Analysis of Proteins and Peptides HPLC Column … Choices... · Page 2 Outline of Talk •...

HPLC Column Choices for the Analysis of Proteins and Peptides

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

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

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

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

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)

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

000855P1.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.

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-

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

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

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

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

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

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

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

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

Break Number 1

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question into the

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during the presentation.

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

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

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.)

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

• 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

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

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

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.

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.

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

8. Myoglobin9. Calmodulin

10. CarbonicAnhydrase

Optimize Resolution: Change Temperature to Improve Resolution

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

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

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.

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.

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

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

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during the presentation.

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)

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

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

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

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.

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

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

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

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

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

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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Analysis of Proteins and Peptides HPLC Column … Choices... · Page 2 Outline of Talk •...

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