Reversed-Phase High Performance Liquid

Chromatography of Proteins

UNIT 8.7

Djuro Josic1 and Spomenka Kovac2

1

Proteomics Core, COBRE Center for Cancer Research Development, Rhode Island Hospital
and Brown University, Providence, Rhode Island
2
Department of Chemistry, J. J. Strossmayer University, Osijek, Croatia

ABSTRACT
Reversed-phase HPLC (RP-HPLC) is one of most important techniques for protein separations and the method of choice for peptide separation. RP-HPLC has been applied on
the nano, micro, and analytical scale, and has also been scaled up for preparative purications, to large industrial scale. Because of its compatibility with mass spectrometry,
RP-HPLC is an indispensable tool in proteomic research. With modern instrumentation
and columns, complex mixtures of peptides and proteins can be separated at attomolar
levels for further analysis. In addition, preparative RP-HPLC is often used for large-scale
purication of proteins. This unit provides protocols for packing and testing a column,
protein separation by use of gradient or step elution, desalting of protein solutions,
and separation of enzymatic digests before mass spectrometric analyses. A protocol is
also provided for cleaning, regenerating, and storing reversed-phase chromatography
C 2010 by John Wiley & Sons, Inc.
columns. Curr. Protoc. Protein Sci. 61:8.7.1-8.7.22. 
Keywords: reversed-phase HPLC r proteins r peptides r LC-MS/MS

INTRODUCTION
Due to its excellent resolving power, convenience, versatility, stability, and reproducibility, reversed-phase high-performance liquid chromatography (RP-HPLC) is one of the
most important techniques for protein separations and the method of choice for peptide
separations. RP-HPLC has been applied on the nano, micro, and analytical scale, and
can also be scaled up for preparative purication on the industrial scale (Aguilar and
Hearn, 1996; Shi et al., 2004). Because of its compatibility with mass spectrometry
(MS), RP-HPLC is an indispensable tool in proteomic research. The increase in resolution offered by LC separation greatly enhances MS detection of sample components, and
high-resolution separation reduces ion suppression in MS (Shi et al., 2004).
Reversed-phase chromatography (RPC) is a separation method based on the hydrophobicity of the protein molecule. In RPC, the hydrophobic stationary phase is based on
silica gel or a synthetic polymer. In recent years, instead of bulk materials for column
packing, polymer- or silica-gel-based monolithic stationary phases have also been used.
Monoliths are separation media in a format that can be compared to a single unit such as
a compact disk, cylinder, or rod that does not contain interparticular voids. During chromatographic separation, all mobile phase ows through the monolithic stationary phase,
and the resulting convective ow greatly accelerates the mass transfer and the separation
speed. Monolithic stationary phases enable reduction of separation time by up to one
order of magnitude (Josic and Clifton, 2007). The stationary phase (or chromatographic
support) bears the hydrophobic ligands, mainly C4 , C8 , or C18 -alkyl chains. The mobile
phase contains water and a water-miscible organic solvent such as methanol, acetonitrile,
or isopropanol. Acid (usually formic, acetic or triuoroacetic acid) is added to the mobile
phase to render the proteins and peptides positively charged, and to reduce undesirable
interactions with the stationary phase. In some cases, mobile phases of intermediate or
Current Protocols in Protein Science 8.7.1-8.7.22, August 2010
Published online August 2010 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471140864.ps0807s61
C 2010 John Wiley & Sons, Inc.
Copyright 

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8.7.1
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even basic pH values can also be used (Yang et al., 2009). With modern instrumentation
and columns, complex mixtures of proteins can be separated in attomolar amounts for
collection and further analysis of the resolved components. On the other hand, preparative
RPC is often used for large-scale purication of proteins.
Reversed-phase chromatographic separation involves loading of a protein mixture onto
a hydrophobic stationary phase in water or a water-solvent mixture, usually containing
very dilute acidtypically 0.1% formic or triuoroacetic acid (TFA). The elution of
bound components occurs by increasing the concentration of organic solvent (usually
acetonitrile or methanol). According to theory, hydrophobic-interaction chromatography
(HIC, UNIT 8.4) and RPC are related techniques, since both are based upon interactions between hydrophobic regions (amino acids) in protein molecules and hydrophobic ligands
of a chromatographic support. However, experimentally these techniques are different in
the following ways:
1. The surface of the RPC supports is usually more hydrophobic than that of the HIC
medium.
2. In HIC, concentrated salt solutions are used for sample application. In RPC, proteins and peptides are dissolved in water or water-organic solvent mixture, usually
containing a dilute acid (see above).
3. Because of stronger hydrophobic interaction, organic solvents are used for RPC
elution. Elution in HIC occurs by decreasing the salt concentration in the mobile
phase.
4. The use of organic solvents in RPC frequently leads to denaturation and loss of biological activity of biopolymers, especially of high-molecular-weight proteins. However,
denaturation and consequent loss of biological activity can be minimized or even reversed by carefully returning the molecule to conditions that favor its native structure
(McNay and Fernandez, 1999).
5. Temperature, especially elevated temperature, is an important variable in controlling
selectivity in RP-HPLC separations of proteins. In routine use, most HIC separations
are performed at room temperature.
RPC provides excellent resolution of complex protein mixtures on an analytical, microanalytical, or nanoanalytical scale. This chromatographic method also has great potential
for high-resolution purication as well as for low-resolution desalting steps, especially
in sample preparation for proteomics applications (Josic and Clifton, 2007). Combined
with other chromatographic methods, especially ion-exchange chromatography, and as a
component (dimension) in so-called multidimensional liquid chromatography (MDLC),
RPC is a key method for separation, identication, and characterization of proteins in
very complex mixtures. MDLC followed by MS (or more frequently MS/MS) is a fast
and reliable method for protein identication and characterization in proteomics (Wagner
et al., 2002).
Sample preparation, selection of chromatographic support, column, and mobile phase,
together with the separation design and optimization, are discussed in the Strategic
Planning section below. Protocols are provided for packing and testing a column (see
Basic Protocol 1), as well as protein separation by use of gradient or step elution (see
Basic Protocol 2 and Basic Protocol 3). Desalting of protein solutions is described in
Basic Protocol 4. Cleaning, regenerating, and storing of RPC columns are described in
the Support Protocol.
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HPLC of Proteins

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STRATEGIC PLANNING
The performance of a typical RPC separation of proteins is depicted in Figure 8.7.1A
and B. In analytical-scale RPC separations, a continuous linear gradient is often used
(Fig. 8.7.1A), while preparative-scale separations typically employ an optimized elution
scheme combining isocratic elution and gradients with different slopes (see Fig. 8.7.1B).
A successful RP-HPLC separation is inuenced by many parameters. Consequently, to
meet the requirements for an application, the RPC separation must be optimized. The

%ACN

TnI

TnT

50
TnC

crude troponin

mAU

400

TnT

200

25
Tm

10

20

30

Elution time (min)

90% TnT

Total area units / min 107

6
5
Column: 280 50 mm I.D.
Sample load: 5700 mg

4
3
2
10% TnT
1

100% IM
100%
TnC

100% Tnl

0
10

30

50

90 110 130
70
Elution time (min)

150

170

190

Figure 8.7.1 Analytical RP-HPLC of crude mixture of proteins from rabbit skeletal muscle:
tropomyosin (Tm) and components of troponin (Tn) complex, comprising troponin T (TnT), troponin I (TnI), and troponin C (TnC). (A) Analytical RP-HPLC. Column, Zorbax SB300 C8 (150

4.6 mm I.D., 5 m particle size, 300 A pore size; Agilent Technologies). Conditions: linear A-B
gradient (1% B/min starting from 25% acetonitrile) at a flow rate of 1 ml/min. Eluent A, water
containing 0.05% TFA; Eluent B, acetonitrile containing 0.05% TFA. (B) Preparative RP-HPLC
(optimized after scaling-up experiments). Column, Bondapack C8 packing, 5 m particle size,

300 A pore size, from Waters, in column of dimensions 280 50 mm I.D. Eluent A, water containing 0.05% TFA; Eluent B, acetonitrile containing 0.05% TFA. Sample load, 5700 mg in 440 ml
water containing 0.05% TFA (13 mg protein/ml); following sample loading at 22 ml/min, a 10-min
isocratic hold (at constant solvent concentration) with water containing 0.05% TFA, followed by
linear A-B gradient (1.7% acetonitrile/min) up to 25% acetonitrile, then 0.1% acetonitrile/min up
to 35% acetonitrile and, finally, 0.5% acetonitrile/min up to 55% acetonitrile. The positions of the
individual components identified following fraction analysis are denoted in histograms. Reprinted
from Mant and Hodges (2002), with permission.
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following are considerations for selecting and optimizing media and conditions for an
RPC separation of proteins and peptides:
1. Select the stationary and mobile phases that provide the best resolution and recovery
under the simplest starting conditions. For routine RP-HPLC separations, TFA and
acetonitrile are the most commonly used components of the mobile phase. For RPC
applications in an LC-MS/MS system, TFA is replaced by another volatile organic
acid, most commonly formic acid.
2. Determine the pH that provides best resolution. It is very important to know that for
separation under basic conditions (pH values higher than 8), only polymer-based chromatographic supports can be used. Silica-based supports (both particles and monoliths)
are not stable at higher pH values.
3. To achieve maximal selectivity, the gradient must be optimized (see also Fig. 8.7.1B).
The gradient slope inuences only distance between peaks, but will not change their
order during the elution.
4. Determine the highest ow rate that provides acceptable separation and back pressure.
5. Steeper gradients and higher ow rates shorten the separation time.
6. If scale-up is planned for the separation, cost factors such as column and mobile phase
prices and waste disposal should be taken in consideration to provide an optimal
process economy.
Choosing the optimal support and column dimensions is crucial for a successful chromatographic separation. A list of some commercially available media for RP-HPLC is
given in Table 8.7.1. The selection of an RPC support must be made empirically. The most
critical factors in choosing the appropriate stationary phase are sample hydrophobicity
and the need for column sanitation. For separation of highly hydrophobic components,
a less hydrophobic stationary phase should be used to facilitate the elution. Proteins that
bind strongly to a more hydrophobic support bind more weakly to a less hydrophobic
medium, and will also be eluted at lower concentrations of organic solvent. If NaOH
is needed for sanitation (e.g., for isolation of therapeutic proteins), only polymer-based
RPC stationary phases can be used.
Table 8.7.1 Some Commercially Available Supports for Reversed-phase HPLC of Proteins and Peptidesa

Protein
separation

Producer

Peptide
Silica- Polymer- Polymer-based Silica-based
Analytical Preparative
separation based based
monolithic
monolithic

Agilent

Yes

Yes

Yes

No

Yes

No

Yes

Yes

Agilent-Varian

Yes

Yes

Yes

Yes

No

No

Yes

Yes

BIASeparations

Yes

Yes

No

No

Yes

No

Yes

Yes b

Dionex

Yes

Yes

Yes

No

Yes

No

Yes

No

GE Healthcare

Yes

Yes

Yes

Yes

No

No

Yes

No

Merck
(Germany)

Yes

Yes

Yes

No

No

Yes

Yes

Yes

Tosoh
Bioscience

Yes

Yes

Yes

Yes

No

No

Yes

No

Waters

Yes

Yes

Yes

No

No

No

Yes

Yes

a Product specications and guides for users of chromatographic columns and bulk supports can be found at manufacturers Web sites.
b Up to 8-liter monolithic polymer-based columns.

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Applications that involve the fractionation of complex samples, such as proteolytic digestions, require extremely high resolution. On the other hand, separation time and economy
are more important factors for preparative applications, such as protein purication, especially in the early stages of the separation scheme. In this case, separation speed, capacity,
and yield may be more important than resolution (see Figs. 8.7.1A and B). Importantly, if
RPC is used in the nal step, the purity of the product is crucial, and maximal resolution
has to be achieved.

Ligand type and degree of substitution

The most common column ligands for RPC are shown in Figure 8.7.2. The ligand is
the group that is immobilized on the chromatographic support, while the ligate is the
component of the sample that interacts with the immobilized ligand. The density and
hydrophobicity of the ligand determine the hydrophobicity of the stationary phase and the
strength of the interaction with the sample. If aliphatic chains are used, the hydrophobicity
of the ligand increases with the chain lengthe.g., a stationary phase with immobilized
C8 chains is more hydrophobic than a stationary phase containing immobilized C4 chains.
If hydrophobic polymer-based supports such as polystyrene/divinyl benzene are used,
the interaction between the sample and the support can also occur on the surface of the
matrix, without the presence of an additional immobilized ligand.
Binding capacity of an RPC support increases with increased number of ligands bound
to its surface. However, after the ligand density reaches a certain level, the binding
capacity of the stationary phase will also reach a plateau because of the steric hindrance
in ligand-ligate interaction. In the case of further increase of ligand density, the strength
of binding between the sample components and the support will continue to increase.
Consequently, elution of more hydrophobic samples (proteins and peptides) bound to
supports with very high ligand density will be difcult or nearly impossible.

Structure and physicochemical characteristics of the chromatographic support

The most important characteristics of the chromatographic support are:
1. Surface chemistry.
2. Density of column-bound ligand.
3. Particle size.
4. Pore size.

SiO

butyl (C4)
SiO

H3C

(CH2)16 CH2 SiO

SiO

N
Figure 8.7.2

SiO

octyl (C8)

octadecyl (C18)

phenyl

cyano

Most common column ligands used in reversed-phase HPLC.

Conventional
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5. Mechanical stability.
The matrix bears the immobilized ligand and also contributes signicantly to the selectivity of the RPC resin. Both silica- and polymer-based, wide-pore materials are the
most commonly used supports for the RPC of proteins. The typical structure of the surface of the silica-based RPC medium is given in Figure 8.7.3A. Almost all silica-based
RPC supports are now end-capped, which means that the residual surface silanol groups
(Si-OH) are replaced by C1 or C2 groups. However, the surface silanols in silica-based
RPC supports cannot be completely eliminated, and they may still interact with polar
solutes and basic components of the sample. Ionizable silanol groups arise not only
from inadequate end-capping: they can also result from column aging or inappropriate
storage of the silica-based column (e.g., over a long period in water). Consequently, the
selectivity of the column can be impaired (Engelhardt et al., 2005). The structure of a
polystyrene-based support is shown in Figure 8.7.3B. This matrix itself is hydrophobic,
and interaction with the sample and subsequent separation can occur on its surface (see
above). Immobilization of a ligand on the surface of polymer-based supports can further
tune their selectivity and increase the binding capacity. The main producers of polymerbased reversed-phase media are Varian and GE Healthcare (see SUPPLIERS APPENDIX).
In order to balance high efciency and short separation time, spherical nonporous or
porous particles with diameters between 1.5 and 5 m, packed in short columns (3 to 5 cm
long), are often used. The efciency, and also the back pressure of the column, increases

C8 chains

H2C
CH2
H2C

CH2
H2C
CH2

CH2
H2C

H2C

H2C

H2C

H2C

CH2
H2C

CH2
H2C
CH2

CH2
H2C

H2C

CH2
H2C end capping
silanol groups
CH2
CH2
CH2
CH2
CH2
CH2
H2C
H2C
H2C
H2C
CH3H2C
C
H
CH
CH
3
CH
Si
Si
3
3
Si CH3 2 Si
Si CH3
H
Si
H
H
H
H
H
HO
O O
O
O
O
O
O
O
O
O
O
O
Si
Si
Si
Si
Si
Si O Si O Si O Si O
Si O Si
O
O
Si
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H2C

CH2

H2C

CH2

H2C

CH2

B
CH2 CH CH2 CH CH2 CH CH2 CH CH2 CH

CH2 CH CH2 CH CH2 CH CH2 CH CH2 CH

CH2 CH CH2 CH CH2 CH CH2 CH CH2 CH

CH2 CH CH2 CH CH2 CH CH2 CH CH2 CH

CH2 CH CH2 CH CH2 CH CH2 CH CH2 CH

Reversed-phase
HPLC of Proteins

Figure 8.7.3 Structure and surface chemistry of supports for reversed-phase HPLC. (A)
Schematic presentation of surface chemistry of an end-capped reversed-phase C8 silica-based
support (reprinted from Engelhardt et al., 2005, with permission). (B) Structure of a polystyrenebased reversed-phase HPLC support (Courtesy of GE Healthcare).

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with smaller particle diameter. The main producer for nonporous, polymer-based RPC
materials is Tosoh Bioscience (see SUPPLIERS APPENDIX). Longer columns (up to 60 cm)
packed with spherical 1.0- to 1.5-m silica gelbased particles are used for very highperformance separation of proteins. These columns are prepared with packing pressures
as high as 4100 bar, and their working back pressure is higher than 1500 bar. This method
is called Ultra High-Pressure Reversed-Phase Liquid Chromatography (UHPRPLC;
also see Eschelbach and Jorgenson, 2006). These columns can be commercially obtained
from Waters Corp.
For large-scale preparative separations, porous particles with larger diameters are used.
Use of chromatographic supports with larger particle size allows use of higher ow rates
at lower back pressure, especially at the early stages of protein or peptide purication.

The standard chromatographic materials with pore diameters around 10 nm (100 A)

are sufcient for separation of peptides with molecular weights less than 2000 Da.
Larger molecules, such as polypeptides and proteins, cannot enter these small pores,
necessitating the
use of wide-pore materials with a pore size between 30 and 200 nm

(300 to 2000 A). Only after development of large-pore materials (rstly silica-based,
followed by polymer-based) did chromatography of high-molecular weight substances
such as proteins and larger polypeptides become possible (Hearn, 1984). However, the
surface of the support and its capacity decreases with increasing pore size. Consequently,
the capacities of porous materials are signicantly higher than those of nonporous silica
gel or polymer-based supports. Nonporous chromatographic materials are also used for
fast analytical high performance separations and for UHPRPLC of proteins (MacNair
et al. 1997; Engelhardt et al. 2005).

Monolithic supports
In proteomic applications and for in-process analysis in biotechnology, RPC columns with
high efciency and short analysis times are required. Hence, high-speed packing materials
are in demand. If porous materials are used, separation speed is limited by mass transfer
between the mobile phase and stationary liquid in the pores of the chromatographic
support. For nonporous materials, the binding of sample components and subsequent
elution occurs on the surface of the support, so separation speed is much less limited
by mass transfer. In order to increase the performance of nonporous chromatographic
material, particles with very small diameter (less than 3 m, e.g., Tosoh Bioscience, for
polymer-based nonporous columns and bulk materials) are used, and high column back
pressure is often the limiting factor. Frequently, monoliths are an alternative to columns
packed with particulate stationary phases. The structure of monolithic materials (see
Fig. 8.7.4A and B) allows very fast mass transfer during chromatographic separation
that is practically not limited by the ow rate. Because of the low back pressure and fast
mass transfer, monolithic columns can be run at very high speed. As a result, separation
time is up to one order of magnitude faster than that of columns packed with bulk
particles (Svec and Huber, 2006; Josic and Clifton, 2007). Similar to columns packed
with spherical particles, monolithic columns are silica- (see Fig. 8.7.4A), or polymerbased (see Fig. 8.7.4B). In reversed-phase mode, monolithic columns are mostly used
for protein and peptide separation in proteomics applications (Josic and Clifton, 2007).
Suppliers for monolithic columns for protein separations are listed in Table 8.7.1.
Instrumentation
Rapid and high-performance separation in reversed-phase mode requires not only a
stationary phase with both minimal mass transfer and kinetic resistance, but also instrumentation capable of generating fast and reproducible gradient elutions and providing
precise temperature control for both the column and mobile phases. Furthermore, the
HPLC instrument must have low overall system dead volume (the volume between the
sample injector and the outlet of the system, including the chromatographic column), a
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8.7.7
Supplement 61

Figure 8.7.4 Structure of HPLC monolithic supports. (A) Silica-based monolith (reprinted with
permission from Luo, et al., 2005. (B) Polymer-based monolith (reprinted with permission from
Lee, et al., 2004).

precise sample introduction device, and a highly sensitive detector with short response
time. For micro- and nano-HPLC separations that are performed mostly in proteomic
applications, the demand for minimal dead volume is extremely stringent.

Reversed-phase
HPLC of Proteins

8.7.8
Supplement 61

For preparative applications, a pump capable of delivering a high ow rate, typically

between 200 and 800 cm/hr, should be chosen. For high-speed analyses, especially if
monolithic supports are used, pumps with higher ow rates will be needed. For microscale and nano-scale separations with ow rates in the nl/min and l/min range, mostly
in proteomic technology where the HPLC system is used for sample preparation or
is a part of the LC-MS(/MS) system, special chromatography equipment is required.
For example, Wagner et al. (2002) used a multidimensional HPLC system for protein
separation with integrated sample preparation. Size-selective sample fractionation was
followed by anion- or cation-exchange chromatography. The nal separation step prior to
MALDI TOF MS was RPC on a short hydrophobic column. For UHPRPLC, fused silica
capillaries paced with nonporous octadecyl RP material are used. These columns have a
length up to 66 cm and were packed with the pressures as high as 4100 bar. The pressures
required to run the system at optimum ow rates are about 1400 bar (MacNair et al., 1997).
Current Protocols in Protein Science

Proteins are detected at 280 nm, and detection of fragments of proteins usually takes
place at 210 to 215 nm. For more specic detections, especially if complex mixtures are
analyzed, a multi-wavelength optical detector, e.g., detecting at 210, 260, 280, and 405
nm, is recommended. For all analytical and large-scale separations, adequate fraction
collectors should be part of the chromatographic system. A computer with appropriate
software for instrument control and data analysis is an integral part of every modern
HPLC system.

Mobile phase
In order to increase the hydrophobicity of sample components, enhance binding to the
chromatographic support and, if necessary, alter the retention time, ion-pairing agents
are added to the mobile phase. Ion-pairing reagents are ionic molecules having a charge
opposite to the charge of the analyte (protein) of interest, as well as a hydrophobic
region. The ion-pairing reagent masks the positive charges of the protein molecule (by
pairing with them), and also enhances its hydrophobicity. Ion-paring agents are most
often acids such as TFA or formic acid. If RPC separation is performed at neutral or
basic pH, ammonium formate or triethylamine is used. It should be stressed that most
RPC separations are performed at low pH (usually between pH 2 and 3) with an acid
as ion-pairing reagent. Reduction of the pH is necessary to minimize the charge of the
C-terminal COO groups. The correct pH can be crucial for a successful separation, and
some separations have to be performed at elevated pH (Yang et al., 2009). During the
gradient elution, an organic modier is added to the eluent (solvent B) to weaken the
interaction of sample components with the hydrophobic support and to increase elution
strength. The organic modier must be miscible in water and UV-transparent to enable
detection of eluting molecules, even at low wavelengths (down to 210 nm or lower
for peptides). Acetonitrile, methanol, ethanol, and isopropanol are the most commonly
used organic modiers. Different organic modiers have also different elution strengths
(Aguilar and Hearn, 1996). Volatile organic modiers, such as methanol or acetonitrile,
enable quick evaporation and concentration or drying of the components collected after
chromatographic separation.
Temperature selectivity
Hancock et al. (1994) and Chen and Horvath (1995) recognized the inuence of temperature on protein and peptide chromatographic separation. Elevated temperature enhances
kinetic and transport properties during the RPC separation. Selectivity changes caused by
changes of temperature were complementary to those effected by changing the solvent
strength. The use of elevated column temperature may also serve as a useful tool to optimize a separation, if the temperature of the stationary phase is controlled. Demonstrated
long-term column stability at the anticipated operating temperature is the main requirement for RPC separations at elevated temperature, and the thermal stability of the given
support has to be determined before the separation method is established. RP-HPLC
separations at temperatures above the atmospheric boiling point have been performed,
but these kinds of separations also require appropriate instruments and columns (Chen
and Horvath, 1995).
Summary
A summary of the roles of different parameters in RP-HPLC separation of proteins and
their inuence on strategic planning is given in Table 8.7.2, e.g., solvent strength and
solvent type are easy to manipulate and can be conveniently adjusted for optimization
of protein separation. Changing ion-paring reagents, column type, and mobile phase
additives is less convenient and, if possible, should be avoided. Changing mobile-phase
pH is also a powerful variable to adjust selectivity, but requires many optimization
experiments. The selectivity effects of temperature and solvent strength are more or less
orthogonal and can also be used to modify the separation (Dolan, 2002).
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Supplement 61

++
++
++
+
+
0

2. Temperature

3. pH

4. Ion-pairing

5. Solvent

6. Column type

7. Mobile phase
additives
0

a ++, very favorable; +, favorable; , unfavorable; ,unfavorable; 0, no inuence.

8. Buffer type and

concentration

++

1. Solvent
hydrophobicity

Experimental
convenience

Ability to change the

separation factor
(alpha)

Variable

++

++

Speed of column
equilibration

Method
robustness

Compatible
with low UV
detection

Impact of different factors

Table 8.7.2 Some Factors that Affect the Choice of Variables for RP-HPLC Method Developmenta

Reversed-phase
HPLC of Proteins

8.7.10

Current Protocols in Protein Science

++

++

Large or small number of

runs required to select
optimum value of variable

Peak tracking
problems

PACKING AND TESTING OF SEMI-PREPARATIVE AND PREPARATIVE

RP-HPLC COLUMNS

BASIC
PROTOCOL 1

Microanalytical and analytical RP-HPLC columns are usually packed by the column
producer, and are ready to use after proper column testing. Prepacked larger columns
for semi-preparative and preparative separation of proteins and peptides are also commercially available, but it is less expensive and less risky to pack such columns in the
laboratory, especially during the development and scaling-up of methods for isolation
and separation. This protocol provides basic rules for packing of semi-preparative and
preparative columns containing silica- or polymer-based reversed-phase materials (e.g.,
C18 -, C8 -, or C4 -silica, polystyrene/divinyl benzene, or other polymer-based beads with
corresponding ligands to provide appropriate surface hydrophobicity) with particle size
larger than 15 m (usually up to 90 m), and pore size larger than 300 A. Large-particlesize supports are generally used for semi-preparative and preparative purications. The
large-pore materials are generally used for separation of macromolecules (see above).
Large-particle materials are also considerably less costly than small-particle materials,
and their use results in lower column back pressure and easier column packing (Mant and
Hodges, 2002). All sizes of monolithic columns, from nanoscale to preparative scale, are
prepared by the producer (Josic and Clifton, 2007).

Materials
Appropriate RPC support (silica- or polymer-based) with a particle size between 15
and 90 m
Packing solution: isopropanol or methanol
Sintered-glass funnel (medium grade) and degasser and/or sonicator
Suction ask
Vacuum source
Chromatography column (e.g., 250 21.2mm i.d.) with corresponding frits
(Tosoh Bioscience or Knauer, http://www.knauer.net)
Adapter with tubing to connect the top of the column to a pump (Tosoh Bioscience,
or Knauer, http://www.knauer.net)
Pump: for smaller columns, an analytical HPLC pump can be used; For
semi-preparative or preparative columns, the use of special pumps (e.g., Agilent,
Knauer) is recommended
1. Equilibrate all materials to room temperature.
2a. To wash the RPC matrix by decantation: Suspend the silica-based gel in isopropanol.
Allow gel to settle, decant the supernatant, and add an excess of isopropanol. Repeat
wash a total of three times.
2b. To wash the RPC matrix by ltration: Suspend the silica-based gel in isopropanol
and pour the suspension into a sintered-glass funnel attached to a suction ask and
vacuum line. After all the solution has passed through the funnel, release the suction,
resuspend the gel in an excess of isopropanol, and restore the suction to lter the
packing solution. Repeat wash three times.
Bulk RPC supports are usually shipped dry. Washing by ltration is a faster method than
washing by settling and decantation.

3. Prepare a slurry consisting of 5% (w/v) silica-based gel in isopropanol.

4. Degas the gel slurry under vacuum for 5 min.
5. Mount the column (with a frit at the lower end) vertically and wash it with isopropanol. Close outlet, leaving a few centimeters of isopropanol in the bottom of the
column.

Conventional
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6. Pour the gel slurry into the column. Try to avoid introduction of air bubbles by
pouring the slurry down a glass rod held against the side of the column.
7. Fill the remainder of the column with packing solution (isopropanol in this case) and
mount the adaptor. Connect the column to the pump.
8. Fill the reservoir with isopropanol. Open the bottom of the column and set the pump
to run at the desired ow rate. Continue passing isopropanol through the system until
a constant bed height and pressure drop is reached, then pass an additional 3 to 5
column volumes of isopropanol at the same ow rate.
IMPORTANT NOTE: The working column pressure should not exceed 70% to 75% of
the packing column pressure.
Different ow rates are used for different supports. For 15-m silica-based support with

300 A pore size, the column should be packed at a pressure of 350 bar (5000 psi).

9. Stop the pump and close the outlet of the column. Disconnect the inlet tubing and
remove the adaptor. Carefully remove any excess gel from the top, mount the upper
frit, and close the column. After testing (see below), the column is ready for use.

Column testing
Testing the packed column is necessary, even for the experienced researcher. For the
beginner, it is not unusual to have to repeat the packing at least once before achieving
success. Testing a column before it is used and regenerated also provides a baseline
for comparison of its performance after a number of runs. It is recommended that
both commercially prepacked and self-made columns be tested before use, and then
again before each additional set of applications (or on some regular schedule, e.g., after
every 10th run). To test a packed column, a low-molecular-weight substance that has no
interaction with the matrix is injected. Acetone is the most frequently used substance for

70
0.72

80
0.97

40

60

0.32

30
20

40

Buffer A (%)

Relative absorbance (mAU)

50

10
20
0
-10

Reversed-phase
HPLC of Proteins

100

0.48

60

10
0

0.2

0.4

0.6

0.8

1
1.2
Time (min)

1.4

1.6

1.8

Figure 8.7.5 Column test for a monolithic, polymer-based, disk-shaped column. Separation of
a mixture of standard proteins. Separation conditions: column, polystyrene-divinylbenzene CIM
disk, 12 mm I.D. 3 mm bed height, column volume 0.34 ml; Eluent A, 20% acetonitrile containing
0.15% TFA; Eluent B, 70% acetonitrile containing 0.15% TFA; gradient, 100% Eluent A for 6 sec,
followed by 0 to 100% Eluent B over 90 sec; isocratic run for 10 sec at 100% B, followed by column
equilibration (100% Eluent A for 20 sec), flow rate 3 ml/min, back pressure 3 bar, detection at 280
nm, room temperature. Sample: test protein solution dissolved in water for HPLC, injection volume,
20 l. Peaks (according to the elution order): impurity from ribonuclease A, ribonuclease A (1.5
mg/ml), cytochrome C (0.5 mg/ml), bovine serum albumin (2.5 mg/ml), ovalbumin (3.0 mg/ml),
and impurity from ovalbumin. Courtesy of Drs. A. Strancar and M. Barut, of BIASeparations.

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column testing. The method for calculating the number of theoretical plates (N), height
equivalent to a theoretical plate (H, or HETP), and asymmetry factor (AS ) is already
comprehensively presented in UNIT 8.4. Here, some additional practical aspects regarding
the testing of the column performance and additional tips for monolithic columns are
described.
Prepacked, ready-to-use particle-based or monolithic columns are delivered with a test
chromatogram. However, low-molecular-weight substances are frequently used for test
chromatograms, even if the column is intended for separation of high-molecular-weight
substances such as polypeptides and proteins. To check if the pore size and support
properties are adequate for protein separation (regarding both column selectivity and
sample recovery), a repeated test with a mixture of standard proteins is recommended.
The conditions for this run (sample loading, ow rate, gradient, temperature) should be
as close as possible to the conditions expected for the separation of protein mixtures
that will be performed with the column. Such a test on a monolithic column is shown in
Figure 8.7.5. This column enables a very fast separation, and the column testing should
be performed using a high ow rate and over a very short separation time. To check
the recovery, separated proteins can be collected, and the protein content in each peak
determined. An additional purity test, e.g., by SDS-PAGE (UNIT 10.1), can also be very
useful.

PILOT EXPERIMENT TO CHOOSE A MATRIX AND DETERMINE

CONDITIONS FOR SAMPLE APPLICATION AND ELUTION FOR RPC
SEPARATION OF PROTEINS

BASIC
PROTOCOL 2

As discussed above, different supports for reversed-phase chromatographic separation

of proteins differ in their hydrophobicity, matrix surface, and pore and particle size. In
this protocol, guidelines for selecting the appropriate support, and conditions for sample
application and elution of target molecules, are discussed. Very small columns and small
amounts of sample and mobile phase are used for these pilot experiments. A small amount
of sample dissolved in starting solution (Eluent A) is applied to different columns (e.g.,
silica-based columns with C4 , C8 , or C18 ligands, or polymer-based RP columns). After
column washing with starting eluent, elution is carried out using a continuous gradient or
step gradient of Eluent B (the organic solvent). Nonbinding species (ow-through) and
fractions eluted during the chromatographic separation are analyzed using an appropriate
assay to identify the protein of interest (e.g., SDS-PAGE, immunoblot, ELISA, or simple
determination of protein concentration). After these analyses, the most appropriate matrix
is identied. If an analytical separation method is being developed, the most selective
matrix, which allows isolation of a target component as a single peak with high purity, is
chosen. For preparative separations, both purity and yield are critical factors.
Method development can be conveniently accomplished with small (50- or 100-l) resin
columns mounted in a 96-well format and employing a robotic liquid-handling system.
This system is fast and reproducible and makes possible both very fast screening of
the resins and method development (Coffman et al., 2008). Mant and Hodges (2002)
developed a strategy for scaling-up and preparative purication of proteins from the
troponin complex from rabbit skeletal muscle. Their experiences can be further used
for choosing an appropriate matrix and determining the optimal binding and elution
conditions for both analytical- and preparative-scale separations of proteins, as well as
for scaling up a separation process.

Materials
Reversed-phase supports (e.g., large-pore silica gel with C4 , C8 , or C18 ligands;
also see Table 8.7.1)

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Starting solution (Eluent A), degassed: 0.1% (v/v) TFA in water or 0.1% (v/v)
formic acid in water (1 liter will be needed)
Protein mixture to be puried (about 5 to 10 mg protein/ml), dissolved in Eluent A
Eluent B, degassed: acetonitrile containing 0.1 % TFA or formic acid (1 liter)
1-ml chromatography columns: several manufacturers, e.g., GE Healthcare and
Tosoh Bioseparations, offer ready-to-use prepacked chromatographic columns
with supports; the hardware (empty column, frits and column cartridge) can also
be reused for packing and testing of new materials; disk-shaped monolithic
columns (BIA Separations, http://www.biaseparations.com) can be purchased
and mounted in corresponding cartridges
Chromatographic system with pumps, gradient mixer, detector, and fraction
collector (e.g., Agilent Technologies, Waters, Knauer, http://www.knauer.net)
1. Pack each column as described in Basic Protocol 1 (or use prepacked 1-ml columns
for screening).
2. Equilibrate each column with 10 column volumes (CV) of Eluent A.
3. Apply 0.1 to 0.5 mg of protein solution dissolved in Eluent A to each column, wash
the column with 5 CV of Eluent A, and collect the unbound fraction (owthrough).
Use a slower ow rate for sample application: e.g., 0.25 ml/min for a 1-ml column.
Column washing and elution can then be performed at 0.5 to 1.0 ml/min.

4. Elute the sample using either a step gradient (e.g., 10%, 20%, 30%, 40%, 50%, 60%,
75%, and 100% Eluent B), or gradient elution from 100% Eluent A to 100% Eluent
B in 30 min.
discusses the general rules for choosing a matrix and determining binding and
elution conditions for hydrophobic interaction chromatography. In principle, these rules
are fully applicable for RP-HPLC, even if method development is performed using a highthroughput robotic liquid-handling system. Applied to reversed-phase chromatography,
these rules are as follows.

UNIT 8.4

1. The most appropriate support is the one that allows the greatest amount of contaminant
material to pass through the column during sample application and initial washing with
Eluent A. If the protein of interest does not bind to the column or binds weakly, choose
the more hydrophobic matrix. The evaluation test should be performed even if the target
protein does not bind or only weakly binds to the column, because a considerable amount
of contaminating material may be bound to the column and hence removed.
2. If the protein of interest does not elute until 100% Eluent B, the concentration of organic
solvent in the starting solution may be increased, or a less hydrophobic matrix should be
used.
BASIC
PROTOCOL 3

Reversed-phase
HPLC of Proteins

ELUTION OF PROTEINS FROM RPC COLUMNS

Continuous gradient elution
Simple linear gradients are the rst choice in pilot experiments for determining initial
conditions for separation of proteins in RP-HPLC. Gradient elutions in all types of adsorption chromatography, such as RPC, HIC, and ion-exchange chromatography, follow
the same rules. UNIT 8.4 gives an overview of the rules for HIC that can be also applied
for RPC separation of proteins.
Important and specic rule for the RPC
The use of water-organic solvents in RPC sometimes results in higher-viscosity mixtures
than the starting viscosity of Eluent A (containing water and ion-pairing reagents) or of
pure Eluent B. The result is increased back pressure during the separation.

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Use of step gradients

Stepwise elution (or step gradient) is preferred in industrial chromatography for largescale preparative applications, because on this scale it is technically simpler than gradient
elution (see also UNIT 8.4). The use of stepwise elution is also preferred if chromatographic
separation is performed on small- (30- to 400-l, disk-shaped), medium-, and large-scale
(8 ml up to 8 liters, cylinder-shaped) monolithic columns (Josic and Strancar, 1997),
because the component of interest is eluted in a more concentrated form. However, it is
important to make sure that there is no coelution of a more strongly bound component.
Artifact peaks can also appear if the next step is started too early, i.e., during a trailing
peak. In order to characterize sample behavior on the column, it is recommended to
optimize a stepwise elution gradient by performing several gradient elutions. As described
in UNIT 8.4, the development of a step gradient includes three stages.
1. A continuous gradient should be optimized to elute all compounds that are less
hydrophobic than the protein of interest while allowing the protein of interest to
remain bound to the column.
The elution strength and gradient volume should be sufcient to wash out all less hydrophobic contaminants, but not high enough to elute the protein of interest.

2. Increase the elution strength to elute the protein of interest with minimal dilution.
More hydrophobic, tightly bound contaminants should not be eluted.

3. Further increase the elution strength to elute the most tightly bound, hydrophobic
contaminants from the column.
4. Once these regions of the chromatogram are identied, a step gradient can be designed (see UNIT 8.4).

DESALTING OF PROTEINS
Rapid desalting and concentration of protein solutions is an important step, e.g., prior
to further mass spectrometric or electrophoretic analysis. Monolithic or particle-based
reversed-phase supports are packed in different-sized pipet tips (usually between 5 and
20 l, e.g., 10-l ZipTip pipet tip, Millipore). This protocol describes the use of ZipTip
10-l pipet tips containing C4 or C18 particle-based reversed-phase media for desalting
and concentration of peptide and protein solutions.

BASIC
PROTOCOL 4

Materials
Protein sample: adjust sample to 0.1% (v/v) triuoroacetic acid (TFA); nal sample
pH should be <4.0
ZipTip (Millipore) C4 or C18 pipet tips with a standard bed volume of 0.6 l for
sample elution in 1 to 4 l
Wetting solution: 100% acetonitrile
Equilibration/wash solution: 0.1% (v/v) TFA in HPLC- or MS-grade H2 O
Elution solution: 0.1% (v/v) TFA in 50% (v/v) acetonitrile (or methanol); for
fractionation of proteins prepare varying concentrations of acetonitrile in H2 O,
e.g., 5%, 10%, 20%, 30%, and 50%) with or without 0.1% TFA
10-l pipettor compatible with the ZipTips
NOTE: To achieve optimal sample uptake and delivery, set the pipettor to 10 l and
attach the tip containing RPC resin. Throughout the desalting procedure, depress and
release the plunger slowly to ensure optimal movement of solution through the resin
bed.

Conventional
Chromatographic
Separations

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NOTE: Because of their lower hydrophobicity, the pipet tips containing the C4 resin are
most applicable for proteins, while the tips containing the more hydrophobic C18 resin
are most suitable for low-molecular-weight proteins and peptides.
1. To achieve the maximum binding, ensure that the nal sample solution has a pH
value lower than 4.0.
Optimal binding of proteins may also require a chaotropic reagent (e.g., 1 to 6 M
guanidineHCl). A chaotropic reagent disrupts the three-dimensional structure of proteins and denatures them. Chaotropic agents interfere with stabilizing intramolecular
protein interactions that are mediated by noncovalent forces. For less hydrophobic proteins (e.g., from serum or cytoplasm), lower concentrations of chaotropic reagent (1 to
2 M) are needed. If the sample contains very hydrophobic proteins (e.g., from cellular
membranes), higher concentration of chaotropic reagents, up to 6 M, are recommended.
If the sample does not already contain chaotropic reagents, add them immediately before
application to the resin. Chaotropic reagents will be removed during the wash steps
following the sample binding.

2. Slowly depress the pipettor plunger to a dead stop. Using the maximum volume
setting (10 l), aspirate 100% acetonitrile (wetting solution) into the tip. Expel the
solution through the tip and discard. Repeat two times.
3. Aspirate three times with equilibration solution (0.1% TFA), and dispense to waste.
4. Fully depress the pipettor plunger to a dead stop, and then slowly aspirate the sample
protein mixture. To attain maximum binding of complex mixtures, aspirate and
dispense the sample for 7 to 10 cycles. Keep the nal dispensed solution for later
analysis.
5. Aspirate equilibration/wash solution (0.1% TFA) into tip, then expel and discard the
wash. Repeat the washing two times.
Additional washing or washing with 5% methanol in 0.1% TFA can improve desalting
efciency.
These wash solutions can be kept for later analysis, such as SDS-PAGE during method
development, but this is not necessary for routine work.

6. Aspirate 5 l of elution solution (0.1% TFA in 50% acetonitrile or methanol).

Aspirate and dispense eluent through the gel in the pipet tip at least three times
without introducing air bubbles into the sample.
CAUTION: Organic solvents that are used as eluents are volatile, and evaporation can
occur rapidly. In this case add more eluent to recover the sample. For direct spotting onto
a MALDI-TOF mass spectrometry target, 0.5 to 4 l desalted and eluted sample can be
directly applied onto the target.
Stepwise elutions with increasing concentrations of organic solvent (see above) may also
be used.
SUPPORT
PROTOCOL

Reversed-phase
HPLC of Proteins

REGENERATION, CLEANING, AND STORAGE OF RPC COLUMNS

Materials
Eluent A: 0.1% (v/v) TFA in H2 O
Eluent B: 0.1% (v/v) TFA in acetonitrile
0.5 to 1.0 M NaOH
Concentrated acetic acid
Storage solvent: 70% methanol, 70% to 80% acetonitrile, or 70% to 80%
isopropanol
Used RPC column

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Regeneration
In order to restore its original functionality, the RPC matrix must be regenerated after
each column run. The best and safest way to ensure the regeneration is to incorporate
this step into the program for each column run. The simplest regeneration procedure is
to wash the column with several (usually ve) column volumes of Eluent B (organic
solvent). The protocol for wash and re-equilibration of an RPC-column is:
1. Wash the column with at least 5 column volumes (CV) of 100% Eluent B to remove
any tightly bound components from the previous run.
2. Apply a decreasing gradient over 2 to 3 CV from 100% Eluent B to 100% Eluent A
to avoid damaging the matrix by a sudden eluent change.
3. Re-equilibrate the column using at least 10 CV of the starting eluent (Eluent A) or
storage solvent (see Storage of RPC columns below).
If a less hydrophobic solvent has been used for the elution (e.g., methanol or acetonitrile), a
stronger solvent such as isopropanol can be applied to remove hydrophobic contaminants
that may still be bound. The most appropriate solvent for column regeneration must be
determined empirically.

Cleaning
Correct sample preparation (e.g., ltration and/or centrifugation), the use of clean eluents
(e.g., Milli-Q puried water or equivalent and HPLC-grade organic solvents), removal
of any solid impurities, and a nal wash with at least 5 CV of Eluent B is the best way
to keep most columns in good condition for a long time (over 200 runs).
Reduced performance, loss of resolution, increasing back pressure, or complete blockage
are indicators that the column needs to be cleaned using more stringent procedures to
remove tightly bound and precipitated components such as lipids and denatured proteins.
Most contaminants bind and precipitate directly after the contact with the resin (or
column frits). This is the reason that some manufacturers recommend a column cleaning
in the reverse ow direction to minimize contact of impurities with the column bed.
The number of column volumes and contact time required for each cleaning may vary
according to the degree of contamination. It is also important to know that silica-based
columns are more sensitive to sudden changes of eluent and pH during the cleaning than
polymer-based columns.

Cleaning protocol
4. Wash the column with at least 10 CV of Eluent A.
5. Wash using the gradient of 20 to 30 CV from 0% to 100% Eluent B.
6. Wash using a gradient of 20 to 30 CV in reverse direction (from 100% to 0%
Eluent B)
7. Wash and equilibrate the column with at least 20 CV of Eluent A. Avoid sudden
change of eluents.
If performance is not restored, repeat the protocol using isopropanol as Eluent B. For
more rigorous cleaning, 10 CV of 90% acetic acid can be used. Re-equilibrate the column
with 10 CV of 0.1% TFA in water; do not leave the column in acetic acid.

Sanitization
Column sanitization is the inactivation and removal of microbial populations such as
bacteria and viruses from the gel matrix. Sanitization is not synonymous with sterilization,
which refers to the killing of a microbial population. In a sanitization step, not only the
microbial population, but often also their harmful products, endotoxins, are removed.

Conventional
Chromatographic
Separations

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Polymer-based reversed-phase columns can be sanitized by exposing the gel to 0.5 to 1.0
M NaOH for 30 to 60 min. This reagent is very effective for sanitizing the matrix. Sodium
hydroxide is also a very efcient cleaning reagent, especially for removal of tightly bound
harmful impurities, such as pyrogenic substances (endotoxins). However, silica-based
RPC columns are not stable at higher pH, and they cannot be sanitized with NaOH.
For silica-based columns, an alternative sanitizing protocol has to be developed, e.g.,
treatment with highly concentrated acetic acid. After treatment with either concentrated
alkaline or acidic solution, the pH has to be adjusted and a large volume of wash solution
may be required. Especially for silica-based RP columns, the pH control after washing
is extremely important. If the column is going to be stored, it should be washed with
sufcient amount of storage solution (see Storage of RPC columns, below).

Storage of RPC columns

For storage, wash the column with at least 5 CV of 70% methanol, 70% to 80% acetonitrile, or 70% to 80% isopropanol, all free from any other additives such as ion-pairing
modiers (e.g., TFA or formic acid). Proper storage conditions are especially critical
for silica-based columns. Store the columns at room temperature and seal them to avoid
drying out of the matrix. Because all organic solvents used for storage are bacteriostatic,
addition of other bacteriostatic agents (such as sodium azide) is not necessary.
COMMENTARY
Background Information

Reversed-phase
HPLC of Proteins

RPC is the leading method for analytical

separation of biomolecules, and RP-HPLC has
become the driving force for further development of chromatography as a fundamental
method for protein separation. After some loss
of popularity in the 1990s (McNay and Fernandez, 1999), RPC became the method of
choice for sample preparation and separation
of peptide digests at micro- and nano-scale
for protein identication by mass spectrometry (Josic and Clifton, 2007). The widespread
practical application of RP-HPLC for protein separation has been accompanied by a
signicant improvement in the understanding of the molecular basis of the retention
process and interaction of protein molecules
with hydrophobic ligands on the surface of
the chromatographic support. According to
Porath et al. (1973), the driving force of hydrophobic interactions is the gain in entropy
arising from the structural changes in the water surrounding the hydrophobic groups. The
displacement of water molecules surrounding the hydrophobic ligands on the matrix
and the hydrophobic residues of the protein leads to the increase in entropy. This
in turn leads to a negative value of G of
the system, which implies that hydrophobic
interaction is thermodynamically favorable.
Horvath and Melander (Molnar, 2005) applied the idea of so-called solvophobic interactions to understand molecular associations
of proteins and other biological molecules in

aqueous organic solvents (Sinanoglu, 1980).

The solvophobic theory is now the most accepted explanation of interactions between
protein molecules and hydrophobic supports in
RPC.
Elution by organic solvent gradient
Acetonitrile is the most frequently used organic solvent for protein elution in RPC. As an
example, Mant and Hodges (2002) developed
an RP-HPLC protocol for purication of all
components of a multi-protein complex from
rabbit skeletal muscle. They were also able
to scale up the analytical separation (40 mg
protein) to a semipreparative (1400 mg)and
preparative (5000 mg)scale separation. A linear gradient starting from 25% acetonitrile
(containing 0.05% TFA) to 55% acetonitrile
in 30 min was used. The main problem during
the development of the scaling-up procedure
was the aggregation and protein solubility. After solving these problems, excellent separation with high yields of puried proteins was
achieved.
For more hydrophobic proteins, other organic solvents such as isopropanol can be used.
Comparative studies with the most frequently
used organic solvents in RPCmethanol,
ethanol, acetonitrile, and isopropanolhave
conrmed that the slope of the retention dependency follows the eluotropic strength of
each solvent. Consequently the relative retention for a particular protein decreases in order:
methanol<ethanol<acetonitrile<isopropanol.

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This means that if the target protein is eluted

late by use of a methanol gradient, a stronger
solvent such as acetonitrile or isopropanol can
be used to shorten the elution time. However,
resolution will not necessarily improve with a
more strongly desorbing solvent. The viscosity
of the organic modier also plays an important
role, because increased viscosity of the mobile
phase also causes higher back pressure in the
column. It has to be taken into consideration
that viscosities of some water-organic solvent
mixtures can be higher than the viscosity of
pure aqueous solvent. Some solvents also have
signicant denaturing effects on protein and
peptide structure. As already mentioned, acetonitrile is the preferred organic modier for
protein separations, but this highly toxic solvent is a more powerful denaturant than alcohols, and also has the largest effect on protein
structure (see Table 8.7.2).
RPC with gradient elution is an excellent
separation method for preparative isolation of
therapeutic polypeptides and small proteins
such as insulin, interferons, and other hormones, and is the optimal analytical method
for protein and peptide separation (for applications, see Aguilar and Hearn, 1996). RPC on
monolithic supports with use of a steep gradient enables protein and peptide separation
in time frames of seconds (Josic and Clifton,
2007).
RPC at neutral and high pH
As with all types of adsorption chromatography, the optimal pH value is one of the
most important parameters to establish. When
silica-based media are used, separations are
mostly performed at pH 2 to 4. If endcapped silica-based supports are used, separations at elevated pH can be performed
(see Mobile phase under Strategic Planning,
above). Under low-pH conditions, good sample solubility and enhanced binding are almost always observed. Additionally, the risk
of so-called mixed-mode retention is also minimized. Mixed-mode retention causes an increase in retention time and signicant peak
broadening, and occurs only with silica-based
RPC media. Mixed-mode retention is caused
by ionic interactions taking place between negatively charged silanol groups exposed on the
surface of the silica medium and positively
charged amino groups of proteins. These interactions occur if the silica-based matrix is
not (or is insufciently) end-capped, or during the aging process of the column (Engelhardt et al., 2005 and Fig. 8.7.3A). Column
aging can often be accelerated by prolonged

exposure of the silica-based matrix to aqueous solution, e.g., with inappropriate storage
conditions (see above).
Separation at higher pH can often improve
both resolution and recovery of separated components. If properly used, the newly developed
end-capped silica-based supports enable separations at pH values up to 8 (Engelhardt et al.,
2005). When using polymer-based media, separations can be performed over a broad pH
range, from 1 to 12. A change in pH can also allow a new selectivity for the sample molecules.
Basic proteins often tail during elution at low
pH, and better resolution can be achieved at
higher pH values. For separations at higher
pH, ammonium acetate and ammonium formate (both for pH ranges between 6 and 10),
and tetramethylammonium chloride and tetrabutylammonium chloride for pH values over
7 (up to 12), can be used. The advantage of
ammonium formate is that this volatile reagent
can be used for HPLC separations in combination with ESI mass spectrometry (Yang et al.,
2009).
RPC and protein hydrophobicity
Hydrophobic RP matrices interact with
nonpolar residues of proteins and peptides,
and partial unfolding of the molecule occurs
during sample binding. Further unfolding and
denaturing of the molecule occurs during the
elution when organic solvents are used. The
use of elevated temperatures can signicantly
improve peak resolution and sometimes recovery, but this causes further unfolding and
denaturing of protein and peptide molecules,
and often loss of biological activity as well.
Some, mostly smaller proteins can be recovered and refolded to restore their activity, so
RPC can be successfully applied for their isolation and purication (see Aguilar and Hearn,
1996; McNay and Fernandez, 1999). Various
hydrophobicity scales for the amino acids have
been published, and they are used to determine
the hydrophobicity of proteins and peptides
and predict their elution in RPC (UNIT 8.4).
Hydrophobic membrane proteins can be difcult to recover from RP columns, and their
separation and further analysis is a challenge
in any chromatographic mode, especially in
RPC (Martosella et al., 2006).
RPC and other analytical techniques
RPC is practically insensitive to high salt
concentration, and mini-columns packed with
small amounts of reversed-phase support are
widely used for desalting and concentration
of protein and peptide solutions (see Basic

Conventional
Chromatographic
Separations

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200

Absorbance at 210 nm (mAU)

crude sample mixture

P

150

100

50

0
0

10

20

30
Time (min)

40

50

60

Figure 8.7.6 Isolation of recombinant protein TM 1-99 (mol. wt. 12,837 Da) from crude sample
mixture by use of an optimized gradient. Target protein (peak P) is eluted in the middle of the gradient as a single peak, and it is separated from less hydrophobic impurities that elute earlier (front
part of the chromatogram) and some more hydrophobic components that elute later. Conditions:
linear gradient (1% Eluent B/min) at a flow rate of 0.3 ml/min. Eluent A: 0.05% TFA in water, Eluent
B: 0.05% TFA in acetonitrile, temperature:
25 C, column: Zorbax 300SB-C8 (150 mm 2.1 mm

I.D.; 3.5 m particle size, 300 An pore size), from Agilent Technologies. Reprinted with permission
from Mills et al. (2006).

Protocol 4) before further analyses. Early

in its application for separation of proteins,
RPC was used in combination with sizeexclusion or ion-exchange chromatography
(Josic et al., 1982). The use of RPC in combination with ion-exchange chromatography
as a part of the automated on-line multidimensional HPLC system for protein and
peptide mapping was demonstrated by Wagner et al. (2002). RPC is also an integrated
part of each LC-tandem mass spectrometry
(MS/MS) system for protein sequencing and
identication (Josic and Clifton, 2007). Automated two-dimensional chromatography, containing one cation-exchange step combined
with the reversed-phase chromatography coupled to MS/MS, is widely used for protein
identication (Wolters et al., 2001).

Critical Parameters and

Troubleshooting

Reversed-phase
HPLC of Proteins

The major advantage of RPC is that the

interaction between the sample and chromatographic material is not impaired by high concentrations of salt. However, it is important
that the sample be completely dissolved in
aqueous buffer, and that the column be properly equilibrated. Incomplete equilibration of
the column or higher content of organic sol-

vent can inuence the retention time and

will have negative effects on reproducibility. Troubleshooting is sometimes different
for polymer- and silica-based columns, and
silica-based RP resins are somewhat more
sensitive, especially to high pH and rapid
changes in solvent composition after gradient
elution and return to starting conditions (column regeneration). On the other hand, silicabased columns are more pressure-resistant
than polymer-based ones.
Sample equilibration
The sample must be completely dissolved
in the starting eluent and free of particles. If
the salt concentration in the sample solution is
high, the starting solution has to be aqueous
to avoid sample precipitation on the column.
RPC is a binding technique, and the sample
load (mass) is of greater signicance than the
sample volume. The amount of sample that
can be applied on a column depends on the
binding capacity of the support and degree of
resolution required.
Column equilibration
The column must be equilibrated using at
least 10 CV of starting eluent (Eluent A) and
should be performed at the temperature at
which the chromatography will be run. The

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time saved for shorter column equilibration

often results in time and material lost for an
unsuccessful chromatographic run.
Optimization of the resolution of a target
component
The methodology for optimization of an
RPC separation is similar to that for HIC, as
described in UNIT 8.4. Instead of changing salt
concentration (in HIC), in RPC the concentration of more hydrophobic organic solvent
B is increased. The almost ideal case for isolation of a pure component (target protein) is
shown in Figure 8.7.6, where the therapeutic
protein is eluted in the middle of the gradient,
far from the less hydrophobic components that
are eluted earlier, and the more hydrophobic
components eluted later by higher concentrations of the hydrophobic solvent B (Mills et al.,
2006).
Ghost peaks
So-called ghost peaks (or ghosting)
are usually caused by poor-quality eluent that
can contain trace amounts of organic impurities. These can bind to the column support,
are concentrated during the chromatographic
run, column equilibration, and sample application, and can appear during the elution as
unidentied, or ghost peaks. Ghost peaks
may also be caused by incomplete elution of
very hydrophobic substances from previous
runs (so-called carry-over or cross-over
contamination). These peaks can be identied
by running a blank gradient, without sample application. Use of pure, HPLC-grade solvents usually prevents the appearance of ghost
peaks.
Baseline drift
The approximately linear, continuous increase (or decrease) of detector response during linear gradient elution is usually a result of
increased absorbancy of the organic modier,
or it can arise from the ion-pairing reagent,
if used. As the proportion of organic solvent
increases, the absorbance properties of the mobile phase may also change, and this can result in further baseline drift. Baseline drifting
can be compensated by using different concentrations of the UV-absorbing ion-pairing
agents in Eluent A and B, and balancing their
concentrations with respect to the UV absorption. It can also be simply memorized by the
computer from a blank run and automatically
corrected.

Time Considerations
Preparative RPC using prepacked columns
can be completed in 30 min to 3 to 4 hr,
depending on the size of the column, the sample, and the ow rate. It will take 2 hr to pack
the column, and an additional 2 to 4 hr will
be necessary for equilibration and testing. The
time between two chromatographic runs can
be up to 6 hr. Cleaning, sanitation (if necessary), and regeneration of the column can
be carried out overnight. Separation at higher
temperature can signicantly shorten the separation time, but it has almost no inuence on
the time required for cleaning, sanitation, and
regeneration.
If short columns, or columns containing
nonporous or monolithic reversed-phase supports, are used, an analytical chromatographic
run can be performed in less than a minute
(see Fig. 8.7.5 and Josic and Clifton, 2007).
Separation at elevated temperature can also
signicantly reduce separation time and also
improve resolution (Hancock et al. 1994, Chen
and Horvath, 1995).

Literature Cited
Aguilar, M.-E. and Hearn, M.T.W. 1996. Highresolution reversed-phase high-performance liquid chromatography of peptides and proteins.
Methods Enzymol. 270:3-26.
Chen, H. and Horvath, C.S. 1995. High-speed highperformance liquid chromatography of peptides
and proteins. J. Chromatogr. A 705:3-20.
Coffman, J.L., Kramarczyk, J.F., and Kelley, B.D.
2008. High-throughput screening of chromatographic separations: I. Method development and
column modeling. Biotech. Bioeng. 100:605618.
Dolan, J.W. 2002. Temperature selectivity in
reversed-phase high performance liquid chromatography. J. Chromatogr. A 965:195-205.
Engelhardt, H., Blay, C., and Saar, J. 2005.
Reversed-phase chromatography: The mystery
of surface silanols. Chromatographia 62:S19S29.
Eschelbach, J.W. and Jorgenson, J.W., 2006. Improved protein recovery in reversed-phase liquid
chromatography by the use of ultrahigh pressure. Anal. Chem. 78:1697-1706.
Hancock, W.S., Chloupek, R.S., Kirkland, J.J., and
Snyder, L.R. 1994. Temperature as a variable
in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples: I. Optimizing the separation of a
growth hormone tryptic digest. J. Chromatogr.
A 686:31-43.
Hearn, M.T.W. 1984. Reversed-phase high performance liquid chromatography. Methods Enzymol. 104:190-212.

Conventional
Chromatographic
Separations

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Josic, D.J. and Clifton, J.G. 2007. Use of monolithic

supports in proteomics technology. J. Chromatogr. A 1144:2-13.
Josic, D.J. and Strancar, A. 1997. Application
of membranes and compact, porous units for
separation of biopolymers. Ind. Eng. Chem. Res.
38:333-342.
Josic, D.J., Reutter, W., and Molnar, I. 1982. HPLC
of membrane bound proteins. In Practical Aspects of Modern HPLC (I. Molnar, ed.) pp. 109121. Walter de Gruyter, Berlin, New York.
Lee, D., Svec, F., and Frechet, J.M. 2004. Photopolymerized monolithic capillary columns for
rapid micro high-performance liquid chromatographic separation of proteins. J. Chromatogr. A
1051:53-61

Porath, J., Sundberg, L., Fornstedt, N., and Olsson, I. 1973. Salting-out in amphiphilic gels as a
new approach to hydrophilic adsorption. Nature
245:465-466.
Shi, Y., Xiang, R., Horvath, C.S., and Wilkins, J.A.
2004. Role of chromatographic techniques in
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Sinanoglu, O. 1980. The solvophobic theory for
the prediction of molecular conformations and
biopolymer bindings in solution with recent direct experimental tests. Int. J. Quant. Chem.
18:381-392.
Svec, F. and Huber, C.G. 2006. Monolithic materials: Promises, challenges, achievements. Anal.
Chem. 78:2101-2107.

Luo, Q., Shen, Y., Hixson, K.K., Zhao, R., Yang,

F., Moore, R.J., Mottaz, H.M., and Smith, R.D.
2005. Preparation of 20 m-i.d. silica-based
monolithic columns and their performance for
proteomics analyses. Anal. Chem. 77:50285035

Wagner, K., Miliotis, T., Marko-Varga, G.,

Bischoff, R., and Unger, K.K. 2002. An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation. Anal. Chem. 74:809820.

MacNair, J., Lewis, K.C., and Jorgenson, J.W. 1997.

Ultrahigh-pressure reversed-phase liquid chromatography in packed capillary columns. Anal.
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Wolters, D.A., Washburn, M.P., and Yates, J.R.

III 2001. An automated multidimensional protein identication technology for shotgun proteomics. Anal. Chem. 73:5683-5690.

Mant, C.T. and Hodges, R.S. 2002. Reversed-phase

liquid chromatography of proteins from rabbit
skeletal troponin, a multi-protein complex. J.
Chromatogr. A 972:101-114.

Yang, Y., Boysen, R.I., Harris, S.J., and Hearn,

M.T.W. 2009. Peptide mapping with mobile phases of intermediate pH value using
capillary reversed-phase high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry. J. Chromatogr. A
1216:3767-3773.

Martosella, J., Zolotarjova, N., Liu, H., Moyer, S.C.,

Perkins, P.D., and Boyes, B.E. 2006. High recovery HPLC separation of lipid rafts for membrane
proteome analysis. J. Proteome Res. 5:13011312.
McNay, J. and Fernandez, E.J. 1999. How does a
protein unfold on a reversed-phase liquid chromatography surface? J. Chromatogr. A 849:135148.
Mills, J.B., Mant, C.T., and Hodges, R.S. 2006. One
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Chromatographia 62:S7-S17.

Key References
Aguilar and Hearn, 1996. See above.
Describes both theoretical and practical aspects
of analytical and preparative reversed-phase chromatography and includes a number of references to
theory as well as practical aspects of this method
in the literature.
Shi et al., 2004. See above.
An excellent review about the use of chromatographic techniques, especially reversed-phase
HPLC in proteomics technology.

Reversed-phase
HPLC of Proteins

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