-
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
1
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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).
2
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
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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)
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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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8
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 (
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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
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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
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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)
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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