RP HPLC

UNIT 8.7Reversed-Phase High Performance LiquidChromatography of ProteinsDjuro Josic1 and Spomenka Kovac21Proteomics Core, COBRE Center for Cancer Research Development, Rhode Island Hospitaland Brown University, Providence, Rhode Island2Department of Chemistry, J. J. Strossmayer University, Osijek, Croatia

ABSTRACTReversed-phase HPLC (RP-HPLC) is one of most important techniques for protein sep-arations and the method of choice for peptide separation. RP-HPLC has been applied onthe nano, micro, and analytical scale, and has also been scaled up for preparative puri-cations, to large industrial scale. Because of its compatibility with mass spectrometry,RP-HPLC is an indispensable tool in proteomic research. With modern instrumentationand columns, complex mixtures of peptides and proteins can be separated at attomolarlevels for further analysis. In addition, preparative RP-HPLC is often used for large-scalepurication 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 isalso provided for cleaning, regenerating, and storing reversed-phase chromatographycolumns. Curr. Protoc. Protein Sci. 61:8.7.1-8.7.22. C 2010 by John Wiley & Sons, Inc.Keywords: reversed-phase HPLC proteins peptides LC-MS/MS

INTRODUCTIONDue to its excellent resolving power, convenience, versatility, stability, and reproducibil-ity, reversed-phase high-performance liquid chromatography (RP-HPLC) is one of themost important techniques for protein separations and the method of choice for peptideseparations. RP-HPLC has been applied on the nano, micro, and analytical scale, andcan also be scaled up for preparative purication on the industrial scale (Aguilar andHearn, 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 resolu-tion offered by LC separation greatly enhances MS detection of sample components, andhigh-resolution separation reduces ion suppression in MS (Shi et al., 2004).Reversed-phase chromatography (RPC) is a separation method based on the hydropho-bicity of the protein molecule. In RPC, the hydrophobic stationary phase is based onsilica gel or a synthetic polymer. In recent years, instead of bulk materials for columnpacking, 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 asa compact disk, cylinder, or rod that does not contain interparticular voids. During chro-matographic separation, all mobile phase ows through the monolithic stationary phase,and the resulting convective ow greatly accelerates the mass transfer and the separationspeed. Monolithic stationary phases enable reduction of separation time by up to oneorder of magnitude (Josic and Clifton, 2007). The stationary phase (or chromatographicsupport) bears the hydrophobic ligands, mainly C4, C8, or C18-alkyl chains. The mobilephase 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 mobilephase to render the proteins and peptides positively charged, and to reduce undesirableinteractions with the stationary phase. In some cases, mobile phases of intermediate or

Current Protocols in Protein Science 8.7.1-8.7.22, August 2010Published online August 2010 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471140864.ps0807s61Copyright C 2010 John Wiley & Sons, Inc.

ConventionalChromato-graphicSeparations

8.7.1Supplement 61

Reversed-phaseHPLC of Proteins

8.7.2Supplement 61 Current Protocols in Protein Science

even basic pH values can also be used (Yang et al., 2009). With modern instrumentationand columns, complex mixtures of proteins can be separated in attomolar amounts forcollection and further analysis of the resolved components. On the other hand, preparativeRPC is often used for large-scale purication of proteins.Reversed-phase chromatographic separation involves loading of a protein mixture ontoa hydrophobic stationary phase in water or a water-solvent mixture, usually containingvery dilute acidtypically 0.1% formic or triuoroacetic acid (TFA). The elution ofbound components occurs by increasing the concentration of organic solvent (usuallyacetonitrile or methanol). According to theory, hydrophobic-interaction chromatography(HIC, UNIT 8.4) and RPC are related techniques, since both are based upon interactions be-tween hydrophobic regions (amino acids) in protein molecules and hydrophobic ligandsof a chromatographic support. However, experimentally these techniques are different inthe following ways:

1. The surface of the RPC supports is usually more hydrophobic than that of the HICmedium.

2. In HIC, concentrated salt solutions are used for sample application. In RPC, pro-teins and peptides are dissolved in water or water-organic solvent mixture, usuallycontaining a dilute acid (see above).

3. Because of stronger hydrophobic interaction, organic solvents are used for RPCelution. Elution in HIC occurs by decreasing the salt concentration in the mobilephase.

4. The use of organic solvents in RPC frequently leads to denaturation and loss of biolog-ical activity of biopolymers, especially of high-molecular-weight proteins. However,denaturation and consequent loss of biological activity can be minimized or even re-versed 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 controllingselectivity in RP-HPLC separations of proteins. In routine use, most HIC separationsare performed at room temperature.

RPC provides excellent resolution of complex protein mixtures on an analytical, micro-analytical, or nanoanalytical scale. This chromatographic method also has great potentialfor high-resolution purication as well as for low-resolution desalting steps, especiallyin sample preparation for proteomics applications (Josic and Clifton, 2007). Combinedwith other chromatographic methods, especially ion-exchange chromatography, and as acomponent (dimension) in so-called multidimensional liquid chromatography (MDLC),RPC is a key method for separation, identication, and characterization of proteins invery complex mixtures. MDLC followed by MS (or more frequently MS/MS) is a fastand reliable method for protein identication and characterization in proteomics (Wagneret al., 2002).Sample preparation, selection of chromatographic support, column, and mobile phase,together with the separation design and optimization, are discussed in the StrategicPlanning section below. Protocols are provided for packing and testing a column (seeBasic Protocol 1), as well as protein separation by use of gradient or step elution (seeBasic Protocol 2 and Basic Protocol 3). Desalting of protein solutions is described inBasic Protocol 4. Cleaning, regenerating, and storing of RPC columns are described inthe Support Protocol.


8.7.3Current Protocols in Protein Science Supplement 61

STRATEGIC PLANNINGThe performance of a typical RPC separation of proteins is depicted in Figure 8.7.1Aand 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 elutionscheme combining isocratic elution and gradients with different slopes (see Fig. 8.7.1B).A successful RP-HPLC separation is inuenced by many parameters. Consequently, tomeet the requirements for an application, the RPC separation must be optimized. The

Elution time (min)

crude troponin

TnT TnI

400

200

mAU

TnTTm

TnC50

25

10 20 30

%ACN

Elution time (min)

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

100% IM10% TnT100% Tnl 100%

TnC

10 30 50 70 90 110 130 150

Tota

l are

a un

its /

min

1

07

170 190

90% TnT7

6

5

4

3

2

1

0

A

B

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), tro-ponin 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, watercontaining 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 con-

taining 0.05% TFA; Eluent B, acetonitrile containing 0.05% TFA. Sample load, 5700 mg in 440 mlwater containing 0.05% TFA (13 mg protein/ml); following sample loading at 22 ml/min, a 10-minisocratic hold (at constant solvent concentration) with water containing 0.05% TFA, followed bylinear A-B gradient (1.7% acetonitrile/min) up to 25% acetonitrile, then 0.1% acetonitrile/min upto 35% acetonitrile and, finally, 0.5% acetonitrile/min up to 55% acetonitrile. The positions of theindividual components identified following fraction analysis are denoted in histograms. Reprintedfrom Mant and Hodges (2002), with permission.


following are considerations for selecting and optimizing media and conditions for anRPC separation of proteins and peptides:1. Select the stationary and mobile phases that provide the best resolution and recoveryunder the simplest starting conditions. For routine RP-HPLC separations, TFA andacetonitrile are the most commonly used components of the mobile phase. For RPCapplications in an LC-MS/MS system, TFA is replaced by another volatile organicacid, most commonly formic acid.

2. Determine the pH that provides best resolution. It is very important to know that forseparation under basic conditions (pH values higher than 8), only polymer-based chro-matographic supports can be used. Silica-based supports (both particles andmonoliths)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 theirorder 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 phaseprices and waste disposal should be taken in consideration to provide an optimalprocess economy.

Choosing the optimal support and column dimensions is crucial for a successful chro-matographic separation. A list of some commercially available media for RP-HPLC isgiven in Table 8.7.1. The selection of an RPC support must bemade empirically. Themostcritical factors in choosing the appropriate stationary phase are sample hydrophobicityand the need for column sanitation. For separation of highly hydrophobic components,a less hydrophobic stationary phase should be used to facilitate the elution. Proteins thatbind strongly to a more hydrophobic support bind more weakly to a less hydrophobicmedium, and will also be eluted at lower concentrations of organic solvent. If NaOHis needed for sanitation (e.g., for isolation of therapeutic proteins), only polymer-basedRPC stationary phases can be used.

Table 8.7.1 Some Commercially Available Supports for Reversed-phase HPLC of Proteins and Peptidesa

Producer ProteinseparationPeptideseparation

Silica-based

Polymer-based

Polymer-basedmonolithic

Silica-basedmonolithic Analytical Preparative

Agilent Yes Yes Yes No Yes No Yes YesAgilent-Varian Yes Yes Yes Yes No No Yes YesBIASeparations Yes Yes No No Yes No Yes Yes bDionex Yes Yes Yes No Yes No Yes NoGE Healthcare Yes Yes Yes Yes No No Yes NoMerck(Germany)

Yes Yes Yes No No Yes Yes Yes

TosohBioscience

Yes Yes Yes Yes No No Yes No

Waters Yes Yes Yes No No No Yes YesaProduct specications and guides for users of chromatographic columns and bulk supports can be found at manufacturers Web sites.bUp to 8-liter monolithic polymer-based columns.



Applications that involve the fractionation of complex samples, such as proteolytic diges-tions, require extremely high resolution. On the other hand, separation time and economyare more important factors for preparative applications, such as protein purication, espe-cially 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, ifRPC is used in the nal step, the purity of the product is crucial, and maximal resolutionhas to be achieved.

Ligand type and degree of substitutionThe most common column ligands for RPC are shown in Figure 8.7.2. The ligand isthe group that is immobilized on the chromatographic support, while the ligate is thecomponent of the sample that interacts with the immobilized ligand. The density andhydrophobicity of the ligand determine the hydrophobicity of the stationary phase and thestrength of the interactionwith the sample. If aliphatic chains are used, the hydrophobicityof the ligand increases with the chain lengthe.g., a stationary phase with immobilizedC8 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 thematrix, without the presence of an additional immobilized ligand.Binding capacity of an RPC support increases with increased number of ligands boundto its surface. However, after the ligand density reaches a certain level, the bindingcapacity of the stationary phase will also reach a plateau because of the steric hindrancein ligand-ligate interaction. In the case of further increase of ligand density, the strengthof binding between the sample components and the support will continue to increase.Consequently, elution of more hydrophobic samples (proteins and peptides) bound tosupports with very high ligand density will be difcult or nearly impossible.

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

SiO

SiO

H3C (CH2)16 CH2 SiO

SiO phenyl

octyl (C8)

octadecyl (C18)

N C SiO cyano

butyl (C4)

Figure 8.7.2 Most common column ligands used in reversed-phase HPLC.



5. Mechanical stability.The matrix bears the immobilized ligand and also contributes signicantly to the se-lectivity of the RPC resin. Both silica- and polymer-based, wide-pore materials are themost commonly used supports for the RPC of proteins. The typical structure of the sur-face of the silica-based RPC medium is given in Figure 8.7.3A. Almost all silica-basedRPC 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-basedRPC supports cannot be completely eliminated, and they may still interact with polarsolutes and basic components of the sample. Ionizable silanol groups arise not onlyfrom inadequate end-capping: they can also result from column aging or inappropriatestorage of the silica-based column (e.g., over a long period in water). Consequently, theselectivity of the column can be impaired (Engelhardt et al., 2005). The structure of apolystyrene-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 (seeabove). Immobilization of a ligand on the surface of polymer-based supports can furthertune their selectivity and increase the binding capacity. The main producers of polymer-based reversed-phase media are Varian and GE Healthcare (see SUPPLIERS APPENDIX).In order to balance high efciency and short separation time, spherical nonporous orporous particles with diameters between 1.5 and 5m, packed in short columns (3 to 5 cmlong), are often used. The efciency, and also the back pressure of the column, increases

C8 chains

H2C

end capping

CH2 CH CH2 CH CH2 CH CH2 CH CH2 CH





B

ACH2

CH2

CH2H2C

H2C

H2C

H2C

O

OH

O O O O O O O O O O O O

silanol groups

CH2

CH2

CH2H2C

H2C

H2C

H2CCH2

CH2

CH2H2C

H2C

H2C

H2CCH2

CH2

CH2H2C

H2C

H2C

H2C

CH2CH2CH2 CH2 CH2CH3

CH2Si CH3

O O O O O O O O O O O OH H

HSi HH

O O O O O O O O O O

H2C H2C H2CH2C H2C

Si Si Si Si Si Si Si Si Si Si Si Si

Si Si SiCH3 CH3 CH3CH3SiH

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-basedsupport (reprinted from Engelhardt et al., 2005, with permission). (B) Structure of a polystyrene-based reversed-phase HPLC support (Courtesy of GE Healthcare).



with smaller particle diameter. The main producer for nonporous, polymer-based RPCmaterials 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 high-performance separation of proteins. These columns are prepared with packing pressuresas high as 4100 bar, and their working back pressure is higher than 1500 bar. This methodis called Ultra High-Pressure Reversed-Phase Liquid Chromatography (UHPRPLC;also see Eschelbach and Jorgenson, 2006). These columns can be commercially obtainedfrom 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 ratesat 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 substancessuch as proteins and larger polypeptides become possible (Hearn, 1984). However, thesurface of the support and its capacity decreases with increasing pore size. Consequently,the capacities of porous materials are signicantly higher than those of nonporous silicagel or polymer-based supports. Nonporous chromatographic materials are also used forfast analytical high performance separations and for UHPRPLC of proteins (MacNairet al. 1997; Engelhardt et al. 2005).

Monolithic supportsIn proteomic applications and for in-process analysis in biotechnology,RPCcolumnswithhigh efciency and short analysis times are required.Hence, high-speed packingmaterialsare in demand. If porous materials are used, separation speed is limited by mass transferbetween the mobile phase and stationary liquid in the pores of the chromatographicsupport. For nonporous materials, the binding of sample components and subsequentelution occurs on the surface of the support, so separation speed is much less limitedby mass transfer. In order to increase the performance of nonporous chromatographicmaterial, particles with very small diameter (less than 3 m, e.g., Tosoh Bioscience, forpolymer-based nonporous columns and bulk materials) are used, and high column backpressure is often the limiting factor. Frequently, monoliths are an alternative to columnspacked with particulate stationary phases. The structure of monolithic materials (seeFig. 8.7.4A and B) allows very fast mass transfer during chromatographic separationthat is practically not limited by the ow rate. Because of the low back pressure and fastmass transfer, monolithic columns can be run at very high speed. As a result, separationtime is up to one order of magnitude faster than that of columns packed with bulkparticles (Svec and Huber, 2006; Josic and Clifton, 2007). Similar to columns packedwith spherical particles, monolithic columns are silica- (see Fig. 8.7.4A), or polymer-based (see Fig. 8.7.4B). In reversed-phase mode, monolithic columns are mostly usedfor 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.

InstrumentationRapid and high-performance separation in reversed-phase mode requires not only astationary phase with both minimal mass transfer and kinetic resistance, but also instru-mentation capable of generating fast and reproducible gradient elutions and providingprecise temperature control for both the column and mobile phases. Furthermore, theHPLC instrument must have low overall system dead volume (the volume between thesample injector and the outlet of the system, including the chromatographic column), a



A

B

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

precise sample introduction device, and a highly sensitive detector with short responsetime. For micro- and nano-HPLC separations that are performed mostly in proteomicapplications, the demand for minimal dead volume is extremely stringent.For preparative applications, a pump capable of delivering a high ow rate, typicallybetween 200 and 800 cm/hr, should be chosen. For high-speed analyses, especially ifmonolithic supports are used, pumps with higher ow rates will be needed. For micro-scale and nano-scale separations with ow rates in the nl/min and l/min range, mostlyin proteomic technology where the HPLC system is used for sample preparation oris 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 proteinseparation with integrated sample preparation. Size-selective sample fractionation wasfollowed by anion- or cation-exchange chromatography. The nal separation step prior toMALDI TOFMS was RPC on a short hydrophobic column. For UHPRPLC, fused silicacapillaries paced with nonporous octadecyl RP material are used. These columns have alength up to 66 cm and were packed with the pressures as high as 4100 bar. The pressuresrequired to run the system at optimumow rates are about 1400 bar (MacNair et al., 1997).



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

Mobile phaseIn order to increase the hydrophobicity of sample components, enhance binding to thechromatographic support and, if necessary, alter the retention time, ion-pairing agentsare added to the mobile phase. Ion-pairing reagents are ionic molecules having a chargeopposite to the charge of the analyte (protein) of interest, as well as a hydrophobicregion. The ion-pairing reagent masks the positive charges of the protein molecule (bypairing with them), and also enhances its hydrophobicity. Ion-paring agents are mostoften acids such as TFA or formic acid. If RPC separation is performed at neutral orbasic pH, ammonium formate or triethylamine is used. It should be stressed that mostRPC separations are performed at low pH (usually between pH 2 and 3) with an acidas ion-pairing reagent. Reduction of the pH is necessary to minimize the charge of theC-terminal COO groups. The correct pH can be crucial for a successful separation, andsome separations have to be performed at elevated pH (Yang et al., 2009). During thegradient elution, an organic modier is added to the eluent (solvent B) to weaken theinteraction of sample components with the hydrophobic support and to increase elutionstrength. The organic modier must be miscible in water and UV-transparent to enabledetection of eluting molecules, even at low wavelengths (down to 210 nm or lowerfor peptides). Acetonitrile, methanol, ethanol, and isopropanol are the most commonlyused 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 afterchromatographic separation.

Temperature selectivityHancock et al. (1994) and Chen and Horvath (1995) recognized the inuence of temper-ature on protein and peptide chromatographic separation. Elevated temperature enhanceskinetic and transport properties during the RPC separation. Selectivity changes caused bychanges of temperature were complementary to those effected by changing the solventstrength. The use of elevated column temperature may also serve as a useful tool to opti-mize a separation, if the temperature of the stationary phase is controlled. Demonstratedlong-term column stability at the anticipated operating temperature is the main require-ment for RPC separations at elevated temperature, and the thermal stability of the givensupport has to be determined before the separation method is established. RP-HPLCseparations at temperatures above the atmospheric boiling point have been performed,but these kinds of separations also require appropriate instruments and columns (Chenand Horvath, 1995).

SummaryA summary of the roles of different parameters in RP-HPLC separation of proteins andtheir inuence on strategic planning is given in Table 8.7.2, e.g., solvent strength andsolvent type are easy to manipulate and can be conveniently adjusted for optimizationof protein separation. Changing ion-paring reagents, column type, and mobile phaseadditives is less convenient and, if possible, should be avoided. Changing mobile-phasepH is also a powerful variable to adjust selectivity, but requires many optimizationexperiments. The selectivity effects of temperature and solvent strength are more or lessorthogonal and can also be used to modify the separation (Dolan, 2002).



Table8.7.2

Som

eFa

ctor

sth

atAf

fect

the

Choi

ceo

fVa

riabl

esfo

rR

P-H

PLC

Met

hod

Dev

elo

pmen

ta

Impactofdifferentfactors

Variable

Abilitytochangethe

separationfactor

(alpha)

Experimental

convenience

Speedofcolumn

equilibration

Method

robustness

Compatible

withlow

UVdetection

Large

orsmallnumberof

runsrequiredtoselect

optimum

value

ofvariable

Peaktracking

problems

1.Solvent

hydrophobicity

+++

+++

+++

+

2.Temperature

+++

+++

+++

+3.pH

++

04.Ion-pairing

++

+

0

05.Solvent

+0

++

00

+6.Colum

ntype

+

0

+

7.Mobilephase

additives

00

0

0

8.Buffertypeand

concentration

0

00

0

a ++,veryfavorable;+,favorable;,unfavorable;,unfavorable;0,noinuence.



BASICPROTOCOL 1

PACKING AND TESTING OF SEMI-PREPARATIVE AND PREPARATIVERP-HPLC COLUMNSMicroanalytical and analytical RP-HPLC columns are usually packed by the columnproducer, and are ready to use after proper column testing. Prepacked larger columnsfor semi-preparative and preparative separation of proteins and peptides are also com-mercially available, but it is less expensive and less risky to pack such columns in thelaboratory, especially during the development and scaling-up of methods for isolationand separation. This protocol provides basic rules for packing of semi-preparative andpreparative columns containing silica- or polymer-based reversed-phase materials (e.g.,C18-, C8-, or C4-silica, polystyrene/divinyl benzene, or other polymer-based beads withcorresponding ligands to provide appropriate surface hydrophobicity) with particle sizelarger than 15 m (usually up to 90 m), and pore size larger than 300 A. Large-particle-size supports are generally used for semi-preparative and preparative purications. Thelarge-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 andHodges, 2002). All sizes of monolithic columns, from nanoscale to preparative scale, areprepared by the producer (Josic and Clifton, 2007).Materials

Appropriate RPC support (silica- or polymer-based) with a particle size between 15and 90 m

Packing solution: isopropanol or methanol

Sintered-glass funnel (medium grade) and degasser and/or sonicatorSuction askVacuum sourceChromatography 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; Forsemi-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 RPCmatrix by decantation: Suspend the silica-based gel in isopropanol.

Allow gel to settle, decant the supernatant, and add an excess of isopropanol. Repeatwash a total of three times.

2b. To wash the RPC matrix by ltration: Suspend the silica-based gel in isopropanoland pour the suspension into a sintered-glass funnel attached to a suction ask andvacuum 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 thepacking solution. Repeat wash three times.Bulk RPC supports are usually shipped dry. Washing by ltration is a faster method thanwashing 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 iso-propanol. Close outlet, leaving a few centimeters of isopropanol in the bottom of thecolumn.



6. Pour the gel slurry into the column. Try to avoid introduction of air bubbles bypouring 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) andmount the adaptor. Connect the column to the pump.

8. Fill the reservoir with isopropanol. Open the bottom of the column and set the pumpto run at the desired ow rate. Continue passing isopropanol through the system untila constant bed height and pressure drop is reached, then pass an additional 3 to 5column volumes of isopropanol at the same ow rate.IMPORTANT NOTE: The working column pressure should not exceed 70% to 75% ofthe packing column pressure.Different ow rates are used for different supports. For 15-m silica-based support with300 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 andremove the adaptor. Carefully remove any excess gel from the top, mount the upperfrit, and close the column. After testing (see below), the column is ready for use.

Column testingTesting the packed column is necessary, even for the experienced researcher. For thebeginner, it is not unusual to have to repeat the packing at least once before achievingsuccess. Testing a column before it is used and regenerated also provides a baselinefor comparison of its performance after a number of runs. It is recommended thatboth commercially prepacked and self-made columns be tested before use, and thenagain before each additional set of applications (or on some regular schedule, e.g., afterevery 10th run). To test a packed column, a low-molecular-weight substance that has nointeraction with the matrix is injected. Acetone is the most frequently used substance for

Rel

ativ

e ab

sorb

ance

(mAU

)

Time (min)0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

70

60

50

40

30

20

10

0

-10

0.32

0.480.72

0.97

100

80

60

40

20

10

Buffer A (%)

Figure 8.7.5 Column test for a monolithic, polymer-based, disk-shaped column. Separation ofa mixture of standard proteins. Separation conditions: column, polystyrene-divinylbenzene CIMdisk, 12 mm I.D. 3 mm bed height, column volume 0.34 ml; Eluent A, 20% acetonitrile containing0.15% 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 columnequilibration (100% Eluent A for 20 sec), flow rate 3 ml/min, back pressure 3 bar, detection at 280nm, 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 (1.5mg/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.



column testing. The method for calculating the number of theoretical plates (N), heightequivalent to a theoretical plate (H, or HETP), and asymmetry factor (AS) is alreadycomprehensively presented in UNIT 8.4. Here, some additional practical aspects regardingthe testing of the column performance and additional tips for monolithic columns aredescribed.Prepacked, ready-to-use particle-based or monolithic columns are delivered with a testchromatogram. However, low-molecular-weight substances are frequently used for testchromatograms, even if the column is intended for separation of high-molecular-weightsubstances such as polypeptides and proteins. To check if the pore size and supportproperties are adequate for protein separation (regarding both column selectivity andsample recovery), a repeated test with a mixture of standard proteins is recommended.The conditions for this run (sample loading, ow rate, gradient, temperature) should beas close as possible to the conditions expected for the separation of protein mixturesthat will be performed with the column. Such a test on a monolithic column is shown inFigure 8.7.5. This column enables a very fast separation, and the column testing shouldbe performed using a high ow rate and over a very short separation time. To checkthe recovery, separated proteins can be collected, and the protein content in each peakdetermined. An additional purity test, e.g., by SDS-PAGE (UNIT 10.1), can also be veryuseful.

BASICPROTOCOL 2

PILOT EXPERIMENT TO CHOOSE A MATRIX AND DETERMINECONDITIONS FOR SAMPLE APPLICATION AND ELUTION FOR RPCSEPARATION OF PROTEINSAs discussed above, different supports for reversed-phase chromatographic separationof proteins differ in their hydrophobicity, matrix surface, and pore and particle size. Inthis protocol, guidelines for selecting the appropriate support, and conditions for sampleapplication and elution of target molecules, are discussed. Very small columns and smallamounts of sample andmobile phase are used for these pilot experiments. A small amountof 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). Aftercolumn washing with starting eluent, elution is carried out using a continuous gradient orstep gradient of Eluent B (the organic solvent). Nonbinding species (ow-through) andfractions eluted during the chromatographic separation are analyzed using an appropriateassay to identify the protein of interest (e.g., SDS-PAGE, immunoblot, ELISA, or simpledetermination of protein concentration). After these analyses, themost appropriatematrixis identied. If an analytical separation method is being developed, the most selectivematrix, which allows isolation of a target component as a single peak with high purity, ischosen. For preparative separations, both purity and yield are critical factors.Method development can be conveniently accomplished with small (50- or 100-l) resincolumns 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 ofthe resins and method development (Coffman et al., 2008). Mant and Hodges (2002)developed a strategy for scaling-up and preparative purication of proteins from thetroponin complex from rabbit skeletal muscle. Their experiences can be further usedfor choosing an appropriate matrix and determining the optimal binding and elutionconditions for both analytical- and preparative-scale separations of proteins, as well asfor 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)



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 AEluent B, degassed: acetonitrile containing 0.1 % TFA or formic acid (1 liter)

1-ml chromatography columns: several manufacturers, e.g., GE Healthcare andTosoh Bioseparations, offer ready-to-use prepacked chromatographic columnswith supports; the hardware (empty column, frits and column cartridge) can alsobe reused for packing and testing of new materials; disk-shaped monolithiccolumns (BIA Separations, http://www.biaseparations.com) can be purchasedand mounted in corresponding cartridges

Chromatographic system with pumps, gradient mixer, detector, and fractioncollector (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 columnsfor 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, washthe 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% EluentB in 30 min.

UNIT 8.4 discusses the general rules for choosing a matrix and determining binding andelution conditions for hydrophobic interaction chromatography. In principle, these rulesare fully applicable for RP-HPLC, even if method development is performed using a high-throughput robotic liquid-handling system. Applied to reversed-phase chromatography,these rules are as follows.1. The most appropriate support is the one that allows the greatest amount of contaminantmaterial to pass through the column during sample application and initial washing withEluent A. If the protein of interest does not bind to the column or binds weakly, choosethe more hydrophobic matrix. The evaluation test should be performed even if the targetprotein does not bind or only weakly binds to the column, because a considerable amountof 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 organicsolvent in the starting solution may be increased, or a less hydrophobic matrix should beused.

BASICPROTOCOL 3

ELUTION OF PROTEINS FROM RPC COLUMNSContinuous gradient elutionSimple linear gradients are the rst choice in pilot experiments for determining initialconditions for separation of proteins in RP-HPLC. Gradient elutions in all types of ad-sorption chromatography, such as RPC, HIC, and ion-exchange chromatography, followthe same rules. UNIT 8.4 gives an overview of the rules for HIC that can be also appliedfor RPC separation of proteins.Important and specic rule for the RPCThe use of water-organic solvents in RPC sometimes results in higher-viscosity mixturesthan the starting viscosity of Eluent A (containing water and ion-pairing reagents) or ofpure Eluent B. The result is increased back pressure during the separation.



Use of step gradientsStepwise elution (or step gradient) is preferred in industrial chromatography for large-scale preparative applications, because on this scale it is technically simpler than gradientelution (see also UNIT 8.4). The use of stepwise elution is also preferred if chromatographicseparation 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 isimportant 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 trailingpeak. In order to characterize sample behavior on the column, it is recommended tooptimize a stepwise elution gradient by performing several gradient elutions.As describedin 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 lesshydrophobic than the protein of interest while allowing the protein of interest toremain bound to the column.The elution strength and gradient volume should be sufcient to wash out all less hy-drophobic 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, hydrophobiccontaminants from the column.

4. Once these regions of the chromatogram are identied, a step gradient can be de-signed (see UNIT 8.4).

BASICPROTOCOL 4

DESALTING OF PROTEINSRapid desalting and concentration of protein solutions is an important step, e.g., priorto further mass spectrometric or electrophoretic analysis. Monolithic or particle-basedreversed-phase supports are packed in different-sized pipet tips (usually between 5 and20 l, e.g., 10-l ZipTip pipet tip, Millipore). This protocol describes the use of ZipTip10-l pipet tips containing C4 or C18 particle-based reversed-phase media for desaltingand concentration of peptide and protein solutions.Materials

Protein sample: adjust sample to 0.1% (v/v) triuoroacetic acid (TFA); nal samplepH should be



NOTE: Because of their lower hydrophobicity, the pipet tips containing the C4 resin aremost applicable for proteins, while the tips containing the more hydrophobic C18 resinare most suitable for low-molecular-weight proteins and peptides.1. To achieve the maximum binding, ensure that the nal sample solution has a pHvalue lower than 4.0.Optimal binding of proteins may also require a chaotropic reagent (e.g., 1 to 6 MguanidineHCl). A chaotropic reagent disrupts the three-dimensional structure of pro-teins and denatures them. Chaotropic agents interfere with stabilizing intramolecularprotein interactions that are mediated by noncovalent forces. For less hydrophobic pro-teins (e.g., from serum or cytoplasm), lower concentrations of chaotropic reagent (1 to2 M) are needed. If the sample contains very hydrophobic proteins (e.g., from cellularmembranes), higher concentration of chaotropic reagents, up to 6 M, are recommended.If the sample does not already contain chaotropic reagents, add them immediately beforeapplication to the resin. Chaotropic reagents will be removed during the wash stepsfollowing the sample binding.

2. Slowly depress the pipettor plunger to a dead stop. Using the maximum volumesetting (10 l), aspirate 100% acetonitrile (wetting solution) into the tip. Expel thesolution 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 sampleprotein mixture. To attain maximum binding of complex mixtures, aspirate anddispense the sample for 7 to 10 cycles. Keep the nal dispensed solution for lateranalysis.

5. Aspirate equilibration/wash solution (0.1% TFA) into tip, then expel and discard thewash. Repeat the washing two times.Additional washing or washing with 5% methanol in 0.1% TFA can improve desaltingefciency.These wash solutions can be kept for later analysis, such as SDS-PAGE during methoddevelopment, 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 timeswithout introducing air bubbles into the sample.CAUTION: Organic solvents that are used as eluents are volatile, and evaporation canoccur rapidly. In this case add more eluent to recover the sample. For direct spotting ontoa MALDI-TOF mass spectrometry target, 0.5 to 4 l desalted and eluted sample can bedirectly applied onto the target.Stepwise elutions with increasing concentrations of organic solvent (see above) may alsobe used.

SUPPORTPROTOCOL

REGENERATION, CLEANING, AND STORAGE OF RPC COLUMNSMaterials

Eluent A: 0.1% (v/v) TFA in H2OEluent B: 0.1% (v/v) TFA in acetonitrile0.5 to 1.0 M NaOHConcentrated acetic acidStorage solvent: 70% methanol, 70% to 80% acetonitrile, or 70% to 80%isopropanol

Used RPC column



RegenerationIn order to restore its original functionality, the RPC matrix must be regenerated aftereach column run. The best and safest way to ensure the regeneration is to incorporatethis step into the program for each column run. The simplest regeneration procedure isto wash the column with several (usually ve) column volumes of Eluent B (organicsolvent). 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 removeany tightly bound components from the previous run.

2. Apply a decreasing gradient over 2 to 3 CV from 100% Eluent B to 100% Eluent Ato 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) orstorage solvent (see Storage of RPC columns below).If a less hydrophobic solvent has been used for the elution (e.g., methanol or acetonitrile), astronger solvent such as isopropanol can be applied to remove hydrophobic contaminantsthat may still be bound. The most appropriate solvent for column regeneration must bedetermined empirically.

CleaningCorrect 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), removalof any solid impurities, and a nal wash with at least 5 CV of Eluent B is the best wayto keep most columns in good condition for a long time (over 200 runs).Reduced performance, loss of resolution, increasing back pressure, or complete blockageare indicators that the column needs to be cleaned using more stringent procedures toremove tightly bound and precipitated components such as lipids and denatured proteins.Most contaminants bind and precipitate directly after the contact with the resin (orcolumn frits). This is the reason that some manufacturers recommend a column cleaningin 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 varyaccording to the degree of contamination. It is also important to know that silica-basedcolumns are more sensitive to sudden changes of eluent and pH during the cleaning thanpolymer-based columns.

Cleaning protocol4. 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 suddenchange of eluents.If performance is not restored, repeat the protocol using isopropanol as Eluent B. Formore rigorous cleaning, 10 CV of 90% acetic acid can be used. Re-equilibrate the columnwith 10 CV of 0.1% TFA in water; do not leave the column in acetic acid.

SanitizationColumn sanitization is the inactivation and removal of microbial populations such asbacteria and viruses from the gelmatrix. Sanitization is not synonymouswith sterilization,which refers to the killing of a microbial population. In a sanitization step, not only themicrobial population, but often also their harmful products, endotoxins, are removed.



Polymer-based reversed-phase columns can be sanitized by exposing the gel to 0.5 to 1.0MNaOH for 30 to 60min. This reagent is very effective for sanitizing the matrix. Sodiumhydroxide is also a very efcient cleaning reagent, especially for removal of tightly boundharmful impurities, such as pyrogenic substances (endotoxins). However, silica-basedRPC 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 concentratedalkaline or acidic solution, the pH has to be adjusted and a large volume of wash solutionmay be required. Especially for silica-based RP columns, the pH control after washingis extremely important. If the column is going to be stored, it should be washed withsufcient amount of storage solution (see Storage of RPC columns, below).Storage of RPC columnsFor storage, wash the column with at least 5 CV of 70% methanol, 70% to 80% acetoni-trile, or 70% to 80% isopropanol, all free from any other additives such as ion-pairingmodiers (e.g., TFA or formic acid). Proper storage conditions are especially criticalfor silica-based columns. Store the columns at room temperature and seal them to avoiddrying 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.

COMMENTARYBackground InformationRPC is the leading method for analytical

separation of biomolecules, and RP-HPLC hasbecome the driving force for further devel-opment of chromatography as a fundamentalmethod for protein separation. After some lossof popularity in the 1990s (McNay and Fer-nandez, 1999), RPC became the method ofchoice for sample preparation and separationof peptide digests at micro- and nano-scalefor protein identication by mass spectrome-try (Josic and Clifton, 2007). The widespreadpractical application of RP-HPLC for pro-tein separation has been accompanied by asignicant improvement in the understand-ing of the molecular basis of the retentionprocess and interaction of protein moleculeswith hydrophobic ligands on the surface ofthe chromatographic support. According toPorath et al. (1973), the driving force of hy-drophobic interactions is the gain in entropyarising from the structural changes in the wa-ter surrounding the hydrophobic groups. Thedisplacement of water molecules surround-ing the hydrophobic ligands on the matrixand the hydrophobic residues of the pro-tein leads to the increase in entropy. Thisin turn leads to a negative value of G ofthe system, which implies that hydrophobicinteraction is thermodynamically favorable.Horvath and Melander (Molnar, 2005) ap-plied the idea of so-called solvophobic inter-actions to understand molecular associationsof proteins and other biological molecules in

aqueous organic solvents (Sinanoglu, 1980).The solvophobic theory is now the most ac-cepted explanation of interactions betweenproteinmolecules and hydrophobic supports inRPC.

Elution by organic solvent gradientAcetonitrile is the most frequently used or-

ganic solvent for protein elution in RPC. As anexample, Mant and Hodges (2002) developedan RP-HPLC protocol for purication of allcomponents of a multi-protein complex fromrabbit skeletal muscle. They were also ableto scale up the analytical separation (40 mgprotein) to a semipreparative (1400 mg)andpreparative (5000mg)scale separation. A lin-ear gradient starting from 25% acetonitrile(containing 0.05% TFA) to 55% acetonitrilein 30 min was used. The main problem duringthe development of the scaling-up procedurewas the aggregation and protein solubility. Af-ter solving these problems, excellent separa-tion with high yields of puried proteins wasachieved.For more hydrophobic proteins, other or-

ganic solvents such as isopropanol can be used.Comparative studies with the most frequentlyused organic solvents in RPCmethanol,ethanol, acetonitrile, and isopropanolhaveconrmed that the slope of the retention de-pendency follows the eluotropic strength ofeach solvent. Consequently the relative reten-tion for a particular protein decreases in order:methanol



Thismeans that if the target protein is elutedlate by use of a methanol gradient, a strongersolvent such as acetonitrile or isopropanol canbe used to shorten the elution time. However,resolution will not necessarily improve with amore strongly desorbing solvent. The viscosityof the organic modier also plays an importantrole, because increased viscosity of the mobilephase also causes higher back pressure in thecolumn. It has to be taken into considerationthat viscosities of some water-organic solventmixtures can be higher than the viscosity ofpure aqueous solvent. Some solvents also havesignicant denaturing effects on protein andpeptide structure. As already mentioned, ace-tonitrile is the preferred organic modier forprotein separations, but this highly toxic sol-vent is a more powerful denaturant than alco-hols, and also has the largest effect on proteinstructure (see Table 8.7.2).RPC with gradient elution is an excellent

separation method for preparative isolation oftherapeutic polypeptides and small proteinssuch as insulin, interferons, and other hor-mones, and is the optimal analytical methodfor protein and peptide separation (for appli-cations, see Aguilar and Hearn, 1996). RPC onmonolithic supports with use of a steep gra-dient enables protein and peptide separationin time frames of seconds (Josic and Clifton,2007).RPC at neutral and high pHAs with all types of adsorption chromatog-

raphy, the optimal pH value is one of themost important parameters to establish. Whensilica-based media are used, separations aremostly performed at pH 2 to 4. If end-capped silica-based supports are used, sep-arations at elevated pH can be performed(see Mobile phase under Strategic Planning,above). Under low-pH conditions, good sam-ple solubility and enhanced binding are al-most always observed. Additionally, the riskof so-called mixed-mode retention is also min-imized. Mixed-mode retention causes an in-crease in retention time and signicant peakbroadening, and occurs only with silica-basedRPC media. Mixed-mode retention is causedby ionic interactions taking place between neg-atively charged silanol groups exposed on thesurface of the silica medium and positivelycharged amino groups of proteins. These in-teractions occur if the silica-based matrix isnot (or is insufciently) end-capped, or dur-ing the aging process of the column (Engel-hardt et al., 2005 and Fig. 8.7.3A). Columnaging can often be accelerated by prolonged

exposure of the silica-based matrix to aque-ous solution, e.g., with inappropriate storageconditions (see above).Separation at higher pH can often improve

both resolution and recovery of separated com-ponents. If properly used, the newly developedend-capped silica-based supports enable sepa-rations at pH values up to 8 (Engelhardt et al.,2005).When using polymer-basedmedia, sep-arations can be performed over a broad pHrange, from1 to 12.A change in pH can also al-low a new selectivity for the samplemolecules.Basic proteins often tail during elution at lowpH, and better resolution can be achieved athigher pH values. For separations at higherpH, ammonium acetate and ammonium for-mate (both for pH ranges between 6 and 10),and tetramethylammonium chloride and tetra-butylammonium chloride for pH values over7 (up to 12), can be used. The advantage ofammonium formate is that this volatile reagentcan be used for HPLC separations in combina-tion with ESI mass spectrometry (Yang et al.,2009).

RPC and protein hydrophobicityHydrophobic RP matrices interact with

nonpolar residues of proteins and peptides,and partial unfolding of the molecule occursduring sample binding. Further unfolding anddenaturing of the molecule occurs during theelution when organic solvents are used. Theuse of elevated temperatures can signicantlyimprove peak resolution and sometimes re-covery, but this causes further unfolding anddenaturing of protein and peptide molecules,and often loss of biological activity as well.Some, mostly smaller proteins can be recov-ered and refolded to restore their activity, soRPC can be successfully applied for their iso-lation and purication (see Aguilar and Hearn,1996; McNay and Fernandez, 1999). Varioushydrophobicity scales for the amino acids havebeen published, and they are used to determinethe hydrophobicity of proteins and peptidesand predict their elution in RPC (UNIT 8.4).Hydrophobic membrane proteins can be dif-cult to recover from RP columns, and theirseparation and further analysis is a challengein any chromatographic mode, especially inRPC (Martosella et al., 2006).

RPC and other analytical techniquesRPC is practically insensitive to high salt

concentration, and mini-columns packed withsmall amounts of reversed-phase support arewidely used for desalting and concentrationof protein and peptide solutions (see Basic



crude sample mixture200

150

100

50

0Ab

sorb

ance

at 2

10 n

m (m

AU)

0 10 20 30 40 50 60Time (min)

P

Figure 8.7.6 Isolation of recombinant protein TM 1-99 (mol. wt. 12,837 Da) from crude samplemixture by use of an optimized gradient. Target protein (peak P) is eluted in the middle of the gra-dient as a single peak, and it is separated from less hydrophobic impurities that elute earlier (frontpart 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, EluentB: 0.05% TFA in acetonitrile, temperature: 25C, column: Zorbax 300SB-C8 (150 mm 2.1 mmI.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. Earlyin its application for separation of proteins,RPC was used in combination with size-exclusion or ion-exchange chromatography(Josic et al., 1982). The use of RPC in com-bination with ion-exchange chromatographyas a part of the automated on-line multi-dimensional HPLC system for protein andpeptide mapping was demonstrated by Wag-ner et al. (2002). RPC is also an integratedpart of each LC-tandem mass spectrometry(MS/MS) system for protein sequencing andidentication (Josic and Clifton, 2007). Auto-mated two-dimensional chromatography, con-taining one cation-exchange step combinedwith the reversed-phase chromatography cou-pled to MS/MS, is widely used for proteinidentication (Wolters et al., 2001).

Critical Parameters andTroubleshootingThe major advantage of RPC is that the

interaction between the sample and chromato-graphic material is not impaired by high con-centrations of salt. However, it is importantthat the sample be completely dissolved inaqueous buffer, and that the column be prop-erly equilibrated. Incomplete equilibration ofthe column or higher content of organic sol-

vent can inuence the retention time andwill have negative effects on reproducibil-ity. Troubleshooting is sometimes differentfor polymer- and silica-based columns, andsilica-based RP resins are somewhat moresensitive, especially to high pH and rapidchanges in solvent composition after gradientelution and return to starting conditions (col-umn regeneration). On the other hand, silica-based columns are more pressure-resistantthan polymer-based ones.Sample equilibrationThe sample must be completely dissolved

in the starting eluent and free of particles. Ifthe salt concentration in the sample solution ishigh, the starting solution has to be aqueousto avoid sample precipitation on the column.RPC is a binding technique, and the sampleload (mass) is of greater signicance than thesample volume. The amount of sample thatcan be applied on a column depends on thebinding capacity of the support and degree ofresolution required.Column equilibrationThe column must be equilibrated using at

least 10 CV of starting eluent (Eluent A) andshould be performed at the temperature atwhich the chromatography will be run. The



time saved for shorter column equilibrationoften results in time and material lost for anunsuccessful chromatographic run.

Optimization of the resolution of a targetcomponentThe methodology for optimization of an

RPC separation is similar to that for HIC, asdescribed in UNIT 8.4. Instead of changing saltconcentration (in HIC), in RPC the concen-tration of more hydrophobic organic solventB is increased. The almost ideal case for iso-lation of a pure component (target protein) isshown in Figure 8.7.6, where the therapeuticprotein is eluted in the middle of the gradient,far from the less hydrophobic components thatare eluted earlier, and the more hydrophobiccomponents eluted later by higher concentra-tions of the hydrophobic solvent B (Mills et al.,2006).

Ghost peaksSo-called ghost peaks (or ghosting)

are usually caused by poor-quality eluent thatcan contain trace amounts of organic impu-rities. These can bind to the column support,are concentrated during the chromatographicrun, column equilibration, and sample appli-cation, and can appear during the elution asunidentied, or ghost peaks. Ghost peaksmay also be caused by incomplete elution ofvery hydrophobic substances from previousruns (so-called carry-over or cross-overcontamination). These peaks can be identiedby running a blank gradient, without sam-ple application. Use of pure, HPLC-grade sol-vents usually prevents the appearance of ghostpeaks.

Baseline driftThe approximately linear, continuous in-

crease (or decrease) of detector response dur-ing linear gradient elution is usually a result ofincreased absorbancy of the organic modier,or it can arise from the ion-pairing reagent,if used. As the proportion of organic solventincreases, the absorbance properties of themo-bile phase may also change, and this can re-sult in further baseline drift. Baseline driftingcan be compensated by using different con-centrations of the UV-absorbing ion-pairingagents in Eluent A and B, and balancing theirconcentrations with respect to the UV absorp-tion. It can also be simply memorized by thecomputer from a blank run and automaticallycorrected.

Time ConsiderationsPreparative RPC using prepacked columns

can be completed in 30 min to 3 to 4 hr,depending on the size of the column, the sam-ple, and the ow rate. It will take 2 hr to packthe column, and an additional 2 to 4 hr willbe necessary for equilibration and testing. Thetime between two chromatographic runs canbe up to 6 hr. Cleaning, sanitation (if nec-essary), and regeneration of the column canbe carried out overnight. Separation at highertemperature can signicantly shorten the sep-aration time, but it has almost no inuence onthe time required for cleaning, sanitation, andregeneration.If short columns, or columns containing

nonporous or monolithic reversed-phase sup-ports, are used, an analytical chromatographicrun can be performed in less than a minute(see Fig. 8.7.5 and Josic and Clifton, 2007).Separation at elevated temperature can alsosignicantly reduce separation time and alsoimprove resolution (Hancock et al. 1994, Chenand Horvath, 1995).

Literature CitedAguilar, M.-E. and Hearn, M.T.W. 1996. High-resolution reversed-phase high-performance liq-uid chromatography of peptides and proteins.Methods Enzymol. 270:3-26.

Chen, H. and Horvath, C.S. 1995. High-speed high-performance liquid chromatography of peptidesand proteins. J. Chromatogr. A 705:3-20.

Coffman, J.L., Kramarczyk, J.F., and Kelley, B.D.2008. High-throughput screening of chromato-graphic separations: I. Method development andcolumn modeling. Biotech. Bioeng. 100:605-618.

Dolan, J.W. 2002. Temperature selectivity inreversed-phase high performance liquid chro-matography. J. Chromatogr. A 965:195-205.

Engelhardt, H., Blay, C., and Saar, J. 2005.Reversed-phase chromatography: The mysteryof surface silanols. Chromatographia 62:S19-S29.

Eschelbach, J.W. and Jorgenson, J.W., 2006. Im-proved protein recovery in reversed-phase liquidchromatography by the use of ultrahigh pres-sure. Anal. Chem. 78:1697-1706.

Hancock, W.S., Chloupek, R.S., Kirkland, J.J., andSnyder, L.R. 1994. Temperature as a variablein reversed-phase high-performance liquid chro-matographic separations of peptide and pro-tein samples: I. Optimizing the separation of agrowth hormone tryptic digest. J. Chromatogr.A 686:31-43.

Hearn, M.T.W. 1984. Reversed-phase high perfor-mance liquid chromatography. Methods Enzy-mol. 104:190-212.



Josic, D.J. andClifton, J.G. 2007. Use ofmonolithicsupports in proteomics technology. J. Chro-matogr. A 1144:2-13.

Josic, D.J. and Strancar, A. 1997. Applicationof membranes and compact, porous units forseparation of biopolymers. Ind. Eng. Chem. Res.38:333-342.

Josic, D.J., Reutter, W., and Molnar, I. 1982. HPLCof membrane bound proteins. In Practical As-pects of Modern HPLC (I. Molnar, ed.) pp. 109-121. Walter de Gruyter, Berlin, New York.

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