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

Page 2
Chromatographic Methods for Peptide/Protein
Separations

• Ion-Exchange • Charge
Chromatography
• Reversed-Phase • Hydrophobic Interaction
Chromatography
• Gel Filtration (SEC) • Molecular Size

• IMAC (immobilized metal • Non-specific affinity

affinity chrom.)
• Affinity Chromatography • (Bio)specificity for a defined
ligand
• Hydrophobic Interaction
Chromatography (HIC) • Hydrophobic interaction under
high salt conditions

Page 3
IMAC: Immobilized Metal Affinity Trp
Chromatography H2N O
NH2

O
HS OH HO
N
Cys H

Matrix with metal

1. Binding of metal chelating group
ions (e.g. iminodiacetic
(Cu2+,Ni 2+, Zn 2+, Co 2+, acid)
Fe2+, Fe3+, Ca2+) NH2

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

Imidazole gradient Application for His peptides

(Cu2+) and Phosphopeptides
(Fe3+)
Page 4
 Affinity Chromatography
Biospecific binding of immobilized ligand and binding partner

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

Column: SynChropak H-Propyl

Mobile Phase: A: 1M (NH4)2SO4, 0.02 M KH2PO4, pH 7
B: 0.01M (NH4)2SO4, 0.02 M KH2PO4, pH 7
Gradient: 0 – 100% B in 30 min.
Flow Rate: 1.0 mL/min
Sample: Proteins
1. RNA polymerase
2. Ovalbumin
3. Lysozyme
4. α-Chymotrypsin
5. Chymotrypsinogen

TSKgel Ether-5PW
TSKgel Phenyl-5PW

• HIC uses a decreasing salt gradient to release the proteins bound to

the column.

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

Column: SynChropak SCX, 4.6 x 100 mm, 6.5 µm

Mobile Phase: A: 0.02 M tris, pH 7
B: 0.02 M tris in 0.5M sodium acetate, pH 7
Gradient: 0 – 100% B in 30 min.
Flow Rate: 1.0 mL/min
Detection: UV 254 nm
Sample: Basic proteins
1. RNA polymerase
2. Chymotrypsinogen
3. Lysozyme

Page 10
 2-D HPLC: Cation Exchange and
Reversed Phase Chromatography
Digest pH < 3 SCX (SEC)
Waste
ICAT

Protein mixture Peptides

1) Load peptides on SCX at 0% salt
2) Elute w/ increments of salt (0.1 M - 1 M) using
injector program
RP

3) a. Collect fractions and re-inject on RP

column (OFF-LINE approach)
or
b. Inject directly on RP column
(ON-LINE approach) Mass Spec
2D approach results in more resolved MS/MS Data
peptides than either single dimension
Eluted and separated peptides
Data Analysis are directly analyzed by MS/MS
Page 11
Gel Filtration (Size Exclusion) Chromatography

1. big molecules
Protein/peptide sample cannot enter pores
contains molecules of
different size fast elution

Column contains
porous particles

Particles act as 2. Small molecules

molecular sieve enter pores
later elution

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

• 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 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

DeLeenheer, A.P. et al. J. Pharm Sci., (1991) 80, 11.

Reprinted with publisher's permission
1 2

• 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.
Page 15
 SEC Separation of Proteins and Peptides
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

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

• 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
Page 16
Effect of Mobile-Phase Ionic Strength on Elution
of a Basic & Hydrophobic Protein

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

Please type your

question into the
Chat Box at any time
during the presentation.

Page 18
 Reversed-Phase Chromatography

Reversed phase: historical term :“nonpolar Peptide sample

hydrocarbon chains are attached to polar groups” retains on nonpolar
hydrocarbon chains
Stationary phase: silica gels with hydrocarbon of column stationary
chains between 1 and 18 carbon atoms (C1 to C18) phase

CH3 Elution by increasing

For peptides: C18 phases
O-Si- (CH2)17-CH3 hydrophobicity:
are most popular
CH3 concentration of
CH3 organic solvent
Mobile phase: organic O-Si- (CH2)17-CH3
solvent; ion-pair reagent CH3 Organic solvent
elutes peptide at
hydrophobic
Elutropic force interaction site
Water>Methanol>Acetonitrile>n-Propanol> THF
Polarity
Fraction collection
Page 19
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 20
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 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

0.18 300SB-C18 (300Å)

0.16 SB-C18 (80Å)
0.14
PW 1/2

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

• Proper pore size selection results in

sharper peaks for large molecules Page 22
ZORBAX 300SB and 300Extend-C18 Columns
for the Analysis of Proteins and Peptides
• 300SB • 300Extend-C18
• Four different bonded- • Uses unique bidentate-C18
phases, 300SB-C18, C8, CN, bonded phase for long
and C3 for selectivity lifetime at high pH
optimization • Double endcapped
• Extremely stable at low pH • Can also be used at low pH
• Use with TFA, Formate and • Ammonium hydroxide mobile
Acetate and ammonium phase good for high pH LC
acetate mobile phases and LC/MS for peptides only
• Stable at high temperature
– up to 80 - 90°C
R1 R1 R1
C18 C18
R Si R R Si R R Si R
Si Si
OH OH
O
O O O O

Silica Support Silica Support

Page 23
Initial Conditions for Separations of
Peptides and Proteins on 300SB Columns

Column: 4.6 x 150 mm, 5- or 3.5 µm 300SB-C8

or 300SB-C3
Mobile Phase: A: 95:5, H2O : ACN with 0.1% TFA
B: 5:95, H2O : ACN with 0.085% TFA
Flow Rate: 1 mL / min.
Temp: 35 - 40°C
Initial Gradient: 0 - 60% B in 60 min.

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

Changing gradient time tG is the

tG = 20 most common way to change
gradient steepness.
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)

• Four different 300SB bonded phases with different alkyl chain

length allow selectivity optimization for proteins.
Page 27
Optimize Resolution – Small Peptide Selectivity
Comparison on 300SB Bonded Phases

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

Flow: 1.0 mL/min

Rapid Resolution 4.6 x 50 mm, 3.5 µm Detection: 215nm
Sample: 1-10µg protein (10µL inj.) in
10-60% B in 10 min. 6M Guanidine HCL, pH7.0

9 min.

0 1 2 3 4 5 6 7 8 9 10

Page 30
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 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

• Recovery may vary depending on bonded phase.

• All 300SB bonded phase generally provide good recovery.
Page 32
Effect of Bonded-Phase Ligand on Recovery of
a Synthetic Lipopeptide

300SB-CN Recovery 59%

300SB-C8 Recovery 75% Conditions:

Column: ZORBAX 300SB, 4.6 x 150 mm, 5 µm
Mobile Phase: A – 0.1% TFA/water, B – 0.1%
TFA/can
Gradient: 10 - 90 % B in 40 min
Flow Rate: 1 mL/min
Temperature: 60°C,
Sample: 15 µL ( 15 µg ) of peptide in 0.1%
300SB-C18 Recovery 89% TFA/DMSO

Retention Time, (min.)

• Recovery on each bonded phase is not always predictable so evaluate

different bonded phases. Page 33
Optimize Recovery by Changing Temperature
ßAP Peptide Separation
Column: ZORBAX 300SB-C18, 4.6 x 150 mm, 5 µm Mobile Phase: A- 0.1%TFA in water, B- 0.09%TFA in acetonitrile
Gradient: 20-45% B in 35 min Flow Rate: 1 mL/min Sample: 10 µl injection of 5 µg peptide in 6M Urea/5% HOAc

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

Solvent Application Comments

0.05-5% TFA in Water General Effective solubilization of many
samples
6 M Guanidine, buffered General Very good for many proteins and
at pH 6-8 peptides
5-80% Acetic Acid or Peptides Frequently used to extract
Formic Acid; peptides from tissues,
0.1-0.5M Perchloric Acid precipitating many proteins and
cellular debris
6 M Urea/ 5% Acetic aid Hydrophobic Useful for membrane proteins,
Peptides, fragments, aggregating systems
Proteins
Water-Miscible Organic Hydrophobic Limit injection volume to avoid
Solvents: Acetonitrile, Peptides, problems; add water, as possible,
Methanol, THF, Dioxane, Polypeptides to improve volume tolerance;
DMSO; +/- TFA; acidify with TFA as required
+/- Water

Page 35
Break Number 2

Please type your

question into the
Chat Box at any time
during the presentation.

Page 36
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 37
Conditions for Separations of Proteins on ZORBAX
300Extend-C18 Columns at High pH

Column: 4.6 x 150 mm, 5 or 3.5 µm

300Extend-C18
Mobile Phase: A: 20 mM NH4OH in water
B: 20 mM NH4OH in 80% ACN
Flow Rate: 1 mL / min.
Temp: 25 - 30°C
Initial Gradient: 5 - 60% B in 30 min.
C18 C18

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

Angiotensin II: Asp-Arg-Val-Tyr-Ile-His-Pro-

5.0E6
TIC (200-1500 m/z) Phe
Calc pI = 6.74

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

B- 10 mM NH4OH in 80%ACN AII Sample: 2.5 µL

8.477

2.0E7 (50 pmol each)

AI
1.0E7

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

TFA Conditions, 80°C

β (1-38)
Aβ β (1-40)
Aβ
A- 0.1% TFA in water β (1-42)
Aβ
B- 0.085% TFA in 80% ACN Amyloid ß Sequences:
29-41%B in 30 min.
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
Glu Val His His Gln
β (1-42)
Aβ Lys Leu17 Val Phe Phe Ala Glu Asp Val
NH4OH Conditions, 25°C β (1-43)
Aβ Gly Ser Asn Lys Gly Ala
β (1-40)
Aβ
A- 20 mM NH4OH in water β (1-38)
Aβ Ile Ile Gly Leu Met Val Gly Gly38 Val
B- 20 mM NH4OH in 80%ACN Val40 Ile Ala42 Thr43-COOH
26-38%B in 30 min.

• 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

5 20% 20 mM NH4OH, pH 10.5

4
Flow Rate: 1.5 mL/min;
3
Aging at 24°C, tests at 40°C
2

0
0.005
1-Chloro-1-nitrobenzene
Trimipramine • Consistent retention and
0.004 plate heights over time
Plate Heights, cm

indicate stable column at

0.003
high pH.
• Long column lifetime
0.002 achieved.

0.001
0 10000 20000 30000 40000 50000

Column Volumes of Mobile Phase

Page 41
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 42
Poroshell Particles Provide More Efficient Peaks
for Peptides and Proteins

• Poroshell 300SB-C18 is for

the separation of large
Porous peptides and proteins
Shell
• Poroshell particles have a
solid silica core with a
SOLID 5 µm superficially porous surface
CORE
with a 300Å pore size
• These particles allow more
efficient mass transfer and
narrower peaks for large
proteins and peptides

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*

mAU F =1 mL/min, 5-100 %B / 20 min

500
400
ß-amylase
300
200
100
0 Zorbax 300SB-C18
1\DATA\PORSHL3\PrSHL010.d\ßamy-ovr.win

-100 12 min
0 2 4 6 8 10 12 14 4.6x150
16 18 , 5 µm
mm min*

• Poroshell technology facilitates ultra-fast HPLC analysis of proteins

Note the similar separation of minor impurities in the 2 and 20 min separations.

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

• 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.

Page 47
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