomer/dimer/aggregates)• Molecular weight characterization – good MW accuracy (<2%)
and good precision (<2% RSD) over wide MW range (1000 –10M) possible
• Expression and folding studies
• Separation of reaction components and products (esp., antibodies, fragments, and conjugates)
• Purification
• Desalting and exchange of sample buffer• Collection of fractions under non-denaturing conditions
• Can be good first dimension for 2D separations
Page 15
SEC of Proteins on ZORBAX GF-250Separation of Albumin Monomer, Dimer and Aggregate
• ZORBAX GF-250/450 columns are specially treated to reduce protein sticking and to deliver long column lifetimes.
• Analyze proteins with molecular weights from 4,000 – 900,000.
Column: ZORBAX GF-250, 9.4 x 250 mmMobile Phase: 0.2M Sodium Phosphate, pH 7.0,
0.1% Sodium Azide, Detection: UV 280 nmSample: 1. Aggregate
2. Albumin dimer3. Albumin
DeLeenheer, A.P. et al. J. Pharm Sci., (1991) 80, 11.Reprinted with publisher's permission
1 2
3
Page 16
min0 5 10 15 20 25 30
mAU
0
100
200
300
400
• To achieve baseline resolution in SEC a molecular weight difference of 1.5 - 2 is needed • Sometimes mixed mechanisms affect the separation – i.e. hydrophobic interactions
SEC Separation of Proteins and Peptides
Sample:1. Thyroglobulin – 660K2. Gamma-Globulin – 150K3. BSA – 66K4. Ovalbumin – 45K5. Carbonic anhydrase – 29K6. Cytochrome C – 12.4K7. Insulin – 5.8K8. Lysozyme – 14.3K9. Tyrosine – 18010. Leucine-Enkephalin - 555
Column: ZORBAX GF-250, 9.4 x 250 mm Mobile Phase: 10 mM Sodium Phosphate, pH 7.4 + 0.4 M NaCl Sample: as listed Flow: 1 mL/min Temperature: Ambient Detection: UV 214,4 nm
Page 17
Column: ZORBAX GF-250, 9.4 x 250 mmMobile Phase: Sodium Phosphate, pH 7.0
concentration as indicatedSample: LysozymeFlow: 1 mL/min Temperature: Ambient
Effect of Mobile-Phase Ionic Strength on Elution of a Basic & Hydrophobic Protein
• Chromatograms were generated at each of the sodium phosphate concentrations indicated.
• Lysozyme (pI=10) increases in retention volume when the sodium phosphate concentration is very high or very low.
• Protein exhibits both basic and hydrophobic properties.
70 mM
100 mM
80 mM
200 mM
1000 mM
600 mM
Page 18
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Page 19
Reversed-Phase Chromatography
Fraction collection
Peptide sample retains on nonpolar hydrocarbon chains of column stationary phase
Elution by increasing hydrophobicity:
concentration of organic solvent
Reversed phase: historical term :“nonpolar hydrocarbon chains are attached to polar groups”
Stationary phase: silica gels with hydrocarbon chains between 1 and 18 carbon atoms (C1 to C18)
CH3
O-Si- (CH2)17-CH3
CH3CH3
O-Si- (CH2)17-CH3
CH3
For peptides: C18 phases are most popular
Mobile phase: organic solvent; ion-pair reagent
Water>Methanol>Acetonitrile>n-Propanol> THFElutropic force
Polarity
Organic solvent elutes peptide at hydrophobic interaction site
Page 20
Strategy for RP-HPLC Method Development of Proteins and Peptides
• Start at low pH (acidic mobile phase) and choose the initial column and conditions
• Initial selection parameters include: pore size (80 or 300Å), mobile phase, bonded phase, particle size, column length and internal diameter
• Obtain best resolution by optimizing:• Gradient steepness, bonded phase, temperature, column
configuration• Obtain best recovery by optimizing:
• Bonded phase, temperature, sample solubility• Evaluate alternate columns/technologies for improved selectivity and
efficiency• Try ZORBAX 300Extend-C18 at higher pH for different selectivity;
LC/MS • Try Poroshell 300SB phases for improved efficiency and shorter
analysis times; LC/MS
Page 21
Reversed Phase Columns for Separations of Proteins and Peptides
• Requirements • Wide pore - 300Å for
unrestricted access to bonded phase
• Multiple bonded phases for selectivity optimization
• Bonded phases with different pH ranges for LC/MS and method confirmation (WCBP)
• Many configurations for LC/MS compatibility, small sample sizes and proteomics
• Columns available• 300StableBond:
C18, C8, C3, CN
• 80A StableBond:C18, C8, Phenyl, CN, C3, AQ for peptides (< 4000-6000 Da)
• Poroshell 300SB – C18, C8, C3
• 300Extend-C18
• Configurations from nano (0.075 mm i.d.) to prep (21.2 mm i.d.)
Page 22
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
300SB-C18 (300Å)SB-C18 (80Å)
PW 1
/2Leu
Enkephali
n
M.W. =
556
Angiotensin
II
M.W. =
1046
Insulin
B
M.W. =
3,49
6
Cyt C
M.W. =
12,32
7
RNase
M.W. =
13,68
4Lys
ozyme
M.W. =
13,90
0
Wide Pore (300Å) Columns are Needed for Proteins and Polypeptides
• Proper pore size selection results in sharper peaks for large molecules
Effect of Pore Size and Molecular Size on Peak Width, Gradient Separations
Page 23
• 300SB• Four different bonded-
phases, 300SB-C18, C8, CN, and C3 for selectivity optimization
• Extremely stable at low pH• Use with TFA, Formate and
Acetate and ammonium acetate mobile phases
• Stable at high temperature – up to 80 - 90°C
ZORBAX 300SB and 300Extend-C18 Columns for the Analysis of Proteins and Peptides
• 300Extend-C18• Uses unique bidentate-C18
bonded phase for long lifetime at high pH
• Double endcapped• Can also be used at low pH • Ammonium hydroxide mobile
phase good for high pH LC and LC/MS for peptides only
O
R RSi
OH
Si
O
SiR R R
OOH
R
R1 R1 R1
SiO
SiO
Silica Support
C18C18
Silica Support
Page 24
Column: 4.6 x 150 mm, 5- or 3.5 µµµµm 300SB-C8 or 300SB-C3
Mobile Phase: A: 95:5, H2O : ACN with 0.1% TFAB: 5:95, H2O : ACN with 0.085% TFA
Flow Rate: 1 mL / min.Temp: 35 - 40°CInitial Gradient: 0 - 60% B in 60 min.
Initial Conditions for Separations of Peptides and Proteins on 300SB Columns
Page 25
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
Demonstration of ZORBAX StableBond Column Lifetime at Low pH
Page 26
0 10 20 30 40
100% B
min.
100% B
100% B
100% B
0% B
0% B
0% B
0% B
tG = 40
tG = 20
tG = 10
tG = 5
Optimize Resolution:Gradient Steepness and Retention
( k*)
Changing gradient time tG is the most common way to change gradient steepness.
Page 27
12,3
4 56
7 8
910
1 24 5
6,7
89
103
1 24 5
68
910
37
Retention Time (min)
0 5 10 15 20 25 30 35 40
1 24 5
68
910
37
Optimize Resolution - Separation of Polypeptides on 300SB Bonded Phases
Columns: ZORBAX StableBond 300SB4.6 x 150 mm, 5 µµµµm
Mobile Phase: Linear Gradient, 25- 70% B in 40 minA: 0.1% TFA in WaterB: 0.09% TFA in 80% ACN/20% water
Flow Rate: 1.0 mL/minTemperature:60°CSample: 3 µg each protein
1. RNase 6. CDR2. Insulin 7. Myoglobin3. Cytochrome C 8. Carbonic Anhydrase4. Lysozyme 9. S-100β5. Parvalbumin 10. S-100α
300SB-C18
300SB-C8
300SB-C3
300SB-CN
• Four different 300SB bonded phases with different alkyl chain length allow selectivity optimization for proteins.
Page 28
Conditions:Columns: ZORBAX 300SB, 4.6 x 150 mm, 5 µmMobile Phase: Gradient, 0 - 26% B in 30min.
A = 0.1% TFA in WaterB = 0.1% TFA in Acetonitrile
Temperature: 40°CSample: 2 µg of each peptideFlow Rate: 1.0 mL / min.Detection: UV-210nm
300SB-C18
300SB-C8
300SB-C3
300SB-CN
Optimize Resolution – Small Peptide Selectivity Comparison on 300SB Bonded Phases
Page 29
8. Myoglobin9. Calmodulin
10. CarbonicAnhydrase
Optimize Resolution: Change Temperature to Improve Resolution
Page 30
0 5 10 15 20 25 30 35 40 45 50
0 2 4 6 8 10 12 14 16 18 20 24 26 28 30
12.
16 22
0 1 2 4 5 6 8 9 1073
1 23 4 5 6 7
89 10
ZORBAX 300SB-C8 4.6 x 250 mm, 5 µm
Rapid Resolution 4.6 x 150 mm, 3.5 µm
Rapid Resolution 4.6 x 50 mm, 3.5 µm
40 min.
24 min.
9 min.
1. Met-enkephalin2. Leu-enkephalin3. Angiotensin II4. Neurotensin5. RNase6. Insulin (Bov)7. Lysozyme8. Calmodulin9. Myoglobin
10. CarbonicAnhydrase
Conditions:
Mobile Phase: A: 95:5, Water : ACN with 0.1% TFAB: 5:95, Water : ACN with 0.085% TFA
Flow: 1.0 mL/minDetection: 215nmSample: 1-10µg protein (10µL inj.) in
6M Guanidine HCL, pH7.010-60% B in 10 min.
10-60% B in 30 min.
10-60% B in 50 min.
Optimize Resolution: Column Length, Particle Size and Gradient Time
Page 31
Optimize Recovery
• Chain length – in general, choose C3 or C4 for proteins and C8 or C18 for peptides (but not always predictable)
• Temperature – higher temperature may improve recovery; be aware of analyte stability
• Hydrophobicity/aggregation – adjust sample solvent to reduce aggregation
• Mobile Phase – adjust acid component (pH)
• Solubility of sample – choose solvent for best solubility working from weakest to strongest
Page 32
50%
60%
70%
80%
90%
100%
110%
300SB-C8 300SB-C3 300SB-CN
ParvalbuminMyoglobinRNase AInsulinLysozymeCarbonic AnhydraseCalmodulin
Columns: 4.6 x 150 mmMobile Phase: 5 - 40% B in 20 min.
A: 0.1% TFA / WaterB: 0.1% TFA / ACN
Flow Rate: 1 mL / min.Temperature: 60°CSample: 4 µg each protein
25 µL injection
Rel
ativ
e R
ecov
ery
to 3
00S
B-C
18Recovery of Polypeptides from ZORBAX 300SB Columns
• Recovery may vary depending on bonded phase.• All 300SB bonded phase generally provide good recovery.
Page 33
Conditions:Column: ZORBAX 300SB, 4.6 x 150 mm, 5 µmMobile Phase: A – 0.1% TFA/water, B – 0.1% TFA/canGradient: 10 - 90 % B in 40 minFlow Rate: 1 mL/minTemperature: 60°C, Sample: 15 µL ( 15 µg ) of peptide in 0.1% TFA/DMSO
300SB-CN
300SB-C8
300SB-C18
Recovery 59%
Recovery 75%
Recovery 89%
Retention Time, (min.)
Effect of Bonded-Phase Ligand on Recovery of a Synthetic Lipopeptide
• Recovery on each bonded phase is not always predictable so evaluate different bonded phases.
Page 34
Column: ZORBAX 300SB-C18, 4.6 x 150 mm, 5 µm Mobile Phase: A- 0.1%TFA in water, B- 0.09%TFA in acetonitrileGradient: 20-45% B in 35 min Flow Rate: 1 mL/min Sample: 10 µl injection of 5 µg peptide in 6M Urea/5% HOAc
Abs
orba
nce
(210
nm
)
25°CßAP(1-38)
ßAP(1-43)* Recovery <10%
40°C
60°C
Time (min.)0
80°C
5 10 15 20 25 30 35 40
Recovery >70%
Optimize Recovery by Changing TemperatureßAP Peptide Separation
Page 35
Solvent Application Comments 0.05-5% TFA in Water General Effective solubilization of many
samples 6 M Guanidine, buffered at pH 6-8
General Very good for many proteins and peptides
5-80% Acetic Acid or Formic Acid; 0.1-0.5M Perchloric Acid
Peptides
Frequently used to extract peptides from tissues, precipitating many proteins and cellular debris
6 M Urea/ 5% Acetic aid Hydrophobic Peptides, Proteins
Useful for membrane proteins, fragments, aggregating systems
Water-Miscible Organic Solvents: Acetonitrile, Methanol, THF, Dioxane, DMSO; +/- TFA; +/- Water
Hydrophobic Peptides, Polypeptides
Limit injection volume to avoid problems; add water, as possible, to improve volume tolerance; acidify with TFA as required
Optimize SolubilitySelected Sample Solvents and Application forProteins and Peptides
Page 36
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Page 37
Evaluate High pH for Improved Selectivity and Resolution
• Changing pH of the mobile phase from low pH to high pH can change selectivity and retention – good for confirmation (orthogonal conditions)
• Special columns are required to work at high pH• ZORBAX 300Extend-C18 column can be used for
high, mid and low pH• Ideal for analysis of proteins and peptides at mid and
high pH• Ammonium hydroxide is ideal mobile phase for LC
and LC/MS (can use instead of TFA)
Page 38
Column: 4.6 x 150 mm, 5 or 3.5 µµµµm300Extend-C18
Mobile Phase: A: 20 mM NH4OH in waterB: 20 mM NH4OH in 80% ACN
Flow Rate: 1 mL / min.Temp: 25 - 30°CInitial Gradient: 5 - 60% B in 30 min.
Conditions for Separations of Proteins on ZORBAX 300Extend-C18 Columns at High pH
SiO
SiO
Silica Support
C18C18
Page 39
min0 2.5 5 7.5 10 12.50
1.0E6
2.0E6
3.0E6
4.0E6
5.0E6
0
1.0E7
2.0E7
3.0E7
4.0E7
5.0E7
12.0
50
13.2
61
min0 2.5 5 7.5 10 12.50
5.49
9
8.47
7
9.62
1
AII + AIII
AI
AI
AIIAIII
TIC (200-1500 m/z)
Column: ZORBAX Extend-C18, 2.1 x 150 mm, 5 µµµµm
Agilent 1100 MSD: Pos. Ion ESI
Vf 70V, Vcap 4.5 KvN2=35psi, 12L/min.325°C
Gradient: 15-50%B / 15 min.0.2 mL/min
Temp: 35°CSample: 2.5 µL
(50 pmol each)
Basic ConditionsA- 10 mM NH4OH in waterB- 10 mM NH4OH in 80%ACN
Acidic ConditionsA- 0.1% TFA in waterB- 0.085% TFA in 80%ACN
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
Angiotensin III: Arg Val Tyr Val His Pro PheCalc pI = 8.75
Angiotensin II: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe Calc pI = 6.74
Page 40
Column: ZORBAX 300Extend-C18 2.1 x 150 mm, 5 µµµµm
Flow Rate: 0.25 mL/min Sample: 5 µL sample
(100 pmol each)
Amyloid ß Sequences:Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His GlnLys Leu17 Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly AlaIle Ile Gly Leu Met Val Gly Gly38 Val Val40 Ile Ala42 Thr43-COOH
Abs
orba
nce
(210
nm
)
TFA Conditions, 25°CA- 0.1% TFA in waterB- 0.085% TFA in 80%ACN33-45%B in 30 min.
TFA Conditions, 80°CA- 0.1% TFA in waterB- 0.085% TFA in 80% ACN29-41%B in 30 min.
Aββββ(1-38)Aββββ(1-40)
Aββββ(1-42)Aββββ(1-43)
Aββββ(1-38) Aββββ(1-40)
Aββββ(1-43)
Aββββ(1-42)
Aββββ(1-42/3)Aββββ(1-40)
Aββββ(1-38)
NH4OH Conditions, 25°CA- 20 mM NH4OH in waterB- 20 mM NH4OH in 80%ACN26-38%B in 30 min.
• High pH and room temperature improve peak shape, recovery and change selectivity.
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
Page 41
Column: ZORBAX Extend-C184.6 x 150 mm, 5 µµµµm
Mobile phase:80% Methanol20% 20 mM NH4OH, pH 10.5
Flow Rate: 1.5 mL/min; Aging at 24°C, tests at 40°C
k Va
lues
0
1
2
3
4
5
6
7
8
9
10
1-Chloro-1-nitrobenzeneTrimipramine
Column Volumes of Mobile Phase
0 10000 20000 30000 40000 50000
Plat
e H
eigh
ts, c
m
0.001
0.002
0.003
0.004
0.005
1-Chloro-1-nitrobenzeneTrimipramine
ZORBAX Extend-C18 is Very Stable at High pHAging of Extend-C18 column in NH4OH at pH 10.5
• Consistent retention and plate heights over time indicate stable column at high pH.
• Long column lifetime achieved.
Page 42
High Sensitivity, High Resolution and High Speed LC and LC/MS of Proteins and Peptides
• Higher speed separations of proteins and peptides are possible
• High throughput and high efficiency protein applications require reduced analysis times
• Higher flow rates can be used to reduce analysis time
• Higher flow rates require improving mass transfer of proteins• High resolution and sensitivity with LC/MS requires the most
efficient peaks with MS-compatible solvents∴ New Poroshell technology makes this possible
Page 43
Porous Shell
SOLID CORE
5 mµ
Poroshell Particles Provide More Efficient Peaks for Peptides and Proteins
• Poroshell 300SB-C18 is for the separation of large peptides and proteins
• Poroshell particles have a solid silica core with a superficially porous surface with a 300Å pore size
• These particles allow more efficient mass transfer and narrower peaks for large proteins and peptides
Page 44
Comparison of Totally Porous and Poroshell 300SB-C18 at High and Low Flow Rates
min1 2 3 4 5
mAU
0
200
400
600
800
min10
0
2 3 4 5
mAU
0
200
400
600
800
min0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
mAU
0
200
400
600
800
min0.10
0
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
mAU
0
200
400
600
800
Flow = 0.5 mL/min(5 - 100 %B / 4 min)
Flow = 3.0 mL/min(5 - 100 %B / 0.67 min)
Totally PorousParticle309 bar
Totally PorousParticle309 bar
SuperficiallyPorous Particle222 bar
SuperficiallyPorous Particle222 bar
Agilent 1100 WPS with AutoBypassColumn: Poroshell 300SB-C18,
2.1 x 75 mm, 5 µµµµmMobile Phase:A= 95% H2O, 5% AcN with 0.1%TFAB= 5% H2O, 95% AcN, with 0.07%TFAGradient: as shownPiston Stroke: 20µLTemp.: 70°C, Det.: UV 215 nm
Page 45
min0 2 4 6 8 10 12 14 16 18
mAU
-1000
100200300400500
min*0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
mAU
-1000
100200300400500
(Poroshell chromatogram - expanded)
min*0 2 4 6 8 10 12 14 16 18
mAU
-1000
100200300400500
1\DATA\PORSHL3\PrSHL010.d\ßamy-ovr.win
Poroshell 300SB-C182.1x75 mm, 5 µm
Zorbax 300SB-C18 4.6x150 mm, 5 µm
F =1 mL/min, 5-100 %B / 20 min
ß-amylase
ß-amylase
ß-amylase
Poroshell chromatogramexpanded
• Poroshell technology facilitates ultra-fast HPLC analysis of proteins Note the similar separation of minor impurities in the 2 and 20 min separations.
12 min
2 min
12 min
0 1 1.4 1.80.6 0.80.40.2 1.2 1.6
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
F =2 mL/min, 5-100 %B / 1 min
Page 46
LC/MS of Proteins with PoroshellFormic acid Provides High Sensitivity
m/z1000 1100 1200 1300 1400 1500 1600 1700 1800 19000
10000
20000
30000
40000
50000
60000
1254
.4
1187
.312
31.2
1146
.4
1303
.612
78.7
1166
.5
1356
.7
1208
.8
1108
.211
26.9
1329
.7
1090
.0
1189
.612
11.9
1445
.2
1128
.7
1257
.3
1385
.5
1233
.2
1414
.4
1149
.1
1306
.8
1168
.4
1331
.8
1110
.3
1360
.0
1510
.915
45.8
1477
.214
48.7
1055
.5
1282
.0
1621
.3
1075
.2
1582
.6
1661
.817
04.1
1749
.1
1023
.3
1796
.7
m/z1000 1200 1400 1600 1800 20000
500
1000
1500
2000
2500
3000
3500
4000
1385
.2
1796
.5
1513
.7
1704
.2
1955
.1
1624
.3
1749
.1
1303
.6
1546
.2
1414
.4
1665
.6
1480
.6
1621
.815
86.3
1388
.213
59.6
1705
.9
2004
.7
1510
.7
1801
.0
1904
.5
2144
.621
71.8
2076
.9
1848
.8
1734
.8
1254
.4
min0.5 1 1.5 2 2.5 3 3.5
0
5000000
10000000
15000000
20000000
25000000
Abundance
Formic Acid
TFA
TIC
Column: Poroshell 300SB-C18, 2.1 x 75 mm, 5 µµµµm 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
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Conclusions
• Ion-exchange chromatography, size-exclusion chromatography and reversed-phase HPLC columns are the most popular choices for the analysis of proteins and peptides.
• Many column options are available for reversed-phase separations, including 300SB, 300Extend-C18 and Poroshell 300SB, providing many choices for optimizing resolution.
• Poroshell columns provide fast analysis or good compatibility with LC/MS and are ideal for the analysis of large polypeptides and proteins.
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