Reverse-Phase Chromatography

Reverse-Phase Chromatography
RP-HPLC for Peptides
Reverse-phase high performance liquid chromatography (RP-HPLC) is an extremely useful
tool for analytical biochemists. However, unlike small molecule HPLC, separations of
proteins and peptides are nearly always performed under gradient conditions. There are
other differences that one needs to be aware of in order to develop RP-HPLC separations
of proteins and peptides as efficiently as possible. The general guidelines given in this short
article may help reduce your method development time.
RP-HPLC of complex peptide or protein mixtures remains a general method of
choice because of the resolution it provides. Unlike small organic molecules whose
chromatographic behavior is described by a finite partitioning equilibrium between the
stationary phase and the mobile phase, proteins and peptides typically do not exhibit
such an effect. Instead, they exhibit an adsorption phenomenon in which the polypeptide
is adsorbed onto the stationary phase and elutes only when the solvent strength of the
mobile phase is sufficient to compete with the hydrophobic forces keeping it there. For
this reason, elution of peptides or proteins from reverse-phase supports is by gradients of
increasing solvent strength. When run under isocratic conditions, peaks for proteins and
peptides are typically much broader than their small molecule counterparts.

Getting Started
Column: A good starting point for separating peptide or polypeptide mixtures is to start
with a C18-bonded silica column designed for these applications. The Discovery BIO Wide
Pore C18 is an ideal choice. Begin with 5 m particles packed into 15 cm L x 2.1 mm I.D.
columns with a mobile phase flow rate of 0.2 mL/min. The 2.1 mm I.D. or narrowbore
column configuration is a good balance between sensitivity (with 4.8-times the sensitivity
of a 4.6 mm I.D. column) and analysis time. If the dwell volume* of your HPLC system
is greater than 500 L, a 4.6 mm I.D. column run at 1.0 mL/min is likely to be a better
dimension to begin with.
Mobile phase: Choice of mobile phase will in part be dictated by the means of detection.
If detection is by mass spectrometry (MS), then the options are more limited and also
generally dont provide optimal chromatography. Commonly used MS-compatible ionic
mobile phase additives are acetate, formate, and carbonate and their corresponding
ammonium salts. The organic component is most often acetonitrile (CH3CN).
If detection is by traditional UV absorption, then mobile phases can be selected that provide
for superior chromatography, which is largely conferred by including ion pairing reagents
like TFA (trifluoroacetic acid) and HFBA (heptafluorobutyric acid). Typical concentrations
are 0.050.1% (v/v). A good starting point is to prepare mobile phase A to be 0.1% TFA

26

sigma.com

ORDER: 800-325-3010

TECHNICAL SERVICE: 800-325-5832

An initial run should begin with a gradient of 10 to 100% B in 45 minutes. An example

of this starting gradient for peptides derived from a protein digest is shown in Figure A.
While this separation does not provide significant resolution of component peptides, there
are means that can be employed to improve the resolution for virtually all parts of the
chromatogram.
The effect of these changes is shown in Figure B where the gradient profile was changed
from 10 to 70% B in 60 minutes. The shallower gradient provided some improvements in
resolution in the first half of the gradient. However, just as significant is the difference in
selectivity in various groups of peaks. This is a fundamental effect due to the differential
responses of the sample components to changes in the gradient slope. Also note that
there is still wasted space at the end of the chromatogram that can be reduced.

Reverse-Phase Chromatography

in water and mobile phase B to be 0.1% TFA in a mixture of 50:50 water:acetonitrile.

Alternatively, having some acetonitrile in both A and B minimizes the refractive index
changes that occur upon mixing and can provide more stable baselines.

To see if a lower initial concentration of acetonitrile improves resolution of early eluting

peaks, a third run is needed. This run has the same slope as previous runs, but in order to
maximize the resolution of early-eluting peaks,
the gradient starts at 100% aqueous. Under
Figure A. Initial Chromatographic Run of
these conditions, the bonded phase is better
Complex Peptide Sample
able to distinguish between small differences in

Column: Discovery BIO Wide Pore C18,
15 cm x 2.1 mm I.D., 5 m (568202-U)
the hydrophobicity of the peptides. Although

Mobile Phase: (A): 0.1% TFA (v/v) in water
this may come at a cost of longer run time
(B): 50:50, (0.1% TFA, v/v in water):
(0.1% TFA, v/v in CH3CN)
and may result in some wasted space in

Flow Rate: 0.208 mL/min

Temp.: 35 C
the initial part of the chromatogram, the

Det.: 215 nm
improved resolution afforded may offset these

Inj.: 10 L

Sample: Carboxymethylated b-Lactoglobulin B
consequences. The chromatogram in Figure C
tryptic digest
shows that this change had the desired effect.

Gradient: Min %A %B
0 90 10
Peaks eluting before 25 minutes were better
45 0 100
resolved than in Figure B.

G001737

Another option to improve resolution is to

increase the run time. Although this reduces
through-put, it may in some cases be the best
way to obtain the required resolution. In Figure
D the run time is increased to 130 minutes:
0 to 65% B in 130 minutes (0.25% CH3CN/
minute) as compared to Figure B where the
slope is 0.5% CH3CN/minute.

Our Innovation, Your Research

Shaping the Future of Life Science

27

Reverse-Phase Chromatography

One last option, but not illustrated here, is

column length. Increased column length may
provide the desired improvements in resolution.
The separations in this article were all run on
15 cm columns, but very often 25 cm columns
are used.
In conclusion, the gradient profile (rate of
change in % CH3CN and the starting and
ending % CH3CN) has enormous power to
increase resolution and decrease analysis time.
By combining gradient methodology with a
highly-efficient RP-HPLC material, like
Discovery BIO Wide Pore C18, one has all the
tools needed to maximize the separation of
peptides and protein digests.

Conditions for Figures B-D

Column: Discovery BIO Wide Pore C18,
15 cm x 2.1 mm I.D., 5 m (568202-U)

Mobile Phase: (A): 0.1% TFA in water
(B): 50:50, (0.1% TFA in water):
(0.1% TFA in CH3CN)

Flow Rate: 0.208 mL/min

Temp.: 35C

Det.: 215 nm

Inj.: 10 L

Sample: Carboxymethylated -Lactoglobulin B
tryptic digest

Figure B. Change Gradient Profile

Gradient: Min %A %B
0 90 10
60 30 70

Reference:

G001738

Geng, X. and Regnier, F.E. 1984. Retention Model for Proteins in

Reversed-Phase Liquid Chromatography. J. Chrom 296, 15.
*Dwell volume is the volume from and including the mixer to the
column inlet. Low pressure mixing systems generally have larger
dwell volumes than high pressure mixing systems.

Figure C. Enhanced Resolution Early in

the Gradient

Gradient: Min %A %B
0 100 0
65 35 65

G001739

Figure D. Further Refine Gradient Slope

Gradient: Min %A %B
0 100 0
130 35 65

G001740

28

sigma.com

ORDER: 800-325-3010

TECHNICAL SERVICE: 800-325-5832

Effect of Stationary Phase on Selectivity of

Reversed-Phase HPLC Separations of Polypeptides

What is Meant by Selectivity?

The resolution equation:

Reverse-Phase Chromatography

RP-HPLC separations of peptides and polypeptides is influenced by the chemistry of the

bonded phase. The objective of most peptide separations is to obtain as much information
about the sample as possible, especially when working with peptide maps. Therefore, it is
to the researchers advantage to run the sample on different stationary phases, like a C18,
C8, and C5.

RS = (1/4) N1/2 {(a-1)/a} {k/(1+k)}

tells us that retention (k), efficiency (N), and selectivity (a) each play a role in a chromatographic separation. There are few improvements that can be made to column efficiency
if one is working with small particles and modern packing materials. Retention also gives
limited options because of the need to keep analysis times as short as possible. However,
selectivity wields great power to increase resolution. Selectivity can be thought of as peak
spacing. The further the peaks are spaced from one another, the better the selectivity.
Selectivity, or separation factor, between peaks 1 and 2 is measured by the equation:
a = k2/k1

where k = (tRt0)/t0
Of course, improvements in selectivity beyond allowing for complete baseline resolution of
all sample components is of no additional benefit. Application Note 166 (T302166) showed
that improvements in selectivity (and thus resolution) for a complex peptide sample can be
achieved by altering the gradient slope and start conditions for the run. These are the most
common strategies for optimizing selectivity with polypeptide samples, but there are other
tools as well. In this short article, we will discuss the effect of stationary phase chemistry on
the selectivity of peptide separations.

How Does the Stationary Phase Effect Selectivity?

Unlike the partitioning mechanism exhibited by small molecules, retention of polypeptide
analytes on a reversed-phase matrix is by differential adsorption to the stationary phase,
primarily due to differences in their hydrophobicity. More hydrophobic peptides are retained
longer by the bonded phase, and vice versa. By reducing the alkyl chain length of the
bonded phase, not only is the hydrophobicity reduced, but also the total surface area that
is in contact with the peptide analytes. For small molecule separations, a C18 and C8 will
usually give the same selectivity, although different retention. However, because of different
Our Innovation, Your Research

Shaping the Future of Life Science

29

Reverse-Phase Chromatography

retention mechanisms for peptide and polypeptide separations, the differences in selectivity
between a C18, C8, and C5 can be dramatic. The same sample run on C18, C8, and C5
phases will yield different information about the sample, an important consideration for
peptide mapping.
There are other factors that affect adsorption to the matrix as well, even when comparing
only linear aliphatic alkyl bonded phases. These other factors involve polar or H-bonding
interactions with the silica surface itself, or the indirect effects of the silica surface chemistry
on the conformation of the bonded phase. Thus, not only differences in the hydrophobicity
of the bonded phase can influence selectivity, but also secondary effects impacted by the
bonding chemistry and surface silanols: bonding density, extent of endcapping of silanols,
and type of bonding (mono, di, or trifunctional).
An example of selectivity differences conferred by bonded phase chemistry is shown in
Figures A and B. Here, a proteolytic digest of apohemoglobin is chromatographed on the
three Discovery BIO Wide Pore reversed-phases C18, C8, and C5. The chromatograms
displayed only represent a portion of the entire run to better illustrate the subtle, but
significant, differences in selectivity conferred by each phase. Each of the phases displays
better selectivity in different parts of the chromatogram. If the goal is purification of a
specific peptide, then this has particular utility. If the goal is the best overall resolution
of the entire sample, then a decision process should be applied which evaluates the
performance of each phase with its optimized method.
In conclusion, different bonded phase chemistries give subtle yet significant differences
in selectivity toward peptides and polypeptides. Running the sample on each of the three
Discovery BIO Wide Pore reversed-phase chemistries will yield different, useful information
about the sample.

30

sigma.com

ORDER: 800-325-3010

TECHNICAL SERVICE: 800-325-5832


Mobile Phase: (A): 95:5, (0.1% TFA (v/v) in water):
(0.1% TFA (v/v) in CH3CN)
(B): 50:50, (0.1% TFA (v/v) in water):
(0.1% TFA (v/v) in CH3CN)

Column: Discovery BIO Wide Pore,
15 cm x 4.6 mm I.D., 5 m particles

Flow Rate: 1.0 mL/min

Temp.: 30 C

Det.: UV, 215 nm

Inj.: 50 L

Sample: tryptic digest of carboxymethylated
apohemoglobin

Gradient: Min %A %B
0 100 0
65 0 10

Figure B. Proteolytic Digest Discovery BIO

Wide Pore C18, C8, and C5
(28-38 minute window)

Mobile Phase: (A): 95:5, (0.1% TFA (v/v) in water):
(0.1% TFA (v/v) in CH3CN)
(B): 50:50, (0.1% TFA (v/v) in water):
(0.1% TFA (v/v) in CH3CN)

Column: Discovery BIO Wide Pore,
15 cm x 4.6 mm, 5 m

Flow Rate: 1.0 mL/min

Temp.: 30 C

Det.: UV, 215 nm

Inj.: 50 L

Sample: tryptic digest of carboxymethylated
apohemoglobin

Gradient: Min %A %B
0 100 0
65 0 100

Discovery BIO Wide Pore C18

Discovery BIO Wide Pore C18

Discovery BIO Wide Pore C8

Discovery BIO Wide Pore C8

Discovery BIO Wide Pore C5

Discovery BIO Wide Pore C5

G001730, 31, 32

Our Innovation, Your Research

Reverse-Phase Chromatography

Figure A. Proteolytic Digest Discovery BIO

Wide Pore C18, C8, and C5
(0-16 minute window)

G001727, 28, 29

Shaping the Future of Life Science

31

Reverse-Phase Chromatography

Products for Reverse-Phase Chromatography

Discovery BIO Wide Pore HPLC columns and capillaries provide sensitive, stable,
reproducible separations of proteins and peptides. The different phase chemistries and
separation modes provide unique selectivity options. Separations are scalable from
analytical to preparative.
Please visit sigma.com/discovery for complete information on our range of highspecification HPLC columns and visit sigma.com/proteomics_literature to request a copy
of the full Discovery BIO catalogue.

Type

Particle Length I.D.

Size (m)
(cm) (mm)

Cat. No.

Reverse-Phase Columns
Discovery BIO Wide Pore C5
Capillary

32

3.0
3.0
3.0
3.0
3.0
3.0
5.0
5.0
5.0
5.0
5.0

5.0
5.0
5.0
10.0
10.0
10.0
5.0
10.0
15.0
15.0
15.0

0.2
0.3
0.5
0.2
0.3
0.5
0.2
0.2
0.2
0.3
0.5

65609-U
65531-U
65520-U
65611-U
65532-U
65521-U
65612-U
65613-U
65614-U
65533-U
65522-U

Microbore

3.0

3.0

5.0

5.0
10.0
15.0

1.0
1.0
1.0

65511-U
65512-U
65513-U

Narrowbore

3.0

3.0

3.0

5.0

5.0

5.0

5.0

5.0
10.0
15.0
5.0
10.0
15.0
25.0

2.1
2.1
2.1
2.1
2.1
2.1
2.1

567226-U
567227-U
567228-U
568400-U
568401-U
568402-U
568403-U

Guards
Pkg of 2
Kit
Pkg of 2
Kit

2.0
2.0
2.0
2.0

2.1
2.1
2.1
2.1

567278-U
567279-U
568470-U
568471-U

sigma.com

3.0
3.0
5.0
5.0

ORDER: 800-325-3010

TECHNICAL SERVICE: 800-325-5832

Type

Particle Length I.D.

Size (m)
(cm) (mm)

Cat. No.

Standard Analytical

3.0

3.0

3.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

10.0

5.0
10.0
15.0
5.0
5.0
10.0
10.0
15.0
15.0
25.0
25.0
25.0

4.6
4.6
4.6
4.0
4.6
4.0
4.6
4.0
4.6
4.0
4.6
4.6

567229-U
567230-U
567231-U
568410-U
568420-U
568411-U
568421-U
568412-U
568422-U
568413-U
568423-U
567232-U

Guards
Pkg of 2
Kit
Pkg of 2
Kit

2.0
2.0
2.0
2.0

4.0
4.0
4.0
4.0

567280-U
567281-U
568472-U
568473-U

Semi-preparative

5.0

10.0

10.0

10.0

25.0
5.0
15.0
25.0

10.0
10.0
10.0
10.0

568430-U
567233-U
567234-U
567235-U

Guards
Pkg of 1

10.0

1.0

10.0

567286-U

Preparative

10.0

10.0

10.0

5.0
15.0
25.0

21.2
21.2
21.2

567236-U
567237-U
567238-U

3.0
3.0
5.0
5.0

Particle Length I.D.

Size (m)
(cm) (mm)

Cat. No.

Discovery BIO Wide Pore C8

5.0
10.0
15.0
5.0
10.0
15.0
25.0

2.1
2.1
2.1
2.1
2.1
2.1
2.1

567213-U
567214-U
567215-U
568300-U
568301-U
568302-U
568303-U

Guards
Pkg of 2
Kit
Pkg of 2
Kit

2.0
2.0
2.0
2.0

2.1
2.1
2.1
2.1

567274-U
567275-U
568370-U
568371-U

Standard Analytical

3.0

3.0

3.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

10.0

5.0
10.0
15.0
5.0
5.0
10.0
10.0
15.0
15.0
25.0
25.0
25.0

4.6
4.6
4.6
4.0
4.6
4.0
4.6
4.0
4.6
4.0
4.6
4.6

567216-U
567217-U
567218-U
568310-U
568320-U
568311-U
568321-U
568312-U
568322-U
568313-U
568323-U
567219-U

Guards
Pkg of 2
Kit
Pkg of 2
Kit

2.0
2.0
2.0
2.0

4.0
4.0
4.0
4.0

567276-U
567277-U
568372-U
568373-U

25.0
5.0
15.0
25.0

10.0
10.0
10.0
10.0

568330-U
567220-U
567221-U
567222-U

10.0

1.0

10.0

567284-U

Preparative

10.0

10.0

10.0

5.0
15.0
25.0

21.2
21.2
21.2

567223-U
567224-U
567225-U

3.0
3.0
5.0
5.0

Semi-preparative

5.0

10.0

10.0

10.0

Particle Length I.D.

Size (m)
(cm) (mm)

Cat. No.

Discovery BIO Wide Pore C18

Narrowbore

3.0

3.0

3.0

5.0

5.0

5.0

5.0
3.0
3.0
5.0
5.0

Type

Capillary

3.0
3.0
3.0
3.0
3.0
3.0
5.0
5.0
5.0
5.0
5.0

5.0
5.0
5.0
10.0
10.0
10.0
5.0
10.0
15.0
15.0
15.0

0.2
0.3
0.5
0.2
0.3
0.5
0.2
0.2
0.2
0.3
0.5

65603-U
65526-U
65517-U
65604-U
65527-U
65518-U
65606-U
65607-U
65608-U
65529-U
65519-U

Microbore

3.0
3.0
5.0
5.0

5.0
10.0
15.0
25.0

1.0
1.0
1.0
1.0

65504-U
65506-U
65508-U
65509-U

Narrowbore

3.0

3.0

3.0

5.0

5.0

5.0

5.0

5.0
10.0
15.0
5.0
10.0
15.0
25.0

2.1
2.1
2.1
2.1
2.1
2.1
2.1

567200-U
567201-U
567202-U
568200-U
568201-U
568202-U
568203-U

Guards
Pkg of 2
Kit
Pkg of 2
Kit

2.0
2.0
2.0
2.0

2.1
2.1
2.1
2.1

567270-U
567271-U
568270-U
568271-U

3.0
3.0
5.0
5.0

Reverse-Phase Chromatography

Type

Guards
Pkg of 1

Our Innovation, Your Research

Shaping the Future of Life Science

33

Particle Length
Type Size (m)

I.D.
(cm)

(mm)

Reverse-Phase Chromatography

Discovery BIO Wide Pore C18 (Cont.)

34

Standard Analytical

3.0

3.0

3.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

5.0

10.0

5.0
10.0
15.0
5.0
5.0
10.0
10.0
15.0
15.0
25.0
25.0
25.0

4.6
4.6
4.6
4.0
4.6
4.0
4.6
4.0
4.6
4.0
4.6
4.6

567203-U
567204-U
567205-U
568210-U
568220-U
568211-U
568221-U
568212-U
568222-U
568213-U
568223-U
567206-U

Guards
Pkg of 2
Kit
Pkg of 2
Kit

2.0
2.0
2.0
2.0

4.0
4.0
4.0
4.0

567272-U
567273-U
568272-U
568273-U

3.0
3.0
5.0
5.0

Cat. No.

Reverse Phase Media

C18 packing pkg of 10g
HYPERPREP C18 SILICA GEL, 30 m, 100 g
Polyamide for column chromatography, 6
Silica gel 90 C18-Reversed phase for
column chromatography, fully endcapped
Silica gel 100 C18-Reversed phase for
column chromatography, fully endcapped
Silica gel 100 C18-Reversed phase for
column chromatography, fully endcapped
Silica gel 100 C18-Reversed phase for
column chromatography, not endcapped
Silica gel 100 C8-Reversed phase for
column chromatography
Silica gel 100 C8-Reversed phase for
column chromatography, fully endcapped

58419
13326
02395
60757
60754
60756
60758
60759
60755

Hydrophobic Interaction
Chromatography (HIC) Columns
TSK-GEL

Semi-preparative

5.0

10.0

10.0

10.0

25.0
5.0
15.0
25.0

10.0
10.0
10.0
10.0

568230-U
567207-U
567208-U
567209-U

Guards
Pkg of 1

10.0

1.0

10.0

567282-U

Preparative

10.0

10.0
10.0

5.0
15.0
25.0

21.2
21.2
21.2

567210-U
567211-U
567212-U

sigma.com

Description

Cat. No.

ORDER: 800-325-3010

TECHNICAL SERVICE: 800-325-5832

Butyl-5PW, 3.5cm 4.6mm I.D., 2.5m

814947
Ether-5PW, 7.5cm 7.5mm I.D., 10m
808641
Phenyl-5PW, 7.5cm 7.5mm I.D., 10m
807573
Guard Column Kit:
Phenyl-5PW 1cm 6.0mm I.D., 20m.
Kit contains one cartridge, a stand-alone holder,
5 mL packing, 5 cm of 1/16 in. tubing, and
2 nuts and ferrules.
807652