The Handbook ofAnalysis and Purification of

Peptides and Proteins byReversed-Phase HPLC

Third Edition, 2002

This handbook presents the basic principles of reversed-phase HPLC for theanalysis and purification of polypeptides. For further details regardingreversed-phase HPLC separations of polypeptides please refer to the technicalreferences at the back of the Handbook or contact the Grace Vydac TechnicalSupport Group.

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2

Mechanism of Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

The Role of the Column in Polypeptide Separations . . . . . . . . . . . . . . . . . . . .8

Analytical Conditions: The Role of the Mobile Phase and Temperature . . . .17

Reversed-Phase HPLC/Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . .26

The Role of Reversed-Phase HPLC in Proteomic Analysis . . . . . . . . . . . . . .30

Examples of the Use of Reversed-Phase HPLC

in the Analysis of Polypeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32

HPLC as a Tool to Purify and Isolate Polypeptides . . . . . . . . . . . . . . . . . . . .40

Viral Inactivation During Reversed-Phase HPLC Purification . . . . . . . . . . . .50

AppendicesAppendix A: Column Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52

Appendix B: The Care and Maintenance of Reversed-Phase Columns . . . . .53

Appendix C: The Effect of Surfactants on Reversed-Phase Separations . . . .56

Appendix D: Ion Exchange Chromatography:

Orthogonal Analytical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58

Appendix E: The Effect of System Hardware

on Reversed-Phase HPLC Polypeptide Separations . . . . . . . . . . . . . . . . . . . .60

Technical References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62

The Grace Vydac Technical Support Group is available for discussions regarding your bio-separation questions.

Please contact us at: Phone 1.800.247.0924 (USA) • 1.760.244.6107 (International)Fax 1.760.244.1984 (USA) • 1.888.244.1984 (International)

www.gracevydac.com

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3

Reversed-Phase High PerformanceLiquid Chromatography (RP-HPLC) has become a widelyused, well-established tool for theanalysis and purification of biomolecules. The reason for the central role that RP-HPLC now plays in analyzing and purifying proteins and peptides is Resolution:RP-HPLC is able to separatepolypeptides of nearly identicalsequences, not only for small peptides such as those obtainedthrough trypsin digestion, but evenfor much larger proteins. Polypeptideswhich differ by a single amino acidresidue can often be separated by RP-HPLC as illustrated in Figure 1showing the separation of insulinvariants.1 Insulin variants havemolecular weights of around 5,300with only slightly different aminoacid sequences, yet most variants can be separated by RP-HPLC. In particular, reversed-phase chromatography is able to separatehuman and rabbit insulin which only differ by a methylene group—rabbit insulin has a threonine where human insulin has a serine!

The scientific literature has many examples where RP-HPLC has been used to separate similar polypeptides. Insulin-like growth factor with an oxidized methionine has been separated from its non-oxidized analogue2 and interleukin-2 muteins

Introduction: Analysis and Purification ofProteins and Peptides by Reversed-Phase HPLC

In the process they demonstrated theresolving power of the technique for similar polypeptides.

RP-HPLC is used for the separationof peptide fragments from enzymaticdigests10-16 and for purification ofnatural and synthetic peptides17.Preparative RP-HPLC is frequentlyused to purifiy synthetic peptides inmilligram and gram quantities46-50.RP-HPLC is used to separate hemoglobin variants34, 35, identifygrain varieties32, study enzyme subunits21 and research cellfunctions33. RP-HPLC is used to purify micro-quantities of peptides for sequencing45 and to purify milligram to kilogram quantities ofbiotechnology-derived polypeptidesfor therapeutic use59-62.

Reversed-Phase HPLC is widely usedin the biopharmaceutical field foranalysis of protein therapeutic products. Enzymatic digests of protein therapeutics are analyzed forprotein identity and to detect geneticchanges and protein degradation(deamidation and oxidation) products.Intact proteins are analyzed by RP-HPLC to verify conformation andto determine degradation products.As the biotechnology revolution hasexpanded so have the technique'sapplications. The number of patentsreferencing VYDAC® reversed-phasecolumns alone has grown exponentially over the past few years as illustrated in Figure 2 (Also see Reference 74).

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have been separated from eachother3. In the latter paper, Kunitaniand colleagues proposed that RP-HPLC retention could provide information on the conformation of retained proteins on the reversed-phase surface. They studiedthirty interleukin-2 muteins and were able to separate muteins thatwere nearly identical. Interleukin inwhich a methionine was oxidizedwas separated from the native formand in other cases single amino acid substitutions were separatedfrom native forms. They concluded that protein conformation was very important in reversed-phase separations and that RP-HPLC couldbe used to study protein conformation.

Figure 1. RP-HPLC separates rabbit andhuman insulin that differ by only a singleamino acid. Column: VYDAC® 214TP54Eluent: 27–30% acetonitrile (ACN) in 0.1%TFA over 25 minutes at 1.5 mL/minute.

Separation of Closely Related Insulin Variants by RP-HPLC

Threonine

Serine

chicken

ovine

bovine

rabbit

human

porcine

rat I

rat II

Figure 2. The number of patents issued by the United States Patent Office in the years 1984–2000 in which VYDAC® Reversed-PhaseHPLC columns are referenced in the patented process.

Number of Patents Issued UsingGrace Vydac Reversed-Phase HPLC Columns

84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99-00

915 13

1928

40 32

5897

140112

172209

289

406

598

54

Understanding the mechanism by which polypeptides interactwith the reversed-phase surface isimportant in understanding RP-HPLCpolypeptide separations. The separationof small molecules involves continuouspartitioning of the molecules betweenthe mobile phase and the hydrophobicstationary phase. Polypeptides, however, are too large to partitioninto the hydrophobic phase; theyadsorb to the hydrophobic surfaceafter entering the column and remainadsorbed until the concentration oforganic modifier reaches the criticalconcentration necessary to cause desorption (Figure 3). They then desorb and interact only slightly with the surface as they elute downthe column4.

Mechanism of Interaction BetweenPolypeptides and RP-HPLC Columns

Important aspects of the adsorption/desorption mechanism of interactions between polypeptides and the hydrophobic phase.

Because the number of organic modifier molecules required to desorb a polypeptide—called the ‘Z’number by Geng and Regnier4—isvery precise, desorption takes placewithin a very narrow window oforganic modifier concentration. This results in complete retentionuntil the critical organic modifierconcentration is reached and suddendesorption of the polypeptide takesplace (Figure 4). The sensitivity ofpolypeptide desorption to preciseconcentrations of organic modifieraccounts for the selectivity of RP-HPLC in the separation ofpolypeptides. The sudden desorptionof polypeptides when the criticalorganic concentration is reached produces sharp peaks. The sensitivity of the ‘Z’ number to protein conformation3 and the sudden desorption at the critical modifier concentration give RP-HPLCthe ability to separate very closely related polypeptides (see Page 2).

Polypeptides may be thought of as “sitting” on the stationary phase, withmost of the molecule exposed to themobile phase and only a part of the molecule—the “hydrophobic foot”—in contact with the RP surface. RP-HPLC separates polypeptidesbased on subtle differences in the “hydrophobic foot” of thepolypeptide being separated.Differences in the “hydrophobicfoot” result from differences inamino acid sequences and differencesin conformation.

Figure 3. Polypeptide enters the column in the mobile phase. The hydrophobic “foot” of the polypeptide adsorbs to the hydrophobic surface of the reversed-phase material where it remains until the organic modifier concentration rises to the critical concentration and desorbs the polypeptide.

Adsorption/Desorption Model of Polypeptide/Reversed-Phase Interaction

Polypeptide enters thecolumn in the mobile phase

Polypeptide adsorbs tothe reversed-phase surface

Polypeptide desorbs fromthe RP surface when theorganic modifier concentrationreaches the critical value

Figure 4. A: The retention of small moleculessuch as biphenyl decreases gradually as theorganic modifier concentration increasesbecause they are retained by partitioning.B: The retention of polypeptides such as lysozyme changes suddenly and drastically as the organic modifier reaches the critical concentration needed to desorb the polypeptide,evidence of the adsorption/desorption model ofpolypeptide-reversed-phase surface interactions.

Molecule Retention Versus Organic Modifier Concentration

0 10 20 30 40 50 60 70 80 90

25

20

15

10

5

0

Biphenyl

0 10 20 30 40 50 60 70 80 90

25

20

15

10

5

0

Biphenyl

B

A

Lysozyme

k1 (r

eten

tion)

Acetonitrile (%)

Acetonitrile (%)

k1 (r

eten

tion)

6

The “hydrophobic foot” of apolypeptide, which is responsible for the separation, is very sensitive to molecular conformation. This sensitivity of RP-HPLC to proteinconformation results in the separationof polypeptides that differ not only inthe hydrophobic foot but elsewherein the molecule as well. Kunitani and Johnson3 found that, due to conformational differences, very similar interleukin-2 muteins couldbe separated, including those differing in an oxidized methionineor in single amino acid substitutions.Geng and Regnier4 found that the ‘Z’number correlates with molecularweight for denatured proteins, however, proteins with intact tertiarystructure elute earlier than expectedbecause only the “hydrophobic foot”is involved in the interaction, whilethe rest of the protein is in contactwith the mobile phase.

The adsorption/desorption steptakes place only once while thepolypeptide is on the column. Afterdesorption, very little interactiontakes place between the polypeptideand the reversed-phase surface andsubsequent interactions have littleaffect on the separation.

A practical consequence of this mechanism of interaction is that polypeptides are very sensitive toorganic modifier concentration. Thesensitivity of polypeptide elution to

the organic modifier concentration is illustrated in Figure 5. Largechanges occur in the retention time of lysozyme with relatively smallchanges in the acetonitrile concentration. The sensitivity of polypeptide retention to subtlechanges in the modifier concentrationmakes isocratic elution difficultbecause the organic modifier concentration must be maintainedvery precisely. Gradient elution isusually preferred for RP-HPLCpolypeptide separations, even if thegradient is very shallow—i.e., asmall change in organic modifierconcentration per unit time.

Figure 5. At 39% ACN, the retention time oflysozyme is nearly 18 minutes. Increasing the ACN concentration to 40% reduces theretention time by more than half, to 7.6 minutes. Increasing the ACN concentration to42% reduces the retention time of lysozymeagain by more than half, to 3.1 mintues.Column: VYDAC® 214TP54 Eluent: ACN at39, 40 and 42% in 0.1% aqueous TFA.

Effect of Acetonitrile Concentration on Elution

0 20

42% ACN

40% ACN

39% ACN

Time (min)

7

Figure 6. The retention behavior on RP-HPLCof many peptides is midway between that of proteins and of small molecules.Pentaphenylalanine, a small peptide, desorbsmore quickly than biphenyl, a small molecule,but more gradually than lysozyme. Small peptides appear to chromatograph by a mixed mechanism.

Retention Behavior of Peptides

0 10 20 30 40 50 60 70 80 90

25

20

15

10

5

0

Lysozyme

Acetonitrile (%)

k' (r

eten

tion)

Pentaphenylalanine

Biphenyl

Shallow gradients can be used very effectively to separate similar polypeptides where isocratic separation would be impractical.

Small peptides appear to chromatograph by a hybrid of partitioning and adsorption. Theydesorb more quickly with changes inorganic modifier concentration thansmall molecules which partition,however they desorb more graduallythan proteins (Figure 6), suggesting a hybrid separation mechanism.Attempts to correlate peptide retentionwith side chain hydrophobicity havebeen partially successful, howevertertiary structure in many peptideslimit interactions to only a portion ofthe molecule and cause discrepanciesin the predictions of most models. It has been shown that the exact location of hydrophobic residues in a helical peptide is important in predicting peptide retention5.

Because large polypeptides diffuse slowly, RP-HPLC results in broader peaks than obtainedwith small molecules. Peak widthsof polypeptides eluted isocraticallyare a function of molecular weight,with large proteins such as

myoglobin having column efficienciesonly 5–10% of the efficienciesobtained with small molecules suchas biphenyl. Gradient elution ofpolypeptides, even with shallow gradients, is preferred, since it resultsin much sharper peaks than isocraticelution. Isocratic elution is rarelyused for polypeptide separations.

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The Role of the Column in PolypeptideSeparations by Reversed-Phase HPLC

Figure 7. This chart indicates the pore size and bonding recommended for variousmolecular weights and hydrophobicities.

Column Selection andCharacteristics of Sample Molecule

Decreasing Hydrophobicity Increasing

Small Pore C18

Large Pore C18

MW100

1,000

10,000

100,000

Large Pore C8

Large Pore C4

The HPLC column provides thehydrophobic surface onto whichthe polypeptides adsorb. Columnsconsist of stainless steel tubes filledwith small diameter, spherical adsorbent particles, generally composed of silica whose surface has been reacted with silane reagentsto make them hydrophobic. Spherical particles of synthetic polymers, suchas polystyrene-divinylbenzene canalso serve as HPLC adsorbents for polypeptides.

Adsorbent Pore Diameter

HPLC adsorbents are porous particles and the majority of the interactive surface is inside thepores. Consequently, polypeptidesmust enter a pore in order to beadsorbed and separated.

For many years, HPLC was performedwith small molecules on particleshaving pores of about 100 Å diameter.Polypeptides chromatographed poorly,in part because many polypeptidesare too large to enter pores of thisdiameter. The development by GraceVydac of large pore (~300 Å) sphericalsilica particles for HPLC heralded the beginning of effective separationsof polypeptides by RP-HPLC. Todaymost polypeptide separations are performed on columns with particleswith pores of about 300 Å, althoughsome peptides (

1110

Angiotensin I—with one histidine—and angiotensin II—with two histidines—both elute earlier relative to the other peptides on the phenylcolumn. When developing peptideseparations, such as those resultingfrom protein digestion, it is best totry several different hydrophobicphases to determine which has thebest selectivity for that particularmixture of peptides. RP-HPLC separation of peptides result fromsubtle interactions of peptides withthe reversed-phase surface. Smallvariations in the reversed-phase surface can affect peptide separationsin small, but important ways. Some

peptide separations are very sensitiveto the density and uniformity of thehydrophobic phase bonded to the silica matrix (Figure 9).

The different reversed-phase adsorbents may offer differentselectivity when separating the peptide fragments from enzymaticdigestion of a protein. Separation of tryptic digest fragments of β-lactoglobulin A on two RP-HPLCcolumns illustrates the subtle effectsthat different phases sometimes haveon reversed-phase separations of peptides (Figure 10). The C4 columnhas slightly less retention and a

somewhat different peptide fragmentelution pattern than the more commonlyused C18 column. Testing differentcolumns is the only practical way ofdetermining which column will givethe best resolution. Selectivity differences between reversed-phasecolumns are used in some laboratoriesto perform two-dimensional peptide separations11.

What is polymeric bonding and how does it affect peptide selectivity?Reversed-phase HPLC adsorbents are usually prepared by bondinghydrocarbon chlorosilanes with onereactive chlorine to the silica matrix.

These form what are calledmonomerically bonded phases, having a single point of attachmentto the silica matrix. Chlorosilaneswith multiple reactive chlorines canalso be used. These form what arecalled polymerically bonded phases,where individual chlorosilanescrosslink and form a silicone polymer on top of the silica matrixwith multiple hydrophobic chainsattached. Although similar inhydrophobicity and separation characteristics, monomerically bonded and polymerically bondedphases can exhibit different selectivities when separating peptides,particularly those resulting fromenzymatic digests of proteins. The different selectivities afford chromatographers additional optionsfor optimizing selectivity and resolution of protein digests andother peptides. An example is given inFigure 11 where a series of syntheticpeptides are separated on amonomerically bonded adsorbent and a polymerically bonded adsorbent. Distinct differences in separation selectivity of the peptides is noted, offering yet another option in column selection when developingpeptide separations.Figure 9. Low carbon load C18 RP-HPLC

column (B) separated two peptides that wereonly partially resolved on a standard carbonload column (A). Columns: A. VYDAC®218TP52-standard C18, 5 µm, 2.1 x 250 mm B. VYDAC® 218LTP52– low carbon load–C18, 5 µm, 2.1 x 250 mm Eluent: 6 mM TFA/4 mMHFBA, 11–95% ACN in 75 min at 0.25 mL/min Sample: Asp–N protein digest. Data courtesy of H. Catipon and T. Salati, Genetics Institute,Andover, MA.

Resolution Improvement withLow Carbon Load Column

A

B

Figure 10. Columns: VYDAC® 218TP54(C18); 214TP54 (C4); Eluent: 0–30 % ACN in 0.1% aqueous TFA over 60 minutes at 1.0 mL/min. Sample: tryptic digest of β-lactoglobulin A.

Separation of a Tryptic Digest on Different Reversed-Phase Columns

C4 (VYDAC 214TP54)

C18 (VYDAC 218TP54)

Figure 11. Columns: VYDAC® 218TP54 polymeric and 238TP54 monomeric (C18, 5 µm,4.6 x 250 mm) Eluent: 10–40% ACN with 0.1% TFA over 30 min. Flowrate: 1.0 mL/min.

The Separation of Synthetic Peptides on Monomerically Bondedand Polymerically Bonded C18Reversed-Phase Columns

Polymerically bonded

Monomerically bonded

1

1 23

4,5

6

7

8

9

10

11

12

13

14

1516

2 43

56 7

8

9

1011,12

13,14

1516

0 10 20 30Minutes

1312

Use of Synthetic Polymer Adsorbents

Although silica-based HPLCcolumns perform well under mild conditions of acidic pH and ambient temperatures, extreme conditions (high pH, high temperature) will degrade silicacolumns. Synthetic polymers suchas polystyrene-divinylbenzene provide a very robust matrix forpolypeptide separations.

Silica-based columns perform verywell under moderate operating conditions of pH and temperature,but there is sometimes a need tooperate at higher than normal pH or temperature or in the presence of high concentrations of chaotropicagents such as guanidine-HCl.Robust synthetic polymer matricessuch as polystyrene-divinylbenzeneare stable under harsh conditions and thus offer practical alternativesto silica. Figure 12 shows the separation of several peptides on a column based on synthetic polystyrene-divinylbenzene.Performance is similar to a silicabased column, thus opening up the possibility of performingpolypeptide separations under relatively harsh conditions on synthetic polymer matrices.

An advantage of separation materialsmade from synthetic polymers is thatthey are not degraded at extremes ofpH. This allows the use of veryacidic or basic solutions as cleaningreagents to remove proteins or othermaterials from columns after chromatography as illustrated inFigure 13. In this example, a columnbased on a polystyrene-divinylbenzenepolymer was washed with strongbase (1 N sodium hydroxide) andwith strong acid (1 N sulfuric acid).Peptides chromatographed beforewashing, after washing with strongbase and after washing with strongacid had similar peak shape, retention and resolution confirmingthat washing the column with strongreagents did not adversely affect column performance.

Column Dimensions: Length The adsorption/desorption of proteinsresponsible for their separation takesplace almost entirely near the top ofthe column. Therefore, column length does not significantly affectseparation and resolution of proteins.Consequently, short columns of 5–15cm length are often used for proteinseparations. Small peptides, such asthose from protease digests, are better separated on longer columnsand columns of 15–25 cm length are often used for the separation of synthetic and natural peptides and enzymatic digest maps. For instance,Stone and Williams found that more peptide fragments from a tryptic digest of carboxymethylatedtransferrin were separated on a column of 250 mm length–104peaks–than on a column of 150mm–80 peaks–or a column of 50mm–65 peaks12.

Column length may affect otheraspects of the separation.

Sample capacitySample capacity is a function of column volume. For columns of equal diameter, longer columns maximize sample capacity.

Column back-pressureColumn back-pressure is directly proportional to the column length.When using more viscous solvents,such as isopropanol, shorter columns will result in more moderate back-pressures.

Figure 12. Separation of peptides on syntheticpolymer column (polystyrene-divinylbenzene). Column: VYDAC® 259VHP5415 (PS-DVB, 5 µm, 4.6 x 150 mm) Eluent: 15–30% ACNover 15 min. with 0.1% TFA. Flowrate: 1.0mL/min. Peptides.1. oxytocin. 2. bradykinin. 3. neurotensin. 4. neurotensin 1–8. 5.angiotensin III. 6. val-4 angiotensin III.

Separation of Several Peptideson a PS-DV3 Column

1

0 5 10 15 min

23

4

5

6

Figure 13. Separation of peptides on a synthetic polymer (polystyrene-divinylbenzene)column before washing with strong reagents(A), after washing with 1N NaOH (B), andafter washing with 1N sulfuric acid (C).Column: VYDAC® 259VHP5415 (PS-DVB, 5µm, 4.6 x 150 mm) Eluent: 15–30% ACN over15 min. with 0.1% TFA. Flowrate: 1.0 mL/min.Peptides. 1. oxytocin. 2. bradykinin. 3.angiotensin II. 4. eledoisin. 5. neurotensin

Peptide Separation Before and AfterExtreme pH Washes

A. Initial Separation

B. Separation afterwashing column with 1N NaOH

C. Separation afterwashing column with 1N H2SO4

1514

Column Dimensions:Diameter

The column diameter does notaffect peak resolution, but it doesaffect sample loading, solvent usageand detection sensitivity. As thediameter of an HPLC column isreduced, the flow rate is decreased,thus lowering the amount of solventused, and the detection sensitivity is increased. Very small diameterHPLC columns are particularlyuseful when coupling HPLC withmass spectrometry (LC/MS).

The standard diameter of analyticalcolumns suitable for analysis of polypeptide samples of 1–200 micrograms is 4.6 mm. Larger diameter columns are used for purification of large amounts ofpolypeptide (see Pages 40–48 onpreparative separations). The use ofsmall diameter columns (0.075 mmto 2.1 mm) has increased in recentyears. Small diameter columns offer:

Reduction in solvent usageFlow rates of as little as a few microliters per minute are used withcapillary and small bore columns(See Appendix A, Page 50). Lowflow rates can significantly reduce theamount of solvent needed forpolypeptide separations.

Increased detection sensitivityPolypeptides elute in smaller volumesof solvent at the reduced flow rates ofsmall bore columns. Detectorresponse increases in proportion to the reduction in flow rate. A narrowbore column with a flow rateof 200 microliters per minute gives a five-fold increase in sensitivitycompared with an analytical columnrun at a flow rate of 1.0 mL/min.

Ability to work with smaller samplesIncreased detection sensitivity meansthat smaller amounts of polypeptidecan be detected. Tryptic digests of aslittle as five nanomoles of proteinhave been separated and collectedusing narrowbore RP-HPLC columns.

Figure 14. Separation of the tryptic digest ofhemoglobin on a microbore RP-HPLC column(Reference 26). Column: C18, 1.0 x 250 mm(VYDAC® 218TP51). Flow rate: 50 µL/min.Eluent: Gradient from 0 to 40% B over 50minutes, where Solvent A is 0.1% TFA in waterand Solvent B is 0.1% TFA in acetonitrile.

Separation of the Tryptic Digest of Hemoglobin on a Microbore (1 mm Diameter) Column

Figure 15. Separation of the tryptic digest ofmyoglobin on a capillary RP-HPLC column.Column: C18 (VYDAC® 218MS), 75 µm i.d.capillary.Flow rate: 0.5 µL/min. Eluent:water/TFA/acetonitrile gradient.

Separation of the Tryptic Digest of Myoglobin on a Capillary (75 µm Diameter) Column

11.88

12.0412.22

13.03

13.24

13.63

13.91

14.56 15.74

16.51

17.79

Figure 16. Separation of the tryptic digest of bovine serum albumin (BSA) on a 300 µm i.d. capillary RP-HPLC Sample: 3 pmole. Column: VYDAC® 218MS5.305 5 µm, 300 Å, polymeric-C18reversed-phase (300 µm i.d. x 50 mm L). Flow: 5 µL/min. Mobile phase: A = 0.1% formic acid,98% water, 2% ACN. B = 0.1% formic acid, 98% ACN, 2% water. Gradient: Hold 3% B from 0 to 5 minutes. Then ramp from 3% B to 50% B at 65 minutes. Final ramp to 75% B at 70 minutes.Detection: MS. (a) Total ion count. (b) Base peak intensity. The base peak is defined as the single mass peak with maximum amplitude at each time in the chromatogram. The base peak chromatogram emphasizes peaks containing a single predominant molecular species and deemphasizes heterogeneous peaks and noise. Data courtesy of Applied Biosystems.

Separation of Tryptic Digest of Bovine Serum Albumin on Capillary RP-HPLC Columns

Max. 8.7e4 cps.

5 10 15 20 25 30 35 40 45 50 55

10%

20%

30%

40%

50%

60%

70%

80%

90%

100% 25.4621.784.81 24.143.30

5.23

21.423.0653.74

27.60

5.59 27.962.56

32.1417.02 32.87 56.82

29.3419.1013.73 45.407.61 47.19

9.64 35.65

Max. 4757.0 cps.

5 10 15 20 25 30 35 40 45 50 55Time (min)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rela

tive

Inte

nsity

(%)

24.14

5.2325.46

21.78

3.30

27.006.54

27.9621.423.06

32.87

Total-Ion Chromatogram (TIC)

Base-Peak Chromatogram (BPC)

(a)

(b)

17

The desorption and elution of polypeptides from RP-HPLCcolumns is accomplished with aqueous solvents containing anorganic modifier and an ion-pairreagent or buffer. The organic modifier solubilizes and desorbs thepolypeptide from the hydrophobicsurface while the ion-pair agent orbuffer sets the eluent pH and interacts with the polypeptide toenhance the separation. Elution isaccomplished by gradually raisingthe concentration of organic solventduring the chromatographic run (solvent gradient). When the solventreaches the precise concentrationnecessary to cause desorption, thepolypeptide is desorbed and elutesfrom the column.

Organic Modifiers

The purpose of the organic solventis to desorb polypeptide moleculesfrom the adsorbent hydrophobicsurface. This is typically done byslowly raising the concentration oforganic solvent (gradient) until thepolypeptides of interest desorb and elute.

Acetonitrile (ACN)Acetonitrile (ACN) is the most commonly used organic modifierbecause:■ It is volatile and easily removed

from collected fractions;

■ It has a low viscosity, minimizing column back-pressure;

■ It has little UV adsorption at low wavelengths;

■ It has a long history of proven reliability in RP-HPLC polypeptide separations.

Isopropanol Isopropanol is often used for large or very hydrophobic proteins. The major disadvantage of isopropanol is itshigh viscosity. To reduce the viscosityof isopropanol while retaining its hydrophobic characteristics, we recommend using a mixture of 50:50 acetonitrile: isopropanol. Adding1–3% isopropanol to acetonitrile hasbeen shown to increase protein recovery in some cases52.

16

Interface with mass spectrometryDirect transfer of the HPLC eluent into the electrospray massspectrometer interface is possiblewith small bore columns and attomole (10-18) levels of individualsample are routinely detected usingsophisticated MS equipment. (See LC/MS, Pages 28–31).

Current Trends in Small Diameter Columns

Narrowbore columnsNarrowbore columns of 2.1 mm i.d.are run at 100–300 microliters perminute. Narrowbore columns are apractical step for most laboratories to take in reducing solvent usage andimproving detection sensitivity. Moststandard HPLC systems can operateat these low flow rates with little orno modification. Narrow borecolumns with flow rates around 200microliters/minute interface well withpneumatically-assisted lectrospraymass spectrometer interfaces.

Microbore and capillary columnsColumns of 1.0 mm diameter andless offer significant reductions insolvent usage and increases in detection sensitivity, however these may require modifications to the HPLC system or the use ofinstruments specifically designed for this purpose.

Capillary columns can be interfacedwith electrospray mass spectrometerinterfaces or even nanoelectrosprayinterfaces after stream splitting.

An article by Davis and Lee providesvaluable information for getting the best performance using microboreand capillary columns44 and is recommended reading for anyoneembarking on the use of small borecolumns. A number of journal articlesdetail the use of mass spectrometerswith capillary columns41–43

(Also see Pages 26–29).

ExamplesMicrobore. Figure 14 illustrates the separation of a tryptic digest of hemoglobin on a microbore (1.0 mm i.d.) column.

Capillary. Figure 15 is an example of the separation of a tryptic digestof myoglobin on a 75 µm i.d. capillary column.

Capillary sample load. Figure 16 illustrates that three picomoles of a tryptic digest of BSA can be separated on a 300 µm i.d. capillary column. Detection was by mass spectrometry.

Analytical Conditions: The Role of the MobilePhase and Temperature in Reversed-Phase

HPLC Polypeptide Separations

Figure 17. Column: C18 (VYDAC® 218TP104)Flow rate: 1 mL/min. Eluent: Gradient slopeas shown. Gradient from 25–50% ACN inaqueous TFA. Sample: Subunits of cytochromec oxidase. Data from reference 21.

Improved Resolution of EnzymeSubunits Using Low Gradient Slope

0.5%/min

0.25%/min

19

The Effect of Gradient Slope on Peptide Selectivity

Because of slight differences in the way that some peptides interactwith the reversed-phase surface, the slope of the solvent gradientmay affect peptide selectivity and,therefore, resolution between peptide pairs.

This effect is best illustrated by the separation of a tryptic digest ofhuman growth hormone at differentgradient times with different gradient slopes. Figure 18 shows theseparation of several tryptic digestfragments from human growth hormone at three different gradientslopes (times). As the slope isdecreased fragments 9 and 10 behave as expected, that is resolutionincreases as the gradient slope isdecreased (increasing gradient time).Fragments 11 and 12, however,behave differently. Resolutiondecreases as the gradient slope isdecreased, indicating a change in the selectivity with changing gradient slope. This effect should be monitored when changing gradient slope by making only modest changes in the gradient slope when developing a method and examining the effect this has on each peptide pair.

Ion-Pairing Reagentsand BuffersThe ion-pairing reagent or buffer sets the eluent pH and interacts with the polypeptide to enhance the separation.

Trifluoroacetic acidThe most common ion-pairingreagent is trifluoroacetic acid (TFA).It is widely used because: ■ It is volatile and easily removed

from collected fractions; ■ It has little UV adsorption at low

wavelengths; ■ It has a long history of proven

reliability in RP-HPLC polypeptide separations.

TFA is normally used at concentrations of about 0.1% (w/v). TFA concentrations up to 0.5% have been useful in solubilizinglarger or more hydrophobic proteinsand lower concentrations are occasionally used for tryptic digestseparations. When chromatographingproteins, using TFA concentrationsbelow 0.1% may degrade peak shape,although new column developmentsallow the use of much lower TFAconcentrations (see Page 28).

Elution gradients with a constantconcentration of TFA sometimesresult in a drifting baseline whenmonitoring at 210–220 nm.

18

Ethanol Ethanol is often used for processscale purifications. Ethanol is a good RP-HPLC solvent, it is readily available at reasonable cost and it isfamiliar to regulatory agencies suchas the FDA. Ethanol has been used toelute hydrophobic, membrane-spanningproteins33 and is used in processpurifications59.

Methanol or other solventsMethanol or other solvents offer littleadvantage over the more commonly used solvents and are not used forpolypeptide separations.

Elution Gradients

Solvent gradients are almost alwaysused to elute polypeptides. Slowly raising the concentration of organic solvent results in the sharpest peaksand best resolution.

Gradient elution is generally preferredfor polypeptide separations. Peakstend to be unacceptably broad in isocratic elution and very low gradient slopes are preferred to isocratic elution. A typical solventgradient has a slope of 0.5 to 1% perminute increase in organic modifierconcentration. Extremely shallow gradients, as low as .05 to 0.1% perminute, can be used to maximize resolution. The gradient slope used toseparate insulin variants in Figure 1(Page 2) was only 0.25% per minute.

Figure 17 illustrates that, for proteins,decreasing the slope of the gradientgenerally improves resolution.

For the best reproducibility and equilibration, avoid extremes inorganic modifier composition. Werecommend beginning gradients atno less than 3 to 5% organic modifierconcentration. Gradients beginningwith less organic modifier may causecolumn equilibration to be long or irreproducible because of the difficulty in "wetting" the surface.We also recommend ending gradientsat no more than 95% organic modifier.High organic concentrations mayremove all traces of water from theorganic phase, also making columnequilibration more difficult.

Figure 18. The effect of gradient time (slope) onpeptide selectivity. Column: C18, 150 x 4.6 mm.Flow rate: 1 mL/min. Eluent: Gradient from0–60% ACN in aqueous 0.1% TFA in A. 45 min.;B. 115 min.; C. 180 min. Sample: tryptic digestof human growth hormone. Fragments 9, 10,11, 12, 13 from the digest. Data from reference 38.

Peptide Separation with DifferentGradient Times

A. 45 minutes

B. 115 minutes

C. 180 minutes

9+10 1112

13

910

910

11+1213

1112

13

2120

The change in dielectric constant asthe solvent environment goes fromaqueous to non-aqueous affects π–πelectron interactions which, in turn,affects the adsorption spectrum in the 190 to 250 nm region, leading to a baseline shift during manyreversed-phase separtions. To reduceor eliminate baseline drift due toTFA spectral adsorption, adjust the wavelength as close to 215 nm as possible and put ~15% less TFA inSolvent B than in Solvent A to compensate for the shift. For example, use 0.1% TFA in Solvent Aand 0.085% TFA in Solvent B.

It is important to use good qualityTFA and to obtain it in smallamounts. Poor quality or aged TFA may have impurities that chromatograph in the reversed-phasesystem, causing spurious peaks toappear (see Appendix B).

The Effect of TFA Concentration on Selectivity

The concentration of trifluoroaceticacid may affect selectivity or resolution of specific peptide pairs.

Although TFA is typically present inthe mobile phase at concentrations of 0.05 to 0.1%, varying the concentration of TFA has a subtleaffect on peptide selectivity as illustrated in Figure 19. This meansthat, for good reproducibility, it is important to control the TFA

concentration very carefully in peptide separation methods. This alsoprovides another tool for optimizingpeptide resolution. After the columnand gradient conditions have beenselected, it is possible to vary theTFA concentration slightly to further optimize resolution betweenpeptide pairs.

Alternate Ion Pairing AgentsAlthough TFA is widely used as the ion pairing reagent, use of otherreagents may result in better resolution or peak shape than TFA. In the separation of five small peptides (Figure 20) phosphate givessharper peaks for some peptides thanTFA and causes a reversal in the elution order of oxytocin andbradykinin. The last three peaks are sharper in phosphate than TFAbecause phosphate interacts withbasic side chains, increasing therigidity of the peptide. Bradykininelutes earlier in phosphate than TFAbecause TFA pairs with the twoarginines in bradykinin resulting inrelatively longer retention. Also, twosmall impurities, hidden in the TFA separation, were revealed by phosphate (Fig. 20B). Hydrochloricacid also reverses the elution order of oxytocin and bradykinin and separates impurities not seen in TFA(Figure 20C).

Heptafluorobutyric acid (HFBA) iseffective in separating basic proteins20

and triethylamine phosphate (TEAP)has been used for preparative separations46, 47, 49. One study foundthat sample capacity was greaterusing TEAP than with TFA32. Formicacid, in concentrations of 10 to 60%,has been used for the chromatographyof very hydrophobic polypeptides.Formic acid is also gaining usage inLC/MS separation of peptides

because TFA reduces the ion signal in the electrospray interface and thevolatile acid, formic acid, has provento be effective in the LC/MS of peptides (See Pages 26–29 for a more detailed discussion of LC/MS).Guo and colleagues compared the use of TFA, HFBA and phosphoricacid in the elution of peptides andfound that each gave somewhat different selectivity8.

Figure 19. Significant differences in the peptide separation pattern due to differencesin TFA concentration are evident. Column: C18(VYDAC® 218TP54). Flow rate: 1 mL/min.Eluent: Gradient from 0–50% ACN in aqueous TFA, concentration as indicated.Sample: Tryptic digest of apotransferrin.Note: Only part of the chromatogram is shown.

The Effect of TFA Concentration on Peptide Selectivity

0.1% TFA

0.3% TFA

Figure 20. Elution of five peptides using TFA(A), Phosphate (B) or HCl (C) as the buffer/ ion-pairing agent. Column: VYDAC® 218TP54(C18, 5 µm, 4.6 x 250 mm). Eluent: 15–30%ACN in 30 min at 1.0 mL/min; plus A. 0.1%TFA B. 20 mM phosphate, pH 2.0 C. 5 mMHCl, pH 2.0 Peptides: 1. oxytocin 2.bradykinin 3. angiotensin II 4. neurotensin5. angiotensin I.

Comparison of TFA and AlternateIon-Pairing Agents/Buffers for theSeparation of Peptides

A. 0.1% TFA

B. 20 mM Phosphate, pH 2.0

C. 5 mM HCI, pH 2.0

2

2

2

1

1

1

3

3

3

4

4

4

5

5

5

23

Developing Conditions for HPLC Separation ofPeptide Fragments from a Protein Digest

Although most enzymatic maps are performed using 0.1% TFA asthe ion-pairing reagent, resolutionmay sometimes be better using a different ion-pairing agent or ahigher pH.

TFA is widely used as an ion-pairingreagent and is the best starting pointfor peptide separations. However,consider the use of buffers such asphosphate or hydrochloric acid orexploring pH effects to optimize peptide separations. To test pHeffects, prepare a 100 mM solution of phosphate—about pH 4.4. Adjust one-third of this to pH 2.0 with phosphoric acid and one-third to pH6.5 with NaOH. Then dilute each to10–20 mM for the eluent buffers.Testing peptide resolution with TFA,each of the three phosphate buffers(pH 2.0, pH 4.4 and pH 6.5) and HCl is an excellent way to find the optimum reagent and pH conditionsto develop a good peptide separation.

22

The Effect of pH on Peptide SeparationsPeptide separations are often sensitive to the eluent pH because of protonation or deprotonation ofacidic or basic side-chains, as illustrated in Figure 21. All five peptides elute earlier at pH 4.4(Figure 21B) than at pH 2.0 (Figure 21A) and the relative retention of peptides changes. This is due to ionization of acidic groupsin the peptides. Bradykinin and oxytocin are well separated at pH 2.0 but co-elute at pH 4.4. Peptideretention at pH 6.5 (Figure 21C) isgreater than at pH 4.4, however theelution order is drastically different.

Angiotensin II, which elutes third at pH 2.0 to 4.4, now elutes first.Neurotensin elutes before oxytocin;bradykinin and neurotensin co-elute. This illustrates that pH can have a dramatic effect on peptide selectivity and can be a useful tool in optimizing peptide separations.

Synthetic polymer-based reversed-phasematerials expand the practical pHrange to nearly pH 14 (See Figure13, Page 13). Peptides elute very differently at high pH than they do atlow pH as illustrated in Figure 22. Ingoing from pH 2 to pH 9 the peptidesin the example change relative elutionorders significantly.

Figure 21. Elution of five peptides at pH 2.0, 4.4 and 6.5 with phosphate as the buffer. Column: VYDAC® 218TP54 (C18, 5 µm, 4.6 x 250 mm). Eluent: 15–30% ACN in 30 min at 1.0mL/min; plus A. 20 mM phosphate, pH 2.0 B. 20 mM phosphate, pH 4.4 C. 20 mM phosphate, pH6.5 Peptides: 1. bradykinin 2. oxytocin 3. angiotensin II 4. neurotensin 5. angiotensin I.

The Effect of pH on Peptide Separations

1

2

3

4

5

1+2

3

4

53

1+4

2

5

A. pH 2.0 B. pH 4.4 C. pH 6.5

Figure 22. Column: VYDAC® 259VHP5415(PS-DVB, 5 µm, 4.6 x 150 mm) Eluent:15–30% ACN over 15 min. with A. 0.1% TFA,pH 2. B. 15 mM NaOH, pH 9. Flowrate: 1.0mL/min. Peptides: 1. oxytocin. 2. bradykinin. 3. neurotensin. 4. neurotensin 1-8. 5.angiotensin III. 6. val-4 angiotensin III.

Separation of Peptides on SyntheticPolymer (Polystyrene-Divinylbenzene)Column at Low and High pH

A. pH 2

B. pH 9

1

1

2

2

3

3

4

4

5

5

6

6

25

Sample solubilityHigh flow rates may improve the solubility of hydrophobicpolypeptides although this alsoincreases the amount of solvent to be removed from the purified sample.

Column back-pressureColumn back-pressure is directlyrelated to flow rate. The higher the flow rate the higher the column back-pressure.

GradientChanges in eluent flow rate may subtly affect gradient slope andshape, depending on the hardwareconfiguration used. Since polypeptideseparations are sensitive to gradientconditions, flow rate adjustmentsmay change the resolution due to the effects on the gradient shape.

The Effect of Temperature on Peptide Separations

Column temperature affects solvent viscosity, column back pressure andretention times. It may also affect peptide selectivity.

Temperature is an important separation parameter when chromatographing peptides andshould be optimized in any HPLCmethod for the separation of peptides.This is illustrated in Figure23 by the separation of fragmentsfrom a tryptic digest of humangrowth hormone39. At 20˚C fragments 11, 12 and 13 nearly co-elute. As the temperature is raisedfragment 13 is more retained than fragments 11 and 12, resulting ingood resolution between the threepeptides at 40˚C. At 60˚C, however,fragments 11 and 12 co-elute, showing the change in selectivity as the temperature is raised. At 20˚Cfragment 15 elutes before fragment14, at 40˚C they nearly co-elute andat 60˚C fragment 14 elutes first andthe two are well separated. Theseresults illustrate the significantimpact that temperature may have on peptide selectivity.

24

Mobile Phase Flow Rate

Flow rate has little effect onpolypeptide separations. The desorption of polypeptides from the reversed-phase surface, andhence resolution, is not affected by the flow rate.

Polypeptide desorption is the resultof reaching a precise organic modifierconcentration. Protein resolution,therefore, is relatively independent of mobile phase flow rate.

The resolution of small peptides maybe affected by the eluent flow ratebecause their behavior on RP-HPLCcolumns is between that of proteinsand small molecules (see Page 4).Stone and Williams found that thenumber of peptide fragments separated from a tryptic digest ofcarboxymethylated transferrindepended on the eluent flow rate12. On an analytical HPLC column,fewer than 80 peptide fragmentswere resolved at a flow rate of 0.2mL/min, compared to 116 fragmentsbeing resolved at 0.8 mL/min. From flow rates of 0.5 mL/min to 1.0 mL/min there was little difference in the number of peptidefragments resolved.

It should be noted that, when refining a separation of small peptides where resolution is limited,slight improvements in resolutionmay be gained through minorchanges in the eluent flow rate. Theflow rate may also influence otheraspects of a separation such as:

Detection sensitivityLow flow rates elute polypeptides in small volumes of solvent and, consequently, adsorption and sensitivity increase. The major reason that narrowbore HPLCcolumns increase detection sensitivity is because they are run at low flow rates and polypeptidesare eluted in small volumes of solvent.

Figure 23. Column: C18, 4.6 x 150 mm. Flow rate: 1 mL/min. Eluent: Gradient from0–60% ACN in aqueous .1% TFA in 60 min.Temperature: As indicated. Sample: Trypticdigest of human growth hormone. Data fromReference 39.

The Effect of Temperature on theSeparation of Peptide Fragments

11

11

11

12

12

12

15

15

15

14

14

14

13

13

13

20°C

40°C

60°C

27

LC/MS Uses Short Columns for Rapid Analysis

The trend in LC/MS toward fasteranalyses with reduced resolutionhas led to the use of relatively shortcolumns with very fast gradients.

The trend toward reduced resolutionand faster separations has led to theuse of short columns packed withsmaller particle adsorbents than normal. The most commonly usedparticle size in short columns is threemicrometers. Using three micrometercolumns of five to ten centimeterlength with fast gradients enablespolypeptide separations to be completed in just a few minutes.Figure 24 shows the separation offive proteins in less than five minutes using a 50 mm long column packed with 3 µm particlesusing a fast gradient.

26

The development of the electrospray interface to couplemass spectrometry with HPLC hascaused a virtual explosion in the use of LC/MS in the analysis ofpolypeptides. RP-HPLC peptidemaps are routinely monitored by anon-line mass spectrometer, obtainingpeptide molecular weights and causing fragmentation of peptides to obtain sequence information.

The combination of mass spectrometrywith HPLC reduces the need for chromatographic resolution because of the resolving capacity of the mass spectrometer. Analysis times are generally short to best utilize the sophisticated mass spectrometer.Detection sensitivity is often much better with mass spectrometry than with UV detection.

Reversed-Phase HPLC/Mass Spectrometry for the Analysis of Polypeptides

Figure 25. Column: C18, 5 µm, 4.6 x 250 mm(VYDAC® 218MS54). Flow rate: 1.5 mL/min.Eluent: Gradient from 5–19% ACN in aqueous0.1% TFA. Sample: 1. neurotensin (1–8 frag) 2. oxytocin 3. angiotensin II 4. neurotensin.

The Use of Low Concentrationsof TFA for Peptide Separations

0.1% TFA

0.05% TFA

0.02% TFA

21

3

4

21

3

4

21

3

4

0.01% TFA21

34

0 10 20 30Minutes

Figure 24. Column: C18, 3 µm 4.6 x 50 mm (VYDAC® 238TP3405). Flow rate: 4.0 ml/min. Eluent: Gradient from 20–45% ACN in aqueous 0.1% TFA in 4 min. Sample: protein standards. (1) ribonuclease, (2) insulin, (3) cytochrome c, (4) BSA and (5) myoglobin.

Rapid Separation of Proteins Using Short (50 mm) Column

12

3

4

5

0 1 2 3 4 5

Minutes

29

identification of peptide fragments ofproteins generated by enzymaticdigests. The example in Figure 27shows the separation of a trypticdigest of bovine serum albumin followed by mass spectrometricanalysis. The eluent from the column was monitored by on-linemass spectrometry, measuring thetotal ion current (Figure 27, top).When the current exceeded a threshold

value the mass spectrum wasobtained on the eluting peak and itsmolecular weight was reported. Theeluting peak was then fragmented ina triple quadrupole mass analyzerproducing product ions of the peptidewhich were used to generate asequence of the peptide (Figure 27, bottom). The peptide fragments can also be matched with a protein orDNA database to identify the protein.

28

Reducing or Eliminating TFA in the Mobile Phase

TFA forms such strong complexes with polypeptides that electrospray signal, and hence detection sensitivity, is reduced when TFA ispresent at concentrations typicalfor polypeptide separations.

The reduction of electrospray signalby TFA has led to the use of ion-pairreagents such as formic acid andacetic acid for polypeptide separations.These ion-pair reagents, however, donot always give good resolution.Recent developments in HPLCcolumns have resulted in columnswith good polypeptide peak shapesusing very low concentrations of TFA.

In some cases the TFA may be completely replaced with formic oracetic acid while retaining good resolution. Figure 25 shows the separation of several peptides on an HPLC column specially developed to allow the use of verylow concentrations of TFA. Goodpeak shapes are maintained on thiscolumn with only 0.01% TFA. Itshould be noted, however, that theTFA concentration does affect peptide selectivity.

Figure 26 demonstrates that, withcolumns developed for use with low concentrations of TFA, it issometimes possible to eliminate the TFA entirely, relying on ion pairreagents such as acetic acid.

HPLC columns developed for lowTFA use enable the use of a widerselection of ion-pairing reagents tooptimize resolution of peptides.Peptide separations can now be done with acetic acid or formic acid acid replacing the trifluoroaceticacid. Mixtures of ion-pair reagentscan also be used to optimize a peptide separation.

Example of Peptide Isolation and SequencingReversed-phase HPLC using capillary columns with very smallsample loads coupled with massspectrometry has become a powerful tool for the isolation and

Figure 26. Columns: Columns developed forpeptide separations in the absence of TFA. Top: C4 (VYDAC® 214MS54); Bottom: C18(VYDAC® 218MS54;). Flow rate: 1 ml/min.Eluent. Gradient from 0–30% ACN in 5 mMHOAc. Sample: Tryptic digest of apotransferrin.

Tryptic Map Replacing TFA with Acetic Acid (No TFA)

0 60Minutes

C4 (214MS54)

C18 (218MS54)

Figure 27. Reversed-phase separation of tryptic digest peptides of bovine serum albumin (BSA) followed by MS determination of molecular weights of each peptide followed in turn by MS fragmentation of each peptide providing data to enable sequencing of the separated peptide.Sample: 3 pmole of a tryptic digest of bovine serum albumin. Column: VYDAC® 218MS5.305, 5 µm,300 Å, polymeric-C18 reversed-phase (300 µm i.d. x 50 mm). Flow rate: 5 µL/min. Mobile phase:A = 0.1% formic acid, 98% water, 2% ACN. B = 0.1% formic acid, 98% ACN, 2% water. Gradient: Hold 3% B from 0 to 5 minutes. Then ramp from 3% B to 50% B at 65 minutes. Final ramp to 75% B at 70 minutes. Detection: Triple quadrupole MS. Data from Reference 75.

Identification of Peptide Fragment of Proteins from Enzymatic Digest

14514013513012512011511010510095908580757065605550454035302520151050

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

0 5 10 15 20 25 30 35 40 45 50 55

10080604020

MS-MS of peak at 21.78 minutes

m/z, amu

Total-Ion Chromatogram (TIC) TOF MS Data

Rel.

Int.

(%)

Inte

nsity

, cou

nts

187.1308

72.0807

86.0946I,L

R

y1 a2175.1184

b2 y2

b3325.1907

465.5980

361.2241y3

3.06

3.304.81

5.23

7.61

5.59

9.64 13.7317.02

21.42

21.78 25.46

27.60

29.34 32.14

35.6532.87 45.40

53.74

56.8247.19

2.56

450.7655

490.2675

y5589.3375

552.3654

541.6491

517.3188b4 b5

693.3699

702.4187y6

b7

b6

y7803.4648

813.4511

747.4450

y8

938.5566

1001.5854y9

b101051.6245y10 b11

1088.6237y11

b9

K V P Q V S T P T L V E V S R

31

Scientists from the ProteinCharacterization and ProteomicsLaboratory at the University ofCincinnati College of Medicinereported using a capillary (300 µmi.d. x 100 mm) reversed-phase columntogether with a triple-quadrupolemass spectrometer for detection andidentification of expressed sequencetags to identify gene products inPseudomonas aeruginosa (Exampleshown in Figure 29). One objectiveof this work was to identify proteins

which could be therapeutic targets formediation of P. aeruginosa biofilmsthat do not respond to conventionalantibiotic therapy and are involved in a number of human diseasesincluding cystic fibrosis. The proteinswere first extracted and separated bySDS-PAGE. Bands of interest weredigested and subjected to RP-HPLCseparation followed by MS and tandem MS to obtain data for protein database searching76.

30

Proteomics is the study of cellularprocesses by identification and quantitation of expressed proteins.Proteomics seeks to catalogue allexpressed proteins in prokaryote or differentiated eukaryote cells and is used to compare protein expressionin two states, for instance comparingprotein expression in normal cells anddiseased cells or in diseased cells andcells treated with a therapeutic drug.

Proteomic methodologies have traditionally used two-dimensionalgel electrophoresis to separate andisolate cellular proteins. The separatedproteins are then protease digestedand the resulting peptides are analyzed by Matrix-Assisted LaserDesorption Ionization (MALDI) massspectrometry. The results are comparedto protein and DNA databases foridentification of the isolated proteins.

Newer proteomic techniques involvethe chromatographic separation ofpeptide fragments generated by protease digests of whole cell lysates.This approach produces very largenumbers of peptide fragments whichrequire high resolution techniques to resolve. Two-dimensional chromatography, consisting of separation of the peptide fragmentsby ion exchange chromatography followed by separation of the ionexchange fractions by RP-HPLC, hasbeen recently described43. The peptide fragments separated by thetwo chromatography steps are then analyzed by electrospray ionizationand tandem mass spectrometry. TheMS results are compared to DNA or protein databases for identification(Figure 28).

The Role of Reversed-Phase HPLCin Proteomic Analysis

Figure 28.

Proteomic Analysis of Cellular Proteins by Two-dimensionalChromatography and Tandem Mass Spectrometry

Cellularproteins

Database Identification of cellular proteins

Mass spectra ofpeptides and MS

fragmentationproduct ions

Tandem MassspectrometryPeptide

fragmentsIon

ExchangeReversed-

phase

Figure 29.

Proteomic Analysis of Pseudomonas aeruginosa

1.4e8

1.2e8

1e8

8e7

6e7

4e7

2e7

0

2.8e6

2.4e6

2.0e6

1.6e6

1.2e6

8.0e5

4.0e5

0

Inte

nsi

ty,

cps

Inte

nsi

ty,

cps

m/z, amu

m/z, amu

Time, min

Q3 Survey Scan

(a)

(a)

(b)

Survey ScanMass Spectrum

36.79

36.9938.21

40.2342.97

43.37 45.80

44.79

47.32

48.74

50.9751.17

51.58

52.39

54.3155.02

55.33

56.04

57.76

35.68

34.0632.94

31.42

30.41

29.80

29.50

28.49

26 28 30 32 34 36 38 4240 44 46 48 50 52 54 56 58 60

Survey scan mass spectrum

Product ion mass spectrum

LC/MS Elongation factor Tu,Gene PA4265Band 16Pseudomonas aeruginosa

400

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

1.20e4

1.00e4

8000

6000

4000

2000

0

Inte

nsi

ty,

cps

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

Precursor ion selected by IDA

M+2H816.2

597.4

85.8157.3175.3

185.0

213.0

229.0312.0

342.3

371.3

425.3443.3

479.3538.3

563.3

597.5

711.5

692.3 741.8855.8

874.3 960.5

y91075.5

y101188.8

y111290.5

y121419.0

1401.3

y12 –18

677.1755.1

766.6843.3

927.4942.0

1034.9 1125.8 1631.3M+H

Max. 2.8e6 cps

Max. 1.3e4 cps

L V E T L D S Y I P E P V R

a2b2

y3

y5

y7 y8

a7 –18

294.0

33

Protein DigestsThe study and analysis of proteinshave long involved protease digestionto produce small peptide fragments,which can then be sequenced orwhich provide important informationon the character and nature of theprotein. Although many proteaseshave been used, trypsin, whichcleaves a polypeptide backbone at the carboxy-terminus of lysine orarginine, has been the most popularprotease. Digestion typically involvesdenaturation of the protein in thepresence of high concentrations of a chaotropic agent such as guanidine-HCl (6 M) or urea (8 M)together with the addition of a reducing agent to reduce the disulfide bonds present in the protein. The free cysteines are usually carboxymethylated to prevent reformation of disulfide bonds.Digestion may be performed at roomtemperatures or higher temperatureswhich reduce the time required forthe digestion. The resulting fragmentsof the protein, averaging about 10amino acids each, can be separatedby RP-HPLC under conditions suchas those shown in Figure 31. In thisinstance a monoclonal antibody wasdigested and the resulting fragmentschromatographed on a C18 column(VYDAC® 218TP54) using a gradient from 0 to 40% acetonitrilecontaining 0.1% TFA.

The defect causing sickle cell anemiais the replacement of glutamic acidby valine in position 6 in the hemglobin protein. Tryptic digestscan reveal amino acid changes in aprotein by the effect the change hason the tryptic fragment containingthat position. As illustrated in Figure32 comparing the tryptic maps ofnormal hemoglobin and sickle cellhemoglobin, the substitution of valinefor glutamic acid causes the peptidefragment containing position 6 toshift to longer retention becausevaline is more hydrophobic than glutamic acid26.

32

Reversed-phase HPLC hasbecome a principle analyticaltechnique in the separation andanalysis of proteins and peptides. Itis widely used in research studyingnatural proteins and peptides and inthe analysis of protein therapeuticproducts in the pharmaceutical industry.This section will focus on a numberof applications and uses, with typicalspecific analytical conditions, toincrease understanding of how to putinto practice the previous sectionswhich have focused on laying a foun-dation of theory and practical aspectsof the RP-HPLC separation ofpolypeptides.

Natural and Synthetic PeptidesRP-HPLC has long been important in the separation and isolation of natural and synthetic peptides. C18columns are most commonly used inthe isolation of peptides as illustratedin Figure 30 in the separation of twonaturally occuring cardioacceleratorypeptides17. Elution conditions aregenerally gradients from low to moderate concentrations of acetonitrile and use 0.1% TFA.

RP-HPLC was used to separate peptides related to Alzheimer's disease18 and is widely used to purify synthetic peptides (Page 49).

Examples of the Use of Reversed-PhaseHPLC in the Analysis of Polypeptides

Figure 30. RP-HPLC was used to separate two octapeptides with cardioacceleratory activity froman extract of Periplaneta Americana—american cockroach. Column: VYDAC® 218TP54 (C18, 5 µm,4.6 x 250 mm) Eluent: Hold at 18% ACN for 10 min; 18–30% ACN from 10–70 min, 30–60% ACNfrom 70–100 min; all with 0.1% TFA Data from Reference 17.

RP-HPLC Separation of Natural Peptides

cardioactive peptides

Figure 31. Column: VYDAC® 218TP54 (C18, 5 µm, 4.6 x 250 mm) Eluent: Gradient from0–40% acetonitrile with 0.1% TFA over 65 minutes. Data from Reference 27.

RP-HPLC Separation of the TrypticDigest of a Monoclonal Antibody

0 10 20 30 40 50 60 min

35

Peptide Maps to Identify GlycopeptidesThe LC/MS analysis of a trypticdigest provides information about thestructure of a protein. It is possible,among other things, to identify thesite of glycosylation (addition of anoligosaccharide) of a protein. Duringthe RP-HPLC separation of the peptide fragments, the mass spectrometer is switching betweenmeasurement of the mass (m/z) of theintact peptide and fragmenting thepeptide through collisionally-induceddissociation, measuring the mass(m/z) of the resulting fragments ofthe peptide40. In particular if anoligosaccharide is present, certain“diagnostic ions” are produced byfragmentation which have m/z of 168and 366. By requesting a combinedtrace of the ion currents produced bythese two ions, an “oligosaccharide-specific” trace is produced (Figure34). This identifies which peptide the glycan (oligosaccharide) isattached to and the site of attachmentcan be identified.

Protein AnalysisWhile peptide digests are often used to study protein structure, intact proteins can be separated and analyzed by RP-HPLC, providinginformation about the intact protein.RP-HPLC is sensitive to both protein modifications, such as deamidation or oxidation, and to protein conformation.

34

One of the most common degradations to occur with proteintherapeutics is the conversion of anasparagine residue to either asparticacid or isoaspartic acid, termeddeamidation14. Deamidation oftenresults in the loss of biological activity. A common means of determining deamidation is to digestthe protein with trypsin and to look

for new peptide fragments elutingslightly later than fragments whichare known to contain asparagine.Under acidic conditions aspartic acidis slightly more hydrophobic thanasparagine, thus a fragment containingthe aspartic acid deamidation productwill elute slightly later than a fragment containing asparagine.

Figure 32. Hemoglobin from normal and sicklecell subjects was tryptic digested and analyzedby RP-HPLC. Peptide 4 contains position six,which is mutated from glutamic acid to valinein sickle cell anemia subjects.Column:VYDAC® 218TP51 (C18, 5 µm 1.0 x 250 mm)Eluent: 0–40% ACN over 50 min, with 0.1%TFA, at 50 mL/min. Data from Reference 26.

Tryptic Maps of Normal Hemoglobinand Sickle Cell Hemoglobin

1

1

2

3

5

7

8

910

12 13

14

15

6

2

3

4

56

7

8

9

10

11

12

13

14

15

17

18

19

2021

22

11

16

4

NormalHemoglobin

“Sickle cell”hemoglobin

16

17

18

19

2021

22

Figure 34. Glycosylated peptides in a peptidemap can be identified by the monitoring of“carbohydrate diagnostic ions” by on-linemass spectrometry. Column: VYDAC®218TP54 (C18, 5 µm 4.6 x 250 mm) Eluent:0–40% ACN over 65 min, with 0.1% TFA, at 1.0 mL/min. Data from Reference 40.

Glycosylated Peptidesin a Peptide Map

Carbohydrate - specific ion trace (168 + 366)

Total MS Ion Current

UV Detection Trace at 214 nm

Figure 33. RP-HPLC separation of peptidefragments from tryptic digests of normal bovine somatotropin (BST) withasparagine at position 99 and deamidated BSTwith the asparagine replaced by isoaspartate.Column: VYDAC® 218TP54 (C18, 5 µm, 4.6 x 250 mm) Eluent: 0–15% ACN over 20min, 15–21% ACN over 12 min, 21–48% ACNover 27 min, 48–75% ACN over 4 min, all with 0.1% TFA, at 2.0 mL/min. Data fromReference 14.

RP-HPLC Used in the Study of Protein Deamidation

normal(asparagine)

modified(isoaspartate)

37

changes in reversed-phase elution. In Figure 37, the retention of aninsulin-like growth factor is shiftedwhen two adjacent disulfide bondsare switched37.

In Figure 38, RP-HPLC is used to monitor a recombinant protein production process. Aggregates of theprotein elute later than the monomer,

carbamylated protein (caused by theuse of urea) elutes as a shoulder onthe native protein peak, oxidized(methionine) protein elutes before thenative form, the desGlyPro clippedprotein elutes earlier than the nativeprotein and misfolded IGF elutes earlier yet. Reversed-phase is able toidentify and quantitate a number ofprotein modifications25.

36

Deamidation and OxidationProtein deamidation results in conversion of an asparagine to anaspartic acid (or isoaspartic acid),thus adding an acidic group to theprotein. At neutral pH the proteintherefore becomes somewhat morehydrophilic. Separating proteins at neutral pH can identify protein degradation deamidation products as illustrated in Figure 35. Humangrowth hormone elutes after thedeamidation products because they are less hydrophobic under theseconditions31.

Methionine residues in proteins can oxidize through metal catalysis, oxygen and light. Most proteins losebiological activity when oxidized.Oxidation causes a protein to becomemore hydrophilic and oxidized proteins elute before the native formin RP-HPLC, as shown in Figure 36.In this instance oxidized forms of acoagulation factor are well separatedfrom the native protein30. Becausereversed-phase HPLC is very sensitive to the “hydrophobic foot”of a protein, even slight changes inprotein conformation can result in

Figure 35. The protein therapeutic recombinanthuman growth hormone deamidates during storage. Deamidation is detected byReversed-Phase HPLC at slightly alkaline pH.Column: VYDAC® 214TP54 (C4, 5 µm, 4.6 x250 mm) Eluent: 29% Isopropanol, 10 mM Tris-HCl, pH 7.5. Data from Reference 31.

Detection of Deamidationby RP-HPLC

deamidationdegradation

products

humangrowth

hormone

Figure 36. Separation of oxidized forms ofcoagulant factor VIIa from the native protein.Column: VYDAC® 214TP54 (C4, 5 µm 4.6 x250 mm) Eluent: 37–47% ACN over 30 min,with 0.1% TFA. Data from Reference 30.

Separation of Oxidized Forms ofCoagulent Factor from Native Protein

MetO(298) and

MetO(306)

dioxidized

MetO(298)

MetO(306)

Native rCFVlla

Figure 37. Insulin-like growth factor has twoadjacent disulfide bonds which can “switch”.This changes the conformation of the protein,which, in turn, affects reversed-phase elution.Column: VYDAC® 214TP54 (C18, 5 µm, 4.6 x250 mm) Eluent: 20–38% ACN:IPA (88:2) with0.1% TFA. Data from Reference 37.

RP-HPLC of Insulin-LikeGrowth Factor

10 20 30

Figure 38. Insulin-like growth factor modified during production was analyzed byRP-HPLC, revealing several modified forms.Column: VYDAC® 218TP54 (C18, 5 µm, 4.6 x250 mm) Eluent: A. 0.12% TFA in H2O. B.0.1% TFA in acetonitrile. Gradient 27.5–28.5%B over 9 minutes, followed by 28.5–40% B over4 min., followed by 40–90% B over 90 minutesat 2 mL/min. Data from Reference 25.

RP-HPLC of Modified Insulin-LikeGrowth Factor

misfoldedIGF

desGlyPro

native IGF

aggregate

carbamylated

OxidizedMetASP45

GLU46

CYS47

TYR4

LEU5

GLY7

GLY9

ALA8

CYS6

CYS48

PHE49

ARG50

CYS52

SER51

ASP53

SS

SS

SS

S S

ASP45

GLU46

CYS47

TYR4

LEU5

GLY7

GLY9

ALA8

CYS6

CYS48

PHE49

ARG50

CYS52

SER51

ASP53

SS

SS

SS

S S

39

Viral proteinsWater insoluble poliovirus proteinswere chromatographed by RP-HPLC28.

Ribosomal proteins30S and 50S ribosomal proteins havebeen separated by RP-HPLC using isopropanol as the organic modifier29.

Membrane proteinsA large, 105 kD, transmembrane protein from Neurospora crassa wasdissolved in anhydrous TFA and purified by RP-HPLC using a C4column and a gradient from 60 to100% ethanol containing 0.1% TFA.These results demonstrate that acrude membrane preparation can bedirectly applied to RP-HPLCcolumns to isolate very hydrophobic,integral proteins33.

Hemoglobin variantsA RP-HPLC method using a C4column has been developed for theseparation of globin chains27. Thismethod has been used to study hemoglobin variants in both animalsand humans. RP-HPLC has helped to detect at least fourteen abnormalhematological states in humans andwas used to study a silent mutantinvolving substitution of threoninefor methionine34.

Protein characterizationProteins are routinely purified forsequencing and characterization by RP-HPLC, for example the purification of an acid soluble proteinfrom Clostridium perfringen spores36.

Grain proteinsGrain varieties cannot usually be identified by physical appearance, so methods based on RP-HPLC profiles of soluble proteins have been developed to identify grain varieties (Reference 24). RP-HPLCprofiles of alcohol-soluble endosperm proteins—glutelins—were obtained on C4 columns andused to identify varieties of rice32.

38

Examples ofProtein SeparationsProteins as large as 105 kD33 and 210 kD19 have been separated using RP-HPLC. Examples include:

Protein subunitsEleven subunits of bovine cytochromec oxidase ranging from MW 4962 to56,993 were separated and analyzedby RP-HPLC21 (Figure 39). The insetin Figure 39 illustrates the use ofshallow gradients to improve resolution for critical proteins.

HistonesHistones are a class of basic nuclear proteins that interact with DNA and may regulate gene activity. They have been separated on C4 RP using heptafluorobutyric acid (HFBA) as the ion-pairing agent20.

Protein foldingThe folding of insulin-like growthfactor was studied using RP-HPLC37.Oxidative refolding of reduced IGF-1 resulted in two major peaks on RP-HPLC which had identicallinear sequences but different disulfide pairing.

Figure 39. Eleven subunits of bovine cytochrome c oxidase ranging in MW from 4962 to 56,993 are separated by RP-HPLC. Column: VYDAC® 214TP104 (C4, 10 µm, 4.6 x 250 mm). Eluent: 25–50% ACN over 50 min, then 50–85% ACN over 17.5 min; all with 0.1% TFA. Flow rate: 1.0 mL/min. Inset: 35–45% ACN with 0.1% TFA over 40 min. Data from Reference 21

RP-HPLC Separation of Bovine Cytochrome c Oxidase Subunits

41

silica from the same manufacturingprocess as analytical size silica andbonded by matched chemical procedures have nearly identical protein and peptide selectivity characteristics as analytical scalematerials. The separation of severalproteins on columns of five, ten and fifteen-to-twenty micrometer particlesize materials illustrates this (Figure41). Protein selectivity and retentionare the same on all three materials.The only difference between thematerials of different particle sizes isthat peak widths are broader with thelarger particle materials, causingsome loss in resolution. Large particlematerials—10-to-15, 15-to-20 or 20-to-30 µm—are normally used inlarge scale purification because theyare less costly than small particle materials, they result in lower column back-pressure and they areeasier to pack into large diametercolumns. In addition, in preparative chromatography, the column is nearlyalways “overloaded” in order to maximize sample throughput (see Page 43). When columns are “overloaded”, large particle materials perform nearly as well assmall particle materials, as illustratedin Figure 42. Although peak widthand resolution are much better (2–3times) with five or ten micrometermaterials than with larger particlematerials at low sample loads, at high sample loads using typical“overload” conditions, peak widthsare only about 20 to 50% greater on

the larger particle materials. Theslight resolution advantage of small particles when overloadingcolumns does not compensate for the higher cost and backpressure and practical difficulties of workingwith small particle materials inprocess applications.

40

RP-HPLC is routinely used in the laboratory to purify microgram to milligram quantities of polypeptides for research purposes. Columns of 50 mm i.d. and greater are used to purify up togram quantities of recombinant proteins for use in clinical trials orfor marketed products. Scaling upseparations in the laboratory usuallyinvolves the use of standard solventsand ion-pairing agents or buffers,choosing column dimensions withthe necessary sample load characteristics (see Appendix A), andoptimization of the elution gradient.

Scaling up laboratory separations to process scale involves not onlyincreasing the size of the column and the elution flow rate, but mayalso involve a change in elution solvents, use of different ion-pairingagents or buffers, and a change ingradient conditions.

In all cases, scaling up laboratory separations is simplified by the availability of separation materialsfor large scale columns that havenearly identical separation characteristics as the columns thatare routinely used in laboratory scale separations.

Selecting Separation Materials

Process scale reversed-phase separation materials are availablewith nearly the same separationcharacteristics as analytical RP columns.

VYDAC® 300 Å silica is produced in particle sizes from less than five to nearly thirty micrometers (Figure40). Physical sizing procedures areused to isolate fractions of five andten micrometers particles for use inanalytical and laboratory scalepreparative separations.

Silica fractions with larger average particle size and broader ranges are separated for preparative and processscale applications. Process-scalereversed-phase materials based on

HPLC as a Tool to Purify andIsolate Polypeptides

Figure 40. Silica is produced in particle sizes from less than five to nearly thirtymicrometers and particle size fractions are isolated for analytical, preparative and process applications.

Particle Size Distribution of VYDAC® TP-300 Å Pore Size Silica

Figure 41. Protein selectivity is the same on RP materials of different particle sizes. Theonly difference between materials of different particle sizes is that peak width increases andresolution decreases as particle size increases.Column materials: A. VYDAC® 214TP, 5 µm B. VYDAC® 214TP, 10 µm C. VYDAC® 214TP,15–20 µm Mobile phase: 24–95 % ACN with0.1% TFA over 30 min at 1.5 mL/min.

Separation of Proteins on RP-HPLCColumns of Different Particle Size

A

B

C

43

Process-scale Purification: MoreThan Five Grams of Peptide

Elution solventThe organic solvents commonly used inlaboratory scale chromatography poseproblems of cost, disposal or safetyin a process environment. Solventssuch as ethanol are more practical forprocess chromatography. Ethanol isrelatively non-toxic, non-flammablewhen mixed with water, is available atlow cost and is known and understoodby regulatory agencies such as theFDA. Ethanol is presently used inlarge scale process purifications59.

Ion-pairing agent or bufferIon pairing agents commonly usedfor analytical chromatography are less practical for process scale chromatography. Alternate ion-pairingagents or buffers useful for processchromatography include acetic acid—which also converts the polypeptide tothe acetate form, useful in formulations—and phosphate. Acetate is presentlyused in the purification of severalbiotechnology derived polypeptidetherapeutics61.

Gradient characteristicsThe comments in the laboratory scalepurification section regarding scaling up elution gradients to largercolumns apply to process scalepurifications (see above). Very shallow gradients in the region where the polypeptide of interestelutes are common.

How Much Polypeptide Can Be Purified in a Single Chromatographic Run?When the purpose of the RP-HPLC separation is to collect purified polypeptide for further use, theamount of sample that can be loadedonto a column while maintaining satisfactory purity is very important.The approach to preparative purifications is generally to load themaximum amount of polypeptide that can be loaded while balancingthree important factors:

ThroughputThe amount of purified polypeptide produced in a given time period. Whilelow sample loads yield maximumresolution, only small quantities arepurified per chromatographic run and throughput is low.

PurityThe purity of the polypeptideexpressed in percent of total weightof final purified product. Purepolypeptides are obtained by avoiding overlap with adjacent peaks although this may limit theamount of sample that can be loadedonto the column.

42

Scaling-up Elution ConditionsThe three key factors to consider in scaling up polypeptide separations arethe elution solvent, the ion-pairingreagent or buffer, and the gradientcharacteristics.

Elution solventLaboratory scale purifications generally use the same organic modifier, namely acetonitrile, as analytical chromatography.

Ion-pairing agent or bufferLaboratory scale purifications generally use the same ion-pairingagents or buffers as analytical chromatography.

Gradient characteristicsTo retain the resolution obtained onan analytical column while increasingcolumn diameter, the gradient shapemust be maintained by keeping theratio of the gradient volume to thecolumn volume constant. For example,a 22 mm diameter column has about23 times the volume of a 4.6 mmdiameter column of the same length(22 divided by 4.6, squared). A 1.0 mL/min gradient over 30 minutes on an analytical column hasa volume of 30 mL. To transfer themethod to a 22 mm column, the gradient volume should be increased23 times to 690 mL. The flow ratecan be increased 23 times whilemaintaining the gradient time constant or the flow rate can be

partially increased while lengtheningthe gradient time. For instance, a flow rate of 23 mL/min for 30 minutes would result in a gradientvolume of 690 mL. However, a flow rate of 10 mL/min for 69 minwould give the same gradient volume, hence the same gradientshape and sample resolution. Ineither case the separation would becomparable to that obtained on ananalytical column. In practice thegradient is often made more shallow—i.e., a smaller increase inorganic modifier concentration perunit time—to increase resolution,particularly for the main polypeptideto be collected.

Figure 42. Although peak widths are much narrower with small particle materials at lowsample loads, there is little difference in peakwidths at high loads, where the column is“overloaded”. Column materials: VYDAC®214TP, 5 µm; VYDAC® 214TP, 10 µm; VYDAC®214TP, 15–20 µm; VYDAC® 214TP, 20–30 µmEluent: 24–95 % ACN in 0.1% aqueous TFA over 30 min at 1.5 mL/min; Protein:ribonuclease.

Protein Loading Capacity of RP-HPLCMaterials of Different Particle Size

20–30 micron

15–20 micron

10 micron

5 micron

0 200 400 600 800 1000

160140120100806040200Pe

ak W

idth

at H

alf H

eigh

t (m

m)

45

In Figure 44, injections of 25, 100,200, 500 and 1,000 micrograms ofribonuclease and lysozyme illustratethe effect on resolution of increasingpeak width resulting from increasingsample loads. At 25 and 100 µginjections—in the region of optimumresolution—resolution betweenribonuclease and the small impuritypreceding it remains constant (Figure44A, B). Resolution begins to decreasebetween ribonuclease and the impurityabove 100 µg—the “overload” point.The 200 µg load shows a definiteincrease in peak width and consequentloss of resolution (Figure 44C). At500 mg there is considerable loss inresolution (Figure 44D) and at 1,000 µgthe impurity peak completely mergeswith the ribonuclease peak (Figure 44E).

Resolution between lysozyme and thepreceding impurity peaks remains constant to about 200 µg, after which resolution is slowly lost. At 500 µg(Figure 44D) the impurity peaksappear only as shoulders on thelysozyme peak and by 1,000 µg(Figure 44E) the impurity peaks havecompletely merged with the lysozymepeak. Resolution between the proteinand impurity peak can be improvedby running a more shallow gradient.

Since resolution between the two, wellseparated, major peaks—ribonucleaseand lysozyme—remains good even atthe 1,000 µg sample load and peakshape is not seriously degraded, veryhigh sample loads are possible forwell separated peaks.

There are many examples in the literature of practical purification of polypeptides at high loadinglevels46–50. In one case 1.2 grams of a synthetic peptide mixture werepurified on a 5 x 30 cm column46. In a personal communication it wasreported that 5 grams of syntheticpeptide were purified on a 5 x 25 cmcolumn in two steps.

44

YieldThe percent of polypeptide purifiedas a percent of the total amount ofpolypeptide present in the originalsample. Maximizing resolutionenables recovery of most of theloaded polypeptide while removing impurities. If resolution is poor thenonly the center of a peak is collected,reducing yield.

There are three measures of samplecapacity on a RP-HPLC column: ■ the loading capacity with

optimum resolution;■ the practical sample loading

capacity;■ and, the maximum amount of

polypeptide the column will bind.

Sample Loading Capacitywith Optimum Resolution

In chromatography the loadinglimit of a column is normallydefined as the maximum amount of analyte that can be chromatographed with no morethan a 10% increase in peak width.

Peak width and resolution remain constant up to the “overload” pointwhich, for analytical (4.6 mm diameter)columns, is about 100 to 200 µg formost polypeptides (Figure 43).Loading samples greater than thisamount results in broadened peaksand decreased resolution.

Practical Loading CapacityPreparative separations require maximizing throughput by balancing resolution, yield and purity. Oftenimproving yield comes at a cost ofreduced purity or reduced throughput.In practice this generally requires“overloading” the column—that is,injecting polypeptide samples greaterthan the sample capacity defined byoptimum resolution. As the sampleload is increased, polypeptide peakwidths increase (Figures 43 and 44),however peak shape remains reasonablysymmetrical. This often allows theloading of samples 10 to 50 times thenominal sample capacity while stillretaining acceptable resolution.

Figure 43. Peak width is constant with sampleloads up to 200 µg. Above 200 µg—the “overload” point—the peak width graduallyincreases. The practical loading region forribonuclease is 200 to 5000 µg. Column:VYDAC® 214TP54 (C4, 5 µm, 4.6 x 250 mm)Eluent: 24–95% ACN with 0.1% TFA over 30 minutes Sample: ribonuclease.

Sample Loading Curve forRibonuclease on Analytical Column

Overload Point

Practical loading range(column is overloaded)

Peak

Wid

th (m

m)

0 400 800 1200 1600

160

140

120

100

80

60

40

20

0

Figure 44. A. 25 µg each protein B. 100 µgeach protein C. 200 µg each protein D. 500 µgeach protein E. 1000 µg each protein Column:VYDAC® 214TP54 (C4, 5 µm, 4.6 x 250 mm)Eluent: 25–50% ACN in 0.1% TFA over 25minutes at 1.5 mL/min. Sample: ribonucleaseand lysozyme.

Effect of Sample Load on ProteinPeak Shape and Resolution

ribonuclease

impurity impurity

lysosyme

47

Biological Activity and Reversed-Phase HPLC

Biological activity of proteinsdepends on tertiary structure and permanent disruption of tertiary structure eliminates biological activity.

RP-HPLC may disrupt protein tertiary structure because of thehydrophobic solvents used for elutionor because of the interaction of theprotein with the hydrophobic surface ofthe material. The amount of biologicalactivity lost depends on the stability ofthe protein and on the elution conditionsused. The loss of biological activitycan be minimized by proper post-chromatographic treatment.

Small peptides and very stable proteinsare less likely to lose biologicalactivity than large enzymes. Somespecific points to keep in mind are:

Protein denaturationDenaturation of proteins onhydrophobic surfaces is kineticallyslow. Reducing the residence time ofthe protein in the column generallyreduces the loss of biological activity.

Solvent effectsSome solvents are less likely to cause a loss of biological activitythan others. Isopropanol is the bestsolvent for retaining biological activity. Ethanol and methanol areslightly worse and acetonitrile causes the greatest loss of biological activity.

Stabilizing factorsStabilizing factors, such as enzyme cofactors, added to the chromatographic eluent, may stabilize proteins and reduce the loss of biological activity.

The most important factor in maintaining or regaining biologicalactivity is post-column sample treatment. Dissolution of a collectedprotein in a stabilizing buffer oftenallows the protein to re-fold. Anexample is HIV protease (Figure 45)56.

46

Maximum Polypeptide Binding CapacityThe maximum binding capacity of a polypeptide on a reversed-phasecolumn depends on the size andcharacteristics of the polypeptide.Small peptides have binding capacities of about 10 mg of peptideper gram of separation material—25 mg on a 4.6 x 250 mm column.Proteins have slightly higher bindingcapacities between 10 and 20 mg of protein per gram of separationmaterial, depending on the ratio ofthe area of the hydrophobic foot tothe total molecular weight.

Although sample loads near the maximum binding capacity of a column provide little resolution, they are useful for simple, fastdesalting of polypeptide samples.

Ways to OptimizeThroughput and Resolution

Sample concentrationResolution between closely elutingpolypeptides may be affected bysample concentration. Dilute samplesappear to spread out over the columnsurface better than concent