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Third Edition, 2002

T his handbook presents the basic principles of reversed-phase HPLC for the
analysis and purification of polypeptides. For further details regarding
reversed-phase HPLC separations of polypeptides please refer to the technical
references at the back of the Handbook or contact the Grace Vydac Technical
Support Group.

Table of Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Mechanism of Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

The Handbook of 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
Analysis and Purification of The Role of Reversed-Phase HPLC in Proteomic Analysis . . . . . . . . . . . . . .30
Examples of the Use of Reversed-Phase HPLC
Peptides and Proteins by in the Analysis of Polypeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32
HPLC as a Tool to Purify and Isolate Polypeptides . . . . . . . . . . . . . . . . . . . .40
Reversed-Phase HPLC Viral Inactivation During Reversed-Phase HPLC Purification . . . . . . . . . . . .50

Appendix 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)
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Introduction: Analysis and Purification of
Proteins and Peptides by Reversed-Phase HPLC
In the process they demonstrated the
R eversed-Phase High Performance
Liquid Chromatography
(RP-HPLC) has become a widely
have been separated from each
other3. In the latter paper, Kunitani
and colleagues proposed that
resolving power of the technique for
similar polypeptides.
Reversed-Phase HPLC is widely used
in the biopharmaceutical field for
analysis of protein therapeutic
used, well-established tool for the RP-HPLC retention could provide products. Enzymatic digests of
analysis and purification of information on the conformation RP-HPLC is used for the separation protein therapeutics are analyzed for
biomolecules. The reason for the of retained proteins on the of peptide fragments from enzymatic protein identity and to detect genetic
central role that RP-HPLC now reversed-phase surface. They studied digests10-16 and for purification of changes and protein degradation
plays in analyzing and purifying thirty interleukin-2 muteins and natural and synthetic peptides17. (deamidation and oxidation) products.
proteins and peptides is Resolution: were able to separate muteins that Preparative RP-HPLC is frequently Intact proteins are analyzed by
RP-HPLC is able to separate were nearly identical. Interleukin in used to purifiy synthetic peptides in RP-HPLC to verify conformation and
polypeptides of nearly identical which a methionine was oxidized milligram and gram quantities46-50. to determine degradation products.
sequences, not only for small was separated from the native form RP-HPLC is used to separate As the biotechnology revolution has
peptides such as those obtained and in other cases single amino hemoglobin variants34, 35, identify expanded so have the technique's
through trypsin digestion, but even acid substitutions were separated grain varieties32, study enzyme applications. The number of patents
for much larger proteins. Polypeptides from native forms. They concluded subunits21 and research cell referencing VYDAC® reversed-phase
which differ by a single amino acid that protein conformation was functions33. RP-HPLC is used to columns alone has grown
residue can often be separated by very important in reversed-phase purify micro-quantities of peptides exponentially over the past few
RP-HPLC as illustrated in Figure 1 separations and that RP-HPLC could for sequencing45 and to purify years as illustrated in Figure 2
showing the separation of insulin be used to study protein conformation. milligram to kilogram quantities of (Also see Reference 74).
variants.1 Insulin variants have biotechnology-derived polypeptides
molecular weights of around 5,300 for therapeutic use59-62.
with only slightly different amino
acid sequences, yet most variants Separation of Closely Related Number of Patents Issued Using
can be separated by RP-HPLC. Insulin Variants by RP-HPLC Grace Vydac Reversed-Phase
In particular, reversed-phase HPLC Columns
chromatography is able to separate Threonine
human and rabbit insulin which Serine
bovine human
only differ by a methylene group—
ovine 406
rabbit insulin has a threonine chicken

where human insulin has a serine! rat II 289

rat I 172
The scientific literature has many 97

examples where RP-HPLC has been 40 32
used to separate similar polypeptides. 15 13 19
Insulin-like growth factor with an
84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99-00
oxidized methionine has been
separated from its non-oxidized Figure 1. RP-HPLC separates rabbit and Figure 2. The number of patents issued by
analogue2 and interleukin-2 muteins human insulin that differ by only a single the United States Patent Office in the years
amino acid. Column: VYDAC® 214TP54 1984–2000 in which VYDAC® Reversed-Phase
Eluent: 27–30% acetonitrile (ACN) in 0.1% HPLC columns are referenced in the
TFA over 25 minutes at 1.5 mL/minute. patented process.

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Mechanism of Interaction Between
Polypeptides and RP-HPLC Columns
Molecule Retention Versus
U nderstanding the mechanism
by which polypeptides interact
with the reversed-phase surface is
Polypeptides may be thought of as
“sitting” on the stationary phase, with
most of the molecule exposed to the
Important aspects of the
adsorption/desorption mechanism
of interactions between polypeptides
Organic Modifier Concentration

important in understanding RP-HPLC mobile phase and only a part of the and the hydrophobic phase. 25
polypeptide separations. The separation molecule—the “hydrophobic foot”— Biphenyl
of small molecules involves continuous in contact with the RP surface. Because the number of organic 20

partitioning of the molecules between RP-HPLC separates polypeptides modifier molecules required to

k1 (retention)
the mobile phase and the hydrophobic based on subtle differences in desorb a polypeptide—called the ‘Z’
stationary phase. Polypeptides, the “hydrophobic foot” of the number by Geng and Regnier4—is 10
however, are too large to partition polypeptide being separated. very precise, desorption takes place
into the hydrophobic phase; they Differences in the “hydrophobic within a very narrow window of 5
adsorb to the hydrophobic surface foot” result from differences in organic modifier concentration.
after entering the column and remain amino acid sequences and differences This results in complete retention 0 10 20 30 40 50 60 70 80 90
adsorbed until the concentration of in conformation. until the critical organic modifier Acetonitrile (%)
organic modifier reaches the critical concentration is reached and sudden
concentration necessary to cause desorption of the polypeptide takes B
desorption (Figure 3). They then place (Figure 4). The sensitivity of 25
Lysozyme Biphenyl
desorb and interact only slightly polypeptide desorption to precise
with the surface as they elute down concentrations of organic modifier

k1 (retention)
the column4. accounts for the selectivity of 15
RP-HPLC in the separation of
polypeptides. The sudden desorption 10

Adsorption/Desorption Model of of polypeptides when the critical

Polypeptide/Reversed-Phase Interaction organic concentration is reached
produces sharp peaks. The 0
sensitivity of the ‘Z’ number to 0 10 20 30 40 50 60 70 80 90
Polypeptide enters the protein conformation3 and the Acetonitrile (%)
column in the mobile phase
sudden desorption at the critical Figure 4. A: The retention of small molecules
Polypeptide adsorbs to such as biphenyl decreases gradually as the
the reversed-phase surface modifier concentration give RP-HPLC
organic modifier concentration increases
the ability to separate very closely because they are retained by partitioning.
Polypeptide desorbs from
the RP surface when the related polypeptides (see Page 2). B: The retention of polypeptides such as
organic modifier concentration lysozyme changes suddenly and drastically
reaches the critical value as the organic modifier reaches the critical
concentration needed to desorb the polypeptide,
evidence of the adsorption/desorption model of
polypeptide-reversed-phase surface interactions.

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.

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The “hydrophobic foot” of a the organic modifier concentration Shallow gradients can be used Retention Behavior of Peptides
polypeptide, which is responsible is illustrated in Figure 5. Large very effectively to separate similar
for the separation, is very sensitive changes occur in the retention time polypeptides where isocratic
to molecular conformation. This of lysozyme with relatively small separation would be impractical.
sensitivity of RP-HPLC to protein changes in the acetonitrile Lysozyme Biphenyl
conformation results in the separation concentration. The sensitivity of Small peptides appear to 20
of polypeptides that differ not only in polypeptide retention to subtle chromatograph by a hybrid of

k' (retention)
the hydrophobic foot but elsewhere changes in the modifier concentration partitioning and adsorption. They 15

in the molecule as well. Kunitani makes isocratic elution difficult desorb more quickly with changes in
and Johnson3 found that, due to because the organic modifier organic modifier concentration than
conformational differences, very concentration must be maintained small molecules which partition, 5
similar interleukin-2 muteins could very precisely. Gradient elution is however they desorb more gradually
be separated, including those usually preferred for RP-HPLC than proteins (Figure 6), suggesting 0
0 10 20 30 40 50 60 70 80 90
differing in an oxidized methionine polypeptide separations, even if the a hybrid separation mechanism.
Acetonitrile (%)
or in single amino acid substitutions. gradient is very shallow—i.e., a Attempts to correlate peptide retention
Geng and Regnier4 found that the ‘Z’ small change in organic modifier with side chain hydrophobicity have Figure 6. The retention behavior on RP-HPLC
of many peptides is midway between that
number correlates with molecular concentration per unit time. been partially successful, however of proteins and of small molecules.
weight for denatured proteins, tertiary structure in many peptides Pentaphenylalanine, a small peptide, desorbs
more quickly than biphenyl, a small molecule,
however, proteins with intact tertiary limit interactions to only a portion of but more gradually than lysozyme. Small
structure elute earlier than expected the molecule and cause discrepancies peptides appear to chromatograph by a
in the predictions of most models. mixed mechanism.
because only the “hydrophobic foot” Effect of Acetonitrile
is involved in the interaction, while Concentration on Elution It has been shown that the exact
the rest of the protein is in contact location of hydrophobic residues
with the mobile phase. 42% ACN in a helical peptide is important in
predicting peptide retention5. myoglobin having column efficiencies
40% ACN
The adsorption/desorption step only 5–10% of the efficiencies
takes place only once while the Because large polypeptides obtained with small molecules such
polypeptide is on the column. After 39% ACN diffuse slowly, RP-HPLC results as biphenyl. Gradient elution of
desorption, very little interaction in broader peaks than obtained polypeptides, even with shallow
takes place between the polypeptide with small molecules. Peak widths gradients, is preferred, since it results
and the reversed-phase surface and of polypeptides eluted isocratically in much sharper peaks than isocratic
0 Time (min) 20 are a function of molecular weight, elution. Isocratic elution is rarely
subsequent interactions have little
affect on the separation. with large proteins such as used for polypeptide separations.
Figure 5. At 39% ACN, the retention time of
lysozyme is nearly 18 minutes. Increasing
A practical consequence of this the ACN concentration to 40% reduces the
retention time by more than half, to 7.6
mechanism of interaction is that minutes. Increasing the ACN concentration to
polypeptides are very sensitive to 42% reduces the retention time of lysozyme
again by more than half, to 3.1 mintues.
organic modifier concentration. The Column: VYDAC® 214TP54 Eluent: ACN at
sensitivity of polypeptide elution to 39, 40 and 42% in 0.1% aqueous TFA.

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The Role of the Column in Polypeptide
Separations by Reversed-Phase HPLC
Subtle differences in reversed-phase
T he HPLC column provides the
hydrophobic surface onto which
the polypeptides adsorb. Columns
Adsorbent Particle Size
The particle size of the adsorbents
Adsorbent Phase Type
Reversed-phase HPLC adsorbents
surfaces sometimes result in
differences in RP-HPLC selectivity
consist of stainless steel tubes filled in the column affect the narrowness are formed by bonding a hydrophobic for peptides that can be used to
with small diameter, spherical of the eluting peaks. Smaller phase to the silica matrix by means optimize specific peptide separations.
adsorbent particles, generally diameter particles generally produce of chlorosilanes, silicon-based
composed of silica whose surface sharper peaks and better resolution. molecules with chlorine as the As illustrated in Figure 8, peptide
has been reacted with silane reagents reactive group and to which a separation selectivity may be affected
to make them hydrophobic. Spherical Five micrometer materials are hydrocarbon group is attached. by the nature of the hydrophobic
particles of synthetic polymers, such recommended for capillary analytical surface. Selectivity for the five peptides
as polystyrene-divinylbenzene can and small scale preparative separations The hydrocarbon group forming the shown is about the same on the C18
also serve as HPLC adsorbents (columns up to 10 mm i.d.). Larger hydrophobic phase is usually a linear and C4 columns, although the C4
for polypeptides. diameter laboratory columns are aliphatic hydrocarbon of eighteen column has slightly shorter retention.
usually packed with 10 µm materials. (C18), eight (C8) or four (C4) carbons. The phenyl column exhibits shorter
Process chromatography columns of The length of the hydrocarbon chain retention times and a different
Adsorbent Pore Diameter
greater than 22 mm i.d. are normally often makes little difference in the selectivity than the C18 column.
HPLC adsorbents are porous packed with particles of 15 µm or effectiveness of protein separations. Bradykinin, with two phenylalanines,
particles and the majority of the greater and have wider particle size There are guidelines as to which is retained somewhat longer, relative
interactive surface is inside the distributions than the particles phase is likely to be most effective in to the other peptides, on the phenyl
pores. Consequently, polypeptides used in analytical columns separating polypeptides of a given column than on the C18 column.
must enter a pore in order to be (see Pages 40–42). size and hydrophobicity. These are
adsorbed and separated. summarized in Figure 7. C18 columns
Column Selection and are generally preferred for peptides Peptide Separation on Different
For many years, HPLC was performed Characteristics of Sample Molecule and small proteins less than about Reversed-Phase Columns
with small molecules on particles 5,000 daltons. The smallest and most
C18 (VYDAC® 218TP54)
having pores of about 100 Å diameter. Decreasing Hydrophobicity Increasing hydrophilic peptides are often best
Polypeptides chromatographed poorly, MW separated on small pore C18 columns.
100 Small Pore C18
in part because many polypeptides Proteins larger than 5,000 daltons
Large Pore C18 or small polypeptides that are Phenyl (VYDAC® 219TP54)
are too large to enter pores of this 1,000
diameter. The development by Grace Large Pore C8 particularly hydrophobic are best
10,000 chromatographed on C4 columns.
Vydac of large pore (~300 Å) spherical
silica particles for HPLC heralded C8 columns are similar to C18
Large Pore C4 C4 (VYDAC® 214TP54)
the beginning of effective separations columns in their application but
of polypeptides by RP-HPLC. Today sometimes offer a different
most polypeptide separations are 100,000 selectivity or ability to separate
performed on columns with particles particular peptides. Phenyl columns Figure 8. Peptide separation on different
reversed-phase columns Columns: VYDAC®
with pores of about 300 Å, although Figure 7. This chart indicates the pore
are slightly less hydrophobic than 218TP54 (C18); 214TP54 (C4); 219TP54
some peptides (<~2,000 MW) may size and bonding recommended for various C4 columns and may offer unique (phenyl); Eluent: 15–30 % ACN in 0.1%
molecular weights and hydrophobicities. selectivity for some polypeptides. aqueous TFA over 30 minutes at 1.0 mL/min.
also be separated on particles of Sample: 1. oxytocin, 2. bradykinin, 3.
100 Å pores. angiotensin II, 4. neurotensin, 5. angiotensin I.

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Angiotensin I—with one histidine— peptide separations are very sensitive somewhat different peptide fragment These form what are called
and angiotensin II—with two to the density and uniformity of the elution pattern than the more commonly monomerically bonded phases,
histidines—both elute earlier relative hydrophobic phase bonded to the used C18 column. Testing different having a single point of attachment
to the other peptides on the phenyl silica matrix (Figure 9). columns is the only practical way of to the silica matrix. Chlorosilanes
column. When developing peptide determining which column will give with multiple reactive chlorines can
separations, such as those resulting The different reversed-phase the best resolution. Selectivity also be used. These form what are
from protein digestion, it is best to adsorbents may offer different differences between reversed-phase called polymerically bonded phases,
try several different hydrophobic selectivity when separating the columns are used in some laboratories where individual chlorosilanes
phases to determine which has the peptide fragments from enzymatic to perform two-dimensional crosslink and form a silicone
best selectivity for that particular digestion of a protein. Separation peptide separations11. polymer on top of the silica matrix
mixture of peptides. RP-HPLC of tryptic digest fragments of with multiple hydrophobic chains
separation of peptides result from β-lactoglobulin A on two RP-HPLC What is polymeric bonding and how attached. Although similar in
subtle interactions of peptides with columns illustrates the subtle effects does it affect peptide selectivity? hydrophobicity and separation
the reversed-phase surface. Small that different phases sometimes have Reversed-phase HPLC adsorbents characteristics, monomerically
variations in the reversed-phase on reversed-phase separations of are usually prepared by bonding bonded and polymerically bonded
surface can affect peptide separations peptides (Figure 10). The C4 column hydrocarbon chlorosilanes with one phases can exhibit different
in small, but important ways. Some has slightly less retention and a reactive chlorine to the silica matrix. selectivities when separating peptides,
particularly those resulting from
enzymatic digests of proteins.
Resolution Improvement with Separation of a Tryptic Digest on The Separation of Synthetic The different selectivities afford
Low Carbon Load Column Different Reversed-Phase Columns Peptides on Monomerically Bonded chromatographers additional options
and Polymerically Bonded C18 for optimizing selectivity and
Reversed-Phase Columns resolution of protein digests and
C18 (VYDAC 218TP54)
other peptides. An example is given in
A Polymerically bonded
13,14 Figure 11 where a series of synthetic
peptides are separated on a
monomerically bonded adsorbent
1 24 15

C4 (VYDAC 214TP54) 3 6 7 and a polymerically bonded


adsorbent. Distinct differences in
separation selectivity of the peptides
Monomerically bonded
14 is noted, offering yet another option
12 in column selection when developing
Figure 9. Low carbon load C18 RP-HPLC Figure 10. Columns: VYDAC® 218TP54 4,5
peptide separations.
7 11
column (B) separated two peptides that were (C18); 214TP54 (C4); Eluent: 0–30 % ACN 1 2 13
3 6
only partially resolved on a standard carbon in 0.1% aqueous TFA over 60 minutes at
load column (A). Columns: A. VYDAC® 1.0 mL/min. Sample: tryptic digest of
218TP52-standard C18, 5 µm, 2.1 x 250 mm β-lactoglobulin A.
0 10 20 30
B. VYDAC® 218LTP52– low carbon load–C18, Minutes
5 µm, 2.1 x 250 mm Eluent: 6 mM TFA/4 mM
HFBA, 11–95% ACN in 75 min at 0.25 mL/min Figure 11. Columns: VYDAC® 218TP54
Sample: Asp–N protein digest. Data courtesy polymeric and 238TP54 monomeric (C18, 5 µm,
of H. Catipon and T. Salati, Genetics Institute, 4.6 x 250 mm) Eluent: 10–40% ACN with
Andover, MA. 0.1% TFA over 30 min. Flowrate: 1.0 mL/min.

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Use of Synthetic An advantage of separation materials Column Dimensions: Length Column back-pressure
Polymer Adsorbents made from synthetic polymers is that The adsorption/desorption of proteins Column back-pressure is directly
they are not degraded at extremes of responsible for their separation takes proportional to the column length.
Although silica-based HPLC pH. This allows the use of very When using more viscous solvents,
columns perform well under place almost entirely near the top of
acidic or basic solutions as cleaning the column. Therefore, column such as isopropanol, shorter
mild conditions of acidic pH reagents to remove proteins or other columns will result in more
and ambient temperatures, length does not significantly affect
materials from columns after separation and resolution of proteins. moderate back-pressures.
extreme conditions (high pH, high chromatography as illustrated in
temperature) will degrade silica Consequently, short columns of 5–15
Figure 13. In this example, a column cm length are often used for protein
columns. Synthetic polymers such based on a polystyrene-divinylbenzene
as polystyrene-divinylbenzene separations. Small peptides, such as
polymer was washed with strong those from protease digests, are
provide a very robust matrix for base (1 N sodium hydroxide) and Peptide Separation Before and After
polypeptide separations. better separated on longer columns Extreme pH Washes
with strong acid (1 N sulfuric acid). and columns of 15–25 cm length
Peptides chromatographed before are often used for the separation of
Silica-based columns perform very washing, after washing with strong
well under moderate operating synthetic and natural peptides and A. Initial Separation
base and after washing with strong enzymatic digest maps. For instance,
conditions of pH and temperature, acid had similar peak shape,
but there is sometimes a need to Stone and Williams found that
retention and resolution confirming more peptide fragments from a
operate at higher than normal pH that washing the column with strong
or temperature or in the presence tryptic digest of carboxymethylated
reagents did not adversely affect transferrin were separated on a
of high concentrations of chaotropic column performance.
agents such as guanidine-HCl. column of 250 mm length–104 B. Separation after
washing column
Robust synthetic polymer matrices peaks–than on a column of 150 with 1N NaOH
Separation of Several Peptides mm–80 peaks–or a column of 50
such as polystyrene-divinylbenzene on a PS-DV3 Column
are stable under harsh conditions mm–65 peaks12.
and thus offer practical alternatives
to silica. Figure 12 shows the
3 Column length may affect other
2 C. Separation after
separation of several peptides on
5 aspects of the separation. washing column
1 with 1N H2SO4
a column based on synthetic 6
4 Sample capacity
Sample capacity is a function of
Performance is similar to a silica
column volume. For columns of
based column, thus opening up
equal diameter, longer columns
the possibility of performing
maximize sample capacity. Figure 13. Separation of peptides on a
polypeptide separations under 0 5 10 15 min
synthetic polymer (polystyrene-divinylbenzene)
relatively harsh conditions on Figure 12. Separation of peptides on synthetic column before washing with strong reagents
synthetic polymer matrices. polymer column (polystyrene-divinylbenzene). (A), after washing with 1N NaOH (B), and
Column: VYDAC® 259VHP5415 (PS-DVB, after washing with 1N sulfuric acid (C).
5 µm, 4.6 x 150 mm) Eluent: 15–30% ACN Column: VYDAC® 259VHP5415 (PS-DVB, 5
over 15 min. with 0.1% TFA. Flowrate: 1.0 µm, 4.6 x 150 mm) Eluent: 15–30% ACN over
mL/min. Peptides.1. oxytocin. 2. bradykinin. 15 min. with 0.1% TFA. Flowrate: 1.0 mL/min.
3. neurotensin. 4. neurotensin 1–8. 5. Peptides. 1. oxytocin. 2. bradykinin. 3.
angiotensin III. 6. val-4 angiotensin III. angiotensin II. 4. eledoisin. 5. neurotensin

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Column Dimensions: The standard diameter of analytical Increased detection sensitivity Ability to work with smaller samples
Diameter columns suitable for analysis of Polypeptides elute in smaller volumes Increased detection sensitivity means
polypeptide samples of 1–200 of solvent at the reduced flow rates of that smaller amounts of polypeptide
The column diameter does not micrograms is 4.6 mm. Larger small bore columns. Detector can be detected. Tryptic digests of as
affect peak resolution, but it does diameter columns are used for response increases in proportion little as five nanomoles of protein
affect sample loading, solvent usage purification of large amounts of to the reduction in flow rate. A have been separated and collected
and detection sensitivity. As the polypeptide (see Pages 40–48 on narrowbore column with a flow rate using narrowbore RP-HPLC columns.
diameter of an HPLC column is preparative separations). The use of of 200 microliters per minute gives
reduced, the flow rate is decreased, small diameter columns (0.075 mm a five-fold increase in sensitivity
thus lowering the amount of solvent to 2.1 mm) has increased in recent compared with an analytical column
used, and the detection sensitivity years. Small diameter columns offer: run at a flow rate of 1.0 mL/min.
is increased. Very small diameter
HPLC columns are particularly Reduction in solvent usage
useful when coupling HPLC with Flow rates of as little as a few Separation of Tryptic Digest of Bovine Serum
Albumin on Capillary RP-HPLC Columns
mass spectrometry (LC/MS). microliters per minute are used with
capillary and small bore columns 100% 21.78

90% 24.14 Max. 8.7e4 cps.
(See Appendix A, Page 50). Low 3.30 (a)
flow rates can significantly reduce the 70%
Total-Ion Chromatogram (TIC)
3.06 21.42
amount of solvent needed for 60% 27.60
polypeptide separations. 50% 5.59 27.96
40% 32.14
17.02 56.82
Separation of the Tryptic Digest Separation of the Tryptic Digest 19.10

Relative Intensity (%)

20% 13.73 45.40
of Hemoglobin on a Microbore of Myoglobin on a Capillary 10%
9.64 35.65

(1 mm Diameter) Column (75 µm Diameter) Column 5 10 15 20 25 30 35 40 45 50 55

13.91 100%
(b) Max. 4757.0 cps.
60% 21.78 Base-Peak Chromatogram (BPC)
13.24 15.74 27.00
14.56 6.54
21.42 27.96
13.63 20% 3.06
12.04 13.03 16.51 10%
11.88 17.79 0%
5 10 15 20 25 30 35 40 45 50 55
Time (min)

Figure 16. Separation of the tryptic digest of bovine serum albumin (BSA) on a 300 µm i.d.
Figure 14. Separation of the tryptic digest of Figure 15. Separation of the tryptic digest of capillary RP-HPLC Sample: 3 pmole. Column: VYDAC® 218MS5.305 5 µm, 300 Å, polymeric-C18
hemoglobin on a microbore RP-HPLC column myoglobin on a capillary RP-HPLC column. reversed-phase (300 µm i.d. x 50 mm L). Flow: 5 µL/min. Mobile phase: A = 0.1% formic acid,
(Reference 26). Column: C18, 1.0 x 250 mm Column: C18 (VYDAC® 218MS), 75 µm i.d. 98% water, 2% ACN. B = 0.1% formic acid, 98% ACN, 2% water. Gradient: Hold 3% B from
(VYDAC® 218TP51). Flow rate: 50 µL/min. capillary.Flow rate: 0.5 µL/min. Eluent: 0 to 5 minutes. Then ramp from 3% B to 50% B at 65 minutes. Final ramp to 75% B at 70 minutes.
Eluent: Gradient from 0 to 40% B over 50 water/TFA/acetonitrile gradient. Detection: MS. (a) Total ion count. (b) Base peak intensity. The base peak is defined as the
minutes, where Solvent A is 0.1% TFA in water single mass peak with maximum amplitude at each time in the chromatogram. The base peak
and Solvent B is 0.1% TFA in acetonitrile. chromatogram emphasizes peaks containing a single predominant molecular species and
deemphasizes heterogeneous peaks and noise. Data courtesy of Applied Biosystems.
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Analytical Conditions: The Role of the Mobile
Phase and Temperature in Reversed-Phase
HPLC Polypeptide Separations

Interface with mass spectrometry Capillary columns can be interfaced The desorption and elution of ■ It has a low viscosity, minimizing
Direct transfer of the HPLC with electrospray mass spectrometer polypeptides from RP-HPLC column back-pressure;
eluent into the electrospray mass interfaces or even nanoelectrospray columns is accomplished with ■ It has little UV adsorption at low
spectrometer interface is possible interfaces after stream splitting. aqueous solvents containing an wavelengths;
with small bore columns and organic modifier and an ion-pair ■ It has a long history of proven
attomole (10-18) levels of individual An article by Davis and Lee provides reagent or buffer. The organic reliability in RP-HPLC
sample are routinely detected using valuable information for getting the modifier solubilizes and desorbs the polypeptide separations.
sophisticated MS equipment. best performance using microbore polypeptide from the hydrophobic
(See LC/MS, Pages 28–31). and capillary columns44 and is surface while the ion-pair agent or Isopropanol
recommended reading for anyone buffer sets the eluent pH and Isopropanol is often used for large or
Current Trends in embarking on the use of small bore interacts with the polypeptide to very hydrophobic proteins. The major
Small Diameter Columns columns. A number of journal articles enhance the separation. Elution is disadvantage of isopropanol is its
detail the use of mass spectrometers accomplished by gradually raising high viscosity. To reduce the viscosity
with capillary columns41–43 the concentration of organic solvent of isopropanol while retaining its
Narrowbore columns
(Also see Pages 26–29). during the chromatographic run hydrophobic characteristics, we
Narrowbore columns of 2.1 mm i.d.
(solvent gradient). When the solvent recommend using a mixture of 50:50
are run at 100–300 microliters per Examples reaches the precise concentration acetonitrile: isopropanol. Adding
minute. Narrowbore columns are a Microbore. Figure 14 illustrates necessary to cause desorption, the 1–3% isopropanol to acetonitrile has
practical step for most laboratories the separation of a tryptic digest polypeptide is desorbed and elutes been shown to increase protein
to take in reducing solvent usage and of hemoglobin on a microbore from the column. recovery in some cases52.
improving detection sensitivity. Most (1.0 mm i.d.) column.
standard HPLC systems can operate
Organic Modifiers
at these low flow rates with little or Capillary. Figure 15 is an example
no modification. Narrow bore of the separation of a tryptic digest The purpose of the organic solvent
Improved Resolution of Enzyme
columns with flow rates around 200 of myoglobin on a 75 µm i.d. is to desorb polypeptide molecules Subunits Using Low Gradient Slope
microliters/minute interface well with capillary column. from the adsorbent hydrophobic
pneumatically-assisted lectrospray surface. This is typically done by 0.25%/min
mass spectrometer interfaces. Capillary sample load. Figure 16 slowly raising the concentration of
organic solvent (gradient) until the 0.5%/min
illustrates that three picomoles of
Microbore and capillary columns polypeptides of interest desorb
a tryptic digest of BSA can be
Columns of 1.0 mm diameter and and elute.
separated on a 300 µm i.d.
less offer significant reductions in
capillary column. Detection was
solvent usage and increases in Acetonitrile (ACN)
by mass spectrometry.
detection sensitivity, however Acetonitrile (ACN) is the most
these may require modifications commonly used organic modifier Figure 17. Column: C18 (VYDAC® 218TP104)
to the HPLC system or the use of because: Flow rate: 1 mL/min. Eluent: Gradient slope
as shown. Gradient from 25–50% ACN in
instruments specifically designed ■ It is volatile and easily removed aqueous TFA. Sample: Subunits of cytochrome
for this purpose. from collected fractions; c oxidase. Data from reference 21.

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Ethanol Figure 17 illustrates that, for proteins, The Effect of Gradient Slope Ion-Pairing Reagents
Ethanol is often used for process decreasing the slope of the gradient on Peptide Selectivity and Buffers
scale purifications. Ethanol is a generally improves resolution.
Because of slight differences in The ion-pairing reagent or buffer
good RP-HPLC solvent, it is readily sets the eluent pH and interacts
available at reasonable cost and it is For the best reproducibility and the way that some peptides interact
with the reversed-phase surface, with the polypeptide to enhance
familiar to regulatory agencies such equilibration, avoid extremes in
the slope of the solvent gradient the separation.
as the FDA. Ethanol has been used to organic modifier composition. We
elute hydrophobic, membrane-spanning recommend beginning gradients at may affect peptide selectivity and,
Trifluoroacetic acid
proteins33 and is used in process no less than 3 to 5% organic modifier therefore, resolution between
The most common ion-pairing
purifications59. concentration. Gradients beginning peptide pairs.
reagent is trifluoroacetic acid (TFA).
with less organic modifier may cause It is widely used because:
Methanol or other solvents column equilibration to be long This effect is best illustrated by the
separation of a tryptic digest of ■ It is volatile and easily removed
Methanol or other solvents offer little or irreproducible because of the
human growth hormone at different from collected fractions;
advantage over the more commonly difficulty in "wetting" the surface.
gradient times with different ■ It has little UV adsorption at low
used solvents and are not used for We also recommend ending gradients
gradient slopes. Figure 18 shows the wavelengths;
polypeptide separations. at no more than 95% organic modifier.
separation of several tryptic digest ■ It has a long history of proven
High organic concentrations may
remove all traces of water from the fragments from human growth reliability in RP-HPLC
Elution Gradients
organic phase, also making column hormone at three different gradient polypeptide separations.
Solvent gradients are almost always equilibration more difficult. slopes (times). As the slope is
used to elute polypeptides. Slowly decreased fragments 9 and 10 TFA is normally used at
raising the concentration of organic behave as expected, that is resolution concentrations of about 0.1%
solvent results in the sharpest peaks Peptide Separation with Different increases as the gradient slope is (w/v). TFA concentrations up to
Gradient Times 0.5% have been useful in solubilizing
and best resolution. decreased (increasing gradient time).
13 Fragments 11 and 12, however, larger or more hydrophobic proteins
Gradient elution is generally preferred 11 12 behave differently. Resolution and lower concentrations are
A. 45 minutes
for polypeptide separations. Peaks decreases as the gradient slope is occasionally used for tryptic digest
tend to be unacceptably broad in 13 decreased, indicating a change in separations. When chromatographing
isocratic elution and very low 9 11 the selectivity with changing proteins, using TFA concentrations
B. 115 minutes 10
gradient slopes are preferred to gradient slope. This effect should below 0.1% may degrade peak shape,
isocratic elution. A typical solvent 11+12 be monitored when changing although new column developments
gradient has a slope of 0.5 to 1% per 9 gradient slope by making only allow the use of much lower TFA
minute increase in organic modifier C. 180 minutes modest changes in the gradient concentrations (see Page 28).
concentration. Extremely shallow slope when developing a method
gradients, as low as .05 to 0.1% per Figure 18. The effect of gradient time (slope) on and examining the effect this has Elution gradients with a constant
peptide selectivity. Column: C18, 150 x 4.6 mm.
minute, can be used to maximize Flow rate: 1 mL/min. Eluent: Gradient from on each peptide pair. concentration of TFA sometimes
resolution. The gradient slope used to 0–60% ACN in aqueous 0.1% TFA in A. 45 min.; result in a drifting baseline when
B. 115 min.; C. 180 min. Sample: tryptic digest
separate insulin variants in Figure 1 of human growth hormone. Fragments 9, 10, monitoring at 210–220 nm.
(Page 2) was only 0.25% per minute. 11, 12, 13 from the digest. Data from reference 38.

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The change in dielectric constant as concentration very carefully in Alternate Ion Pairing Agents because TFA reduces the ion signal
the solvent environment goes from peptide separation methods. This also Although TFA is widely used as in the electrospray interface and the
aqueous to non-aqueous affects π–π provides another tool for optimizing the ion pairing reagent, use of other volatile acid, formic acid, has proven
electron interactions which, in turn, peptide resolution. After the column reagents may result in better to be effective in the LC/MS of
affects the adsorption spectrum in and gradient conditions have been resolution or peak shape than TFA. peptides (See Pages 26–29 for a
the 190 to 250 nm region, leading selected, it is possible to vary the In the separation of five small more detailed discussion of LC/MS).
to a baseline shift during many TFA concentration slightly to peptides (Figure 20) phosphate gives Guo and colleagues compared the
reversed-phase separtions. To reduce further optimize resolution between sharper peaks for some peptides than use of TFA, HFBA and phosphoric
or eliminate baseline drift due to peptide pairs. TFA and causes a reversal in the acid in the elution of peptides and
TFA spectral adsorption, adjust the elution order of oxytocin and found that each gave somewhat
wavelength as close to 215 nm as bradykinin. The last three peaks different selectivity8.
possible and put ~15% less TFA in are sharper in phosphate than TFA
Solvent B than in Solvent A to because phosphate interacts with
compensate for the shift. For The Effect of TFA Concentration basic side chains, increasing the Comparison of TFA and Alternate
example, use 0.1% TFA in Solvent A on Peptide Selectivity Ion-Pairing Agents/Buffers for the
rigidity of the peptide. Bradykinin Separation of Peptides
and 0.085% TFA in Solvent B. elutes earlier in phosphate than TFA
0.1% TFA because TFA pairs with the two A. 0.1% TFA 1
It is important to use good quality 5
arginines in bradykinin resulting in 3
TFA and to obtain it in small 2 4
relatively longer retention. Also, two
amounts. Poor quality or aged
small impurities, hidden in the
TFA may have impurities that
TFA separation, were revealed by
chromatograph in the reversed-phase B. 20 mM Phosphate, pH 2.0
phosphate (Fig. 20B). Hydrochloric
system, causing spurious peaks to 1 5
0.3% TFA acid also reverses the elution order
appear (see Appendix B). 3
of oxytocin and bradykinin and 4
separates impurities not seen in TFA
The Effect of TFA (Figure 20C).
Concentration on Selectivity
C. 5 mM HCI, pH 2.0

The concentration of trifluoroacetic Heptafluorobutyric acid (HFBA) is 1

acid may affect selectivity or Figure 19. Significant differences in the effective in separating basic proteins20
peptide separation pattern due to differences and triethylamine phosphate (TEAP) 2 4
resolution of specific peptide pairs. in TFA concentration are evident. Column: C18
(VYDAC® 218TP54). Flow rate: 1 mL/min. has been used for preparative
Although TFA is typically present in Eluent: Gradient from 0–50% ACN in separations46, 47, 49. One study found
aqueous TFA, concentration as indicated.
the mobile phase at concentrations Sample: Tryptic digest of apotransferrin. that sample capacity was greater Figure 20. Elution of five peptides using TFA
of 0.05 to 0.1%, varying the Note: Only part of the chromatogram is shown. using TEAP than with TFA32. Formic (A), Phosphate (B) or HCl (C) as the buffer/
concentration of TFA has a subtle acid, in concentrations of 10 to 60%, ion-pairing agent. Column: VYDAC® 218TP54
(C18, 5 µm, 4.6 x 250 mm). Eluent: 15–30%
affect on peptide selectivity as has been used for the chromatography ACN in 30 min at 1.0 mL/min; plus A. 0.1%
illustrated in Figure 19. This means of very hydrophobic polypeptides. TFA B. 20 mM phosphate, pH 2.0 C. 5 mM
HCl, pH 2.0 Peptides: 1. oxytocin 2.
that, for good reproducibility, it is Formic acid is also gaining usage in bradykinin 3. angiotensin II 4. neurotensin
important to control the TFA LC/MS separation of peptides 5. angiotensin I.

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The Effect of pH Angiotensin II, which elutes third Developing Conditions
on Peptide Separations at pH 2.0 to 4.4, now elutes first. for HPLC Separation of
Neurotensin elutes before oxytocin; Peptide Fragments from
Peptide separations are often
bradykinin and neurotensin co-elute.
sensitive to the eluent pH because a Protein Digest
This illustrates that pH can have a
of protonation or deprotonation of
dramatic effect on peptide selectivity Although most enzymatic maps
acidic or basic side-chains, as
and can be a useful tool in are performed using 0.1% TFA as
illustrated in Figure 21. All five
optimizing peptide separations. the ion-pairing reagent, resolution
peptides elute earlier at pH 4.4
may sometimes be better using a
(Figure 21B) than at pH 2.0
Synthetic polymer-based reversed-phase different ion-pairing agent or a
(Figure 21A) and the relative
materials expand the practical pH higher pH.
retention of peptides changes. This
is due to ionization of acidic groups range to nearly pH 14 (See Figure
13, Page 13). Peptides elute very TFA is widely used as an ion-pairing Separation of Peptides on Synthetic
in the peptides. Bradykinin and Polymer (Polystyrene-Divinylbenzene)
differently at high pH than they do at reagent and is the best starting point
oxytocin are well separated at pH Column at Low and High pH
low pH as illustrated in Figure 22. In for peptide separations. However,
2.0 but co-elute at pH 4.4. Peptide
going from pH 2 to pH 9 the peptides consider the use of buffers such as
retention at pH 6.5 (Figure 21C) is A. pH 2 3
in the example change relative elution phosphate or hydrochloric acid or 2
greater than at pH 4.4, however the 5
orders significantly. exploring pH effects to optimize
elution order is drastically different. 1
peptide separations. To test pH 4 6
effects, prepare a 100 mM solution
The Effect of pH on Peptide Separations of phosphate—about pH 4.4. Adjust
one-third of this to pH 2.0 with
phosphoric acid and one-third to pH
6.5 with NaOH. Then dilute each to 6
2 1+2 2 B. pH 9 1
10–20 mM for the eluent buffers. 4
5 5
3 Testing peptide resolution with TFA,
3 1+4
each of the three phosphate buffers 2

1 4 4 (pH 2.0, pH 4.4 and pH 6.5) and

HCl is an excellent way to find the
optimum reagent and pH conditions
to develop a good peptide separation.
A. pH 2.0 B. pH 4.4 C. pH 6.5

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 1.0 Figure 22. Column: VYDAC® 259VHP5415
mL/min; plus A. 20 mM phosphate, pH 2.0 B. 20 mM phosphate, pH 4.4 C. 20 mM phosphate, pH (PS-DVB, 5 µm, 4.6 x 150 mm) Eluent:
6.5 Peptides: 1. bradykinin 2. oxytocin 3. angiotensin II 4. neurotensin 5. angiotensin I. 15–30% ACN over 15 min. with A. 0.1% TFA,
pH 2. B. 15 mM NaOH, pH 9. Flowrate: 1.0
mL/min. Peptides: 1. oxytocin. 2. bradykinin.
3. neurotensin. 4. neurotensin 1-8. 5.
angiotensin III. 6. val-4 angiotensin III.

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Mobile Phase Flow Rate It should be noted that, when Sample solubility The Effect of Temperature
refining a separation of small High flow rates may improve on Peptide Separations
Flow rate has little effect on peptides where resolution is limited, the solubility of hydrophobic
polypeptide separations. The slight improvements in resolution Column temperature affects solvent
polypeptides although this also
desorption of polypeptides from may be gained through minor viscosity, column back pressure and
increases the amount of solvent to
the reversed-phase surface, and changes in the eluent flow rate. The retention times. It may also affect
be removed from the purified sample.
hence resolution, is not affected flow rate may also influence other peptide selectivity.
by the flow rate. aspects of a separation such as: Column back-pressure
Column back-pressure is directly Temperature is an important
Polypeptide desorption is the result Detection sensitivity related to flow rate. The higher separation parameter when
of reaching a precise organic modifier Low flow rates elute polypeptides the flow rate the higher the chromatographing peptides and
concentration. Protein resolution, in small volumes of solvent and, column back-pressure. should be optimized in any HPLC
therefore, is relatively independent consequently, adsorption and method for the separation of
of mobile phase flow rate. sensitivity increase. The major Gradient peptides.This is illustrated in Figure
reason that narrowbore HPLC Changes in eluent flow rate may 23 by the separation of fragments
The resolution of small peptides may columns increase detection subtly affect gradient slope and from a tryptic digest of human
be affected by the eluent flow rate sensitivity is because they are run shape, depending on the hardware growth hormone39. At 20˚C
because their behavior on RP-HPLC at low flow rates and polypeptides configuration used. Since polypeptide fragments 11, 12 and 13 nearly
columns is between that of proteins are eluted in small volumes of solvent. separations are sensitive to gradient co-elute. As the temperature is raised
and small molecules (see Page 4). conditions, flow rate adjustments fragment 13 is more retained than
Stone and Williams found that the may change the resolution due to fragments 11 and 12, resulting in
number of peptide fragments The Effect of Temperature on the
the effects on the gradient shape. good resolution between the three
separated from a tryptic digest of Separation of Peptide Fragments
peptides at 40˚C. At 60˚C, however,
carboxymethylated transferrin fragments 11 and 12 co-elute,
depended on the eluent flow rate12. 13 15
20°C showing the change in selectivity
11 12 14
On an analytical HPLC column, as the temperature is raised. At 20˚C
fewer than 80 peptide fragments fragment 15 elutes before fragment
were resolved at a flow rate of 0.2 14, at 40˚C they nearly co-elute and
mL/min, compared to 116 fragments 40°C 13 15 at 60˚C fragment 14 elutes first and
12 14
being resolved at 0.8 mL/min. 11
the two are well separated. These
From flow rates of 0.5 mL/min results illustrate the significant
to 1.0 mL/min there was little impact that temperature may have
difference in the number of peptide 11 12 15
60°C 13 on peptide selectivity.
fragments resolved.

Figure 23. Column: C18, 4.6 x 150 mm.

Flow rate: 1 mL/min. Eluent: Gradient from
0–60% ACN in aqueous .1% TFA in 60 min.
Temperature: As indicated. Sample: Tryptic
digest of human growth hormone. Data from
Reference 39.

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

T he development of the
electrospray interface to couple
mass spectrometry with HPLC has
The combination of mass spectrometry
with HPLC reduces the need for
chromatographic resolution because
LC/MS Uses Short Columns
for Rapid Analysis

caused a virtual explosion in the of the resolving capacity of the mass The trend in LC/MS toward faster
use of LC/MS in the analysis of spectrometer. Analysis times are analyses with reduced resolution The Use of Low Concentrations
polypeptides. RP-HPLC peptide generally short to best utilize the has led to the use of relatively short of TFA for Peptide Separations
maps are routinely monitored by an sophisticated mass spectrometer. columns with very fast gradients.
on-line mass spectrometer, obtaining Detection sensitivity is often much 1 2 4
The trend toward reduced resolution 0.1% TFA
peptide molecular weights and better with mass spectrometry than 3
causing fragmentation of peptides with UV detection. and faster separations has led to the
to obtain sequence information. use of short columns packed with
smaller particle adsorbents than
normal. The most commonly used
particle size in short columns is three
0.05% TFA
Rapid Separation of Proteins Using Short (50 mm) Column micrometers. Using three micrometer 1 2 4
columns of five to ten centimeter
length with fast gradients enables
3 polypeptide separations to be
2 completed in just a few minutes.
Figure 24 shows the separation of
5 five proteins in less than five 0.02% TFA
1 2 4
minutes using a 50 mm long
column packed with 3 µm particles 3
using a fast gradient.
0 1 2 3 4 5

Figure 24. Column: C18, 3 µm 4.6 x 50 mm (VYDAC® 238TP3405). Flow rate: 4.0 ml/min. 0.01% TFA
Eluent: Gradient from 20–45% ACN in aqueous 0.1% TFA in 4 min. Sample: protein standards. 1 2
(1) ribonuclease, (2) insulin, (3) cytochrome c, (4) BSA and (5) myoglobin.

0 10 20 30

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 aqueous
0.1% TFA. Sample: 1. neurotensin (1–8 frag)
2. oxytocin 3. angiotensin II 4. neurotensin.

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Reducing or Eliminating TFA In some cases the TFA may be identification of peptide fragments of value the mass spectrum was
in the Mobile Phase completely replaced with formic or proteins generated by enzymatic obtained on the eluting peak and its
acetic acid while retaining good digests. The example in Figure 27 molecular weight was reported. The
TFA forms such strong complexes resolution. Figure 25 shows the shows the separation of a tryptic eluting peak was then fragmented in
with polypeptides that electrospray separation of several peptides on digest of bovine serum albumin a triple quadrupole mass analyzer
signal, and hence detection an HPLC column specially followed by mass spectrometric producing product ions of the peptide
sensitivity, is reduced when TFA is developed to allow the use of very analysis. The eluent from the which were used to generate a
present at concentrations typical low concentrations of TFA. Good column was monitored by on-line sequence of the peptide (Figure 27,
for polypeptide separations. peak shapes are maintained on this mass spectrometry, measuring the bottom). The peptide fragments can
column with only 0.01% TFA. It total ion current (Figure 27, top). also be matched with a protein or
The reduction of electrospray signal should be noted, however, that the When the current exceeded a threshold DNA database to identify the protein.
by TFA has led to the use of ion-pair TFA concentration does affect
reagents such as formic acid and peptide selectivity. Identification of Peptide Fragment of Proteins from Enzymatic Digest
acetic acid for polypeptide separations.
Total-Ion Chromatogram (TIC) TOF MS Data
These ion-pair reagents, however, do Figure 26 demonstrates that, with 100

Rel. Int. (%)

4.81 25.46
not always give good resolution. 80
columns developed for use with 60
5.23 21.42 53.74
3.06 27.60
Recent developments in HPLC low concentrations of TFA, it is 40 2.56
17.02 29.34
32.14 56.82
13.73 32.87 45.40 47.19
columns have resulted in columns sometimes possible to eliminate 20 7.61 9.64 35.65

with good polypeptide peak shapes the TFA entirely, relying on ion pair 0 5 10 15 20 25 30 35 40 45 50 55

using very low concentrations of TFA. reagents such as acetic acid. 145
MS-MS of peak at 21.78 minutes
135 589.3375
Tryptic Map Replacing TFA HPLC columns developed for low
with Acetic Acid (No TFA) TFA use enable the use of a wider

selection of ion-pairing reagents to 105
optimize resolution of peptides.

Intensity, counts
Peptide separations can now be 80
C4 (214MS54) 75
done with acetic acid or formic 70 361.2241 450.7655
acid acid replacing the trifluoroacetic 60
I,L 702.4187
acid. Mixtures of ion-pair reagents 45
R b3
40 325.1907 b7
can also be used to optimize a 35
72.0807 b2 y2
541.6491 y8
30 y7 938.5566
peptide separation. 25 y1 a2 517.3188 803.4648
20 175.1184 b4 b5 552.3654 b10
15 465.5980 813.4511 y9
C18 (218MS54) 10
693.3699 y10 b11
Example of Peptide Isolation 5
747.4450 1088.6237

0 Minutes 60
and Sequencing 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
m/z, amu
Reversed-phase HPLC using Figure 27. Reversed-phase separation of tryptic digest peptides of bovine serum albumin (BSA)
Figure 26. Columns: Columns developed for
peptide separations in the absence of TFA. capillary columns with very small followed by MS determination of molecular weights of each peptide followed in turn by MS
Top: C4 (VYDAC® 214MS54); Bottom: C18 fragmentation of each peptide providing data to enable sequencing of the separated peptide.
sample loads coupled with mass Sample: 3 pmole of a tryptic digest of bovine serum albumin. Column: VYDAC® 218MS5.305, 5 µm,
(VYDAC® 218MS54;). Flow rate: 1 ml/min.
Eluent. Gradient from 0–30% ACN in 5 mM spectrometry has become a 300 Å, polymeric-C18 reversed-phase (300 µm i.d. x 50 mm). Flow rate: 5 µL/min. Mobile phase:
HOAc. Sample: Tryptic digest of apotransferrin. powerful tool for the isolation and 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.
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The Role of Reversed-Phase HPLC
in Proteomic Analysis

P roteomics is the study of cellular

processes by identification and
quantitation of expressed proteins.
Newer proteomic techniques involve
the chromatographic separation of
peptide fragments generated by
Scientists from the Protein
Characterization and Proteomics
Laboratory at the University of
which could be therapeutic targets for
mediation of P. aeruginosa biofilms
that do not respond to conventional
Proteomics seeks to catalogue all protease digests of whole cell lysates. Cincinnati College of Medicine antibiotic therapy and are involved
expressed proteins in prokaryote or This approach produces very large reported using a capillary (300 µm in a number of human diseases
differentiated eukaryote cells and is numbers of peptide fragments which i.d. x 100 mm) reversed-phase column including cystic fibrosis. The proteins
used to compare protein expression require high resolution techniques together with a triple-quadrupole were first extracted and separated by
in two states, for instance comparing to resolve. Two-dimensional mass spectrometer for detection and SDS-PAGE. Bands of interest were
protein expression in normal cells and chromatography, consisting of identification of expressed sequence digested and subjected to RP-HPLC
diseased cells or in diseased cells and separation of the peptide fragments tags to identify gene products in separation followed by MS and
cells treated with a therapeutic drug. by ion exchange chromatography Pseudomonas aeruginosa (Example tandem MS to obtain data for
followed by separation of the ion shown in Figure 29). One objective protein database searching76.
Proteomic methodologies have exchange fractions by RP-HPLC, has of this work was to identify proteins
traditionally used two-dimensional been recently described43. The
gel electrophoresis to separate and peptide fragments separated by the Proteomic Analysis of Pseudomonas aeruginosa
isolate cellular proteins. The separated two chromatography steps are then 1.4e8 (a) Survey Scan 48.74

proteins are then protease digested analyzed by electrospray ionization 1.2e8

Mass Spectrum
Q3 Survey Scan 43.37 45.8047.32 51.58
and the resulting peptides are and tandem mass spectrometry. The 51.17

Intensity, cps
1e8 30.41 36.79 44.79 50.97
analyzed by Matrix-Assisted Laser MS results are compared to DNA 54.31
31.42 35.68 40.23
Desorption Ionization (MALDI) mass or protein databases for identification 36.99 42.97 55.33
6e7 38.21
spectrometry. The results are compared (Figure 28). 29.80 32.94 56.04
4e7 34.06
to protein and DNA databases for 57.76
2e7 28.49
identification of the isolated proteins.
26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
Time, min
Proteomic Analysis of Cellular Proteins by Two-dimensional M+2H
Chromatography and Tandem Mass Spectrometry 2.8e6
Precursor ion selected by IDA Max. 2.8e6 cps
Survey scan mass spectrum

Intensity, cps
2.0e6 LC/MS Elongation factor Tu,
1.6e6 Gene PA4265
Band 16
Pseudomonas aeruginosa
Tandem Mass 4.0e5 755.1 843.3
597.4 677.1
Cellular Peptide Ion Reversed- spectrometry 766.6 927.4 1034.9 1125.8 1631.3
proteins fragments Exchange phase 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
m/z, amu
213.0 b2 y5
1.20e4 a2 Product ion mass spectrum Max. 1.3e4 cps
(b) 185.0 597.5
Intensity, cps

8000 371.3
a7 –18 y9
6000 711.5
Mass spectra of y7 y8
1075.5 y12 –18
peptides and MS 4000 342.3 443.3 563.3
874.3 960.5 y10 y11 y12
fragmentation 85.8 175.3 312.0 425.3 538.3 1188.8 1290.5 1419.0
2000 1401.3
Database Identification product ions 157.3
479.3 692.3 741.8
of cellular proteins 0
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
m/z, amu

Figure 28. Figure 29.

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Examples of the Use of Reversed-Phase
HPLC in the Analysis of Polypeptides

R eversed-phase HPLC has

become a principle analytical
technique in the separation and
Natural and
Synthetic Peptides
Protein Digests
The study and analysis of proteins
The defect causing sickle cell anemia
is the replacement of glutamic acid
by valine in position 6 in the
RP-HPLC has long been important have long involved protease digestion
analysis of proteins and peptides. It hemglobin protein. Tryptic digests
in the separation and isolation of to produce small peptide fragments,
is widely used in research studying can reveal amino acid changes in a
natural and synthetic peptides. C18 which can then be sequenced or
natural proteins and peptides and in protein by the effect the change has
columns are most commonly used in which provide important information
the analysis of protein therapeutic on the tryptic fragment containing
the isolation of peptides as illustrated on the character and nature of the
products in the pharmaceutical industry. that position. As illustrated in Figure
in Figure 30 in the separation of two protein. Although many proteases
This section will focus on a number 32 comparing the tryptic maps of
naturally occuring cardioacceleratory have been used, trypsin, which
of applications and uses, with typical normal hemoglobin and sickle cell
peptides17. Elution conditions are cleaves a polypeptide backbone at
specific analytical conditions, to hemoglobin, the substitution of valine
generally gradients from low to the carboxy-terminus of lysine or
increase understanding of how to put for glutamic acid causes the peptide
moderate concentrations of arginine, has been the most popular
into practice the previous sections fragment containing position 6 to
acetonitrile and use 0.1% TFA. protease. Digestion typically involves
which have focused on laying a foun- shift to longer retention because
denaturation of the protein in the
dation of theory and practical aspects valine is more hydrophobic than
RP-HPLC was used to separate presence of high concentrations of
of the RP-HPLC separation of glutamic acid26.
peptides related to Alzheimer's a chaotropic agent such as
disease18 and is widely used to guanidine-HCl (6 M) or urea (8 M)
purify synthetic peptides (Page 49). together with the addition of a
reducing agent to reduce the
disulfide bonds present in the protein.
RP-HPLC Separation of Natural Peptides The free cysteines are usually RP-HPLC Separation of the Tryptic
Digest of a Monoclonal Antibody
carboxymethylated to prevent
reformation of disulfide bonds.
cardioactive peptides
Digestion may be performed at room
temperatures or higher temperatures
which reduce the time required for
the digestion. The resulting fragments
of the protein, averaging about 10
amino acids each, can be separated
by RP-HPLC under conditions such
as those shown in Figure 31. In this
instance a monoclonal antibody was
digested and the resulting fragments
0 10 20 30 40 50 60 min
chromatographed on a C18 column
(VYDAC® 218TP54) using a Figure 31. Column: VYDAC® 218TP54 (C18,
Figure 30. RP-HPLC was used to separate two octapeptides with cardioacceleratory activity from gradient from 0 to 40% acetonitrile 5 µm, 4.6 x 250 mm) Eluent: Gradient from
an extract of Periplaneta Americana—american cockroach. Column: VYDAC® 218TP54 (C18, 5 µm, 0–40% acetonitrile with 0.1% TFA over
4.6 x 250 mm) Eluent: Hold at 18% ACN for 10 min; 18–30% ACN from 10–70 min, 30–60% ACN
containing 0.1% TFA. 65 minutes. Data from Reference 27.
from 70–100 min; all with 0.1% TFA Data from Reference 17.

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One of the most common for new peptide fragments eluting Peptide Maps to Protein Analysis
degradations to occur with protein slightly later than fragments which Identify Glycopeptides While peptide digests are often
therapeutics is the conversion of an are known to contain asparagine.
The LC/MS analysis of a tryptic used to study protein structure,
asparagine residue to either aspartic Under acidic conditions aspartic acid
digest provides information about the intact proteins can be separated and
acid or isoaspartic acid, termed is slightly more hydrophobic than
structure of a protein. It is possible, analyzed by RP-HPLC, providing
deamidation14. Deamidation often asparagine, thus a fragment containing
among other things, to identify the information about the intact protein.
results in the loss of biological the aspartic acid deamidation product
site of glycosylation (addition of an RP-HPLC is sensitive to both
activity. A common means of will elute slightly later than a
oligosaccharide) of a protein. During protein modifications, such as
determining deamidation is to digest fragment containing asparagine.
the RP-HPLC separation of the deamidation or oxidation, and to
the protein with trypsin and to look
peptide fragments, the mass protein conformation.
spectrometer is switching between
Tryptic Maps of Normal Hemoglobin RP-HPLC Used in the measurement of the mass (m/z) of the Glycosylated Peptides
and Sickle Cell Hemoglobin Study of Protein Deamidation intact peptide and fragmenting the in a Peptide Map
peptide through collisionally-induced
9 18 normal dissociation, measuring the mass UV Detection Trace at 214 nm
7 (asparagine) (m/z) of the resulting fragments of
19 modified
Normal 10 (isoaspartate) the peptide40. In particular if an
Hemoglobin 22
oligosaccharide is present, certain Total MS Ion Current
14 17 “diagnostic ions” are produced by
4 13 15 fragmentation which have m/z of 168
2 5 8 11 and 366. By requesting a combined
6 20
16 trace of the ion currents produced by Carbohydrate - specific ion trace (168 + 366)
3 21

these two ions, an “oligosaccharide-

7 22
specific” trace is produced (Figure
34). This identifies which peptide
“Sickle cell” Figure 34. Glycosylated peptides in a peptide
hemoglobin 10
19 the glycan (oligosaccharide) is
14 Figure 33. RP-HPLC separation of peptide map can be identified by the monitoring of
fragments from tryptic digests of attached to and the site of attachment “carbohydrate diagnostic ions” by on-line
5 17
4 normal bovine somatotropin (BST) with can be identified. mass spectrometry. Column: VYDAC®
asparagine at position 99 and deamidated BST 218TP54 (C18, 5 µm 4.6 x 250 mm) Eluent:
12 13
2 11
with the asparagine replaced by isoaspartate. 0–40% ACN over 65 min, with 0.1% TFA, at
8 Column: VYDAC® 218TP54 (C18, 5 µm, 1.0 mL/min. Data from Reference 40.
6 4.6 x 250 mm) Eluent: 0–15% ACN over 20
3 16 20 21
min, 15–21% ACN over 12 min, 21–48% ACN
over 27 min, 48–75% ACN over 4 min, all
Figure 32. Hemoglobin from normal and sickle with 0.1% TFA, at 2.0 mL/min. Data from
cell subjects was tryptic digested and analyzed Reference 14.
by RP-HPLC. Peptide 4 contains position six,
which is mutated from glutamic acid to valine
in 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.

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Deamidation and Oxidation Methionine residues in proteins can changes in reversed-phase elution. carbamylated protein (caused by the
Protein deamidation results in oxidize through metal catalysis, In Figure 37, the retention of an use of urea) elutes as a shoulder on
conversion of an asparagine to an oxygen and light. Most proteins lose insulin-like growth factor is shifted the native protein peak, oxidized
aspartic acid (or isoaspartic acid), biological activity when oxidized. when two adjacent disulfide bonds (methionine) protein elutes before the
thus adding an acidic group to the Oxidation causes a protein to become are switched37. native form, the desGlyPro clipped
protein. At neutral pH the protein more hydrophilic and oxidized protein elutes earlier than the native
therefore becomes somewhat more proteins elute before the native form In Figure 38, RP-HPLC is used to protein and misfolded IGF elutes
hydrophilic. Separating proteins in RP-HPLC, as shown in Figure 36. monitor a recombinant protein earlier yet. Reversed-phase is able to
at neutral pH can identify protein In this instance oxidized forms of a production process. Aggregates of the identify and quantitate a number of
degradation deamidation products coagulation factor are well separated protein elute later than the monomer, protein modifications25.
as illustrated in Figure 35. Human from the native protein30. Because
growth hormone elutes after the reversed-phase HPLC is very
deamidation products because sensitive to the “hydrophobic foot”
they are less hydrophobic under these of a protein, even slight changes in
conditions31. protein conformation can result in

Detection of Deamidation Separation of Oxidized Forms of RP-HPLC of Insulin-Like RP-HPLC of Modified Insulin-Like
by RP-HPLC Coagulent Factor from Native Protein Growth Factor Growth Factor

human MetO
growth (298) Oxidized native IGF
hormone MetO MetO ASP
53 Met

(306) GLU S S SER


(298) and
46 51
46 S

48 49 50

47 48 49 50

S desGlyPro
degradation (306) TYR


products dioxidized aggregate

Native rCF
Figure 35. The protein therapeutic recombinant IGF
10 20 30
human growth hormone deamidates
during storage. Deamidation is detected by carbamylated
Reversed-Phase HPLC at slightly alkaline pH. Figure 36. Separation of oxidized forms of Figure 37. Insulin-like growth factor has two
Column: VYDAC® 214TP54 (C4, 5 µm, 4.6 x coagulant factor VIIa from the native protein. adjacent disulfide bonds which can “switch”. Figure 38. Insulin-like growth factor
250 mm) Eluent: 29% Isopropanol, 10 mM Column: VYDAC® 214TP54 (C4, 5 µm 4.6 x This changes the conformation of the protein, modified during production was analyzed by
Tris-HCl, pH 7.5. Data from Reference 31. 250 mm) Eluent: 37–47% ACN over 30 min, which, in turn, affects reversed-phase elution. RP-HPLC, revealing several modified forms.
with 0.1% TFA. Data from Reference 30. Column: VYDAC® 214TP54 (C18, 5 µm, 4.6 x Column: VYDAC® 218TP54 (C18, 5 µm, 4.6 x
250 mm) Eluent: 20–38% ACN:IPA (88:2) with 250 mm) Eluent: A. 0.12% TFA in H2O. B.
0.1% TFA. Data from Reference 37. 0.1% TFA in acetonitrile. Gradient 27.5–28.5%
B over 9 minutes, followed by 28.5–40% B over
4 min., followed by 40–90% B over 90 minutes
at 2 mL/min. Data from Reference 25.

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Examples of Histones Viral proteins Protein characterization
Protein Separations Histones are a class of basic nuclear Water insoluble poliovirus proteins Proteins are routinely purified for
Proteins as large as 105 kD33 and proteins that interact with DNA and were chromatographed by RP-HPLC28. sequencing and characterization
210 kD19 have been separated using may regulate gene activity. They by RP-HPLC, for example the
RP-HPLC. Examples include: have been separated on C4 RP using Ribosomal proteins purification of an acid soluble protein
heptafluorobutyric acid (HFBA) as 30S and 50S ribosomal proteins have from Clostridium perfringen spores36.
Protein subunits the ion-pairing agent20. been separated by RP-HPLC using
Eleven subunits of bovine cytochrome isopropanol as the organic modifier29. Grain proteins
c oxidase ranging from MW 4962 to Protein folding Grain varieties cannot usually be
56,993 were separated and analyzed The folding of insulin-like growth Membrane proteins identified by physical appearance,
factor was studied using RP-HPLC37. A large, 105 kD, transmembrane so methods based on RP-HPLC
by RP-HPLC21 (Figure 39). The inset
protein from Neurospora crassa was profiles of soluble proteins have
in Figure 39 illustrates the use of Oxidative refolding of reduced
dissolved in anhydrous TFA and been developed to identify grain
shallow gradients to improve IGF-1 resulted in two major peaks
purified by RP-HPLC using a C4 varieties (Reference 24). RP-HPLC
resolution for critical proteins. on RP-HPLC which had identical
column and a gradient from 60 to profiles of alcohol-soluble
linear sequences but different
100% ethanol containing 0.1% TFA. endosperm proteins—glutelins—
disulfide pairing. were obtained on C4 columns and
These results demonstrate that a
crude membrane preparation can be used to identify varieties of rice32.
directly applied to RP-HPLC
RP-HPLC Separation of Bovine Cytochrome c Oxidase Subunits
columns to isolate very hydrophobic,
integral proteins33.

Hemoglobin variants
A RP-HPLC method using a C4
column has been developed for the
separation of globin chains27. This
method has been used to study
hemoglobin variants in both animals
and humans. RP-HPLC has helped
to detect at least fourteen abnormal
hematological states in humans and
was used to study a silent mutant
involving substitution of threonine
for methionine34.

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

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HPLC as a Tool to Purify and
Isolate Polypeptides

R P-HPLC is routinely used in

the laboratory to purify
microgram to milligram quantities
Selecting Separation Materials
Process scale reversed-phase
silica from the same manufacturing
process as analytical size silica and
bonded by matched chemical
the larger particle materials. The
slight resolution advantage of
small particles when overloading
of polypeptides for research separation materials are available procedures have nearly identical columns does not compensate for
purposes. Columns of 50 mm i.d. with nearly the same separation protein and peptide selectivity the higher cost and backpressure
and greater are used to purify up to characteristics as analytical characteristics as analytical scale and practical difficulties of working
gram quantities of recombinant RP columns. materials. The separation of several with small particle materials in
proteins for use in clinical trials or proteins on columns of five, ten and process applications.
for marketed products. Scaling up VYDAC® 300 Å silica is produced fifteen-to-twenty micrometer particle
separations in the laboratory usually in particle sizes from less than five size materials illustrates this (Figure
involves the use of standard solvents to nearly thirty micrometers (Figure 41). Protein selectivity and retention Separation of Proteins on RP-HPLC
and ion-pairing agents or buffers, 40). Physical sizing procedures are are the same on all three materials. Columns of Different Particle Size
choosing column dimensions with used to isolate fractions of five and The only difference between the
the necessary sample load ten micrometers particles for use in materials of different particle sizes is
characteristics (see Appendix A), and analytical and laboratory scale that peak widths are broader with the
optimization of the elution gradient. preparative separations. larger particle materials, causing
some loss in resolution. Large particle A
Scaling up laboratory separations Silica fractions with larger average materials—10-to-15, 15-to-20 or
to process scale involves not only particle size and broader ranges are 20-to-30 µm—are normally used in
increasing the size of the column separated for preparative and process large scale purification because they
and the elution flow rate, but may scale applications. Process-scale are less costly than small particle
also involve a change in elution reversed-phase materials based on materials, they result in lower
solvents, use of different ion-pairing column back-pressure and they are
agents or buffers, and a change in easier to pack into large diameter B
gradient conditions. columns. In addition, in preparative
Particle Size Distribution of chromatography, the column is nearly
In all cases, scaling up laboratory VYDAC® TP-300 Å Pore Size Silica always “overloaded” in order to
separations is simplified by the maximize sample throughput
availability of separation materials (see Page 43). When columns are
for large scale columns that have “overloaded”, large particle
nearly identical separation materials perform nearly as well as
characteristics as the columns that small particle materials, as illustrated
are routinely used in laboratory in Figure 42. Although peak width
scale separations. and resolution are much better (2–3 Figure 41. Protein selectivity is the same on
times) with five or ten micrometer RP materials of different particle sizes. The
materials than with larger particle only difference between materials of different
particle sizes is that peak width increases and
Figure 40. Silica is produced in particle materials at low sample loads, at resolution decreases as particle size increases.
sizes from less than five to nearly thirty high sample loads using typical Column materials: A. VYDAC® 214TP, 5 µm
micrometers and particle size fractions are B. VYDAC® 214TP, 10 µm C. VYDAC® 214TP,
isolated for analytical, preparative and “overload” conditions, peak widths 15–20 µm Mobile phase: 24–95 % ACN with
process applications. are only about 20 to 50% greater on 0.1% TFA over 30 min at 1.5 mL/min.

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Scaling-up Elution Conditions partially increased while lengthening Process-scale Purification: More How Much Polypeptide
The three key factors to consider in the gradient time. For instance, Than Five Grams of Peptide Can Be Purified in a Single
scaling up polypeptide separations are a flow rate of 23 mL/min for Chromatographic Run?
the elution solvent, the ion-pairing 30 minutes would result in a gradient Elution solvent
volume of 690 mL. However, a When the purpose of the RP-HPLC
reagent or buffer, and the gradient The organic solvents commonly used in
flow rate of 10 mL/min for 69 min separation is to collect purified
characteristics. laboratory scale chromatography pose
would give the same gradient polypeptide for further use, the
problems of cost, disposal or safety
volume, hence the same gradient amount of sample that can be loaded
Elution solvent in a process environment. Solvents
onto a column while maintaining
Laboratory scale purifications shape and sample resolution. In such as ethanol are more practical for
either case the separation would be satisfactory purity is very important.
generally use the same organic process chromatography. Ethanol is
comparable to that obtained on an The approach to preparative
modifier, namely acetonitrile, as relatively non-toxic, non-flammable
analytical column. In practice the purifications is generally to load the
analytical chromatography. when mixed with water, is available at
gradient is often made more maximum amount of polypeptide
low cost and is known and understood
shallow—i.e., a smaller increase in that can be loaded while balancing
Ion-pairing agent or buffer by regulatory agencies such as the
organic modifier concentration per three important factors:
Laboratory scale purifications FDA. Ethanol is presently used in
generally use the same ion-pairing unit time—to increase resolution, large scale process purifications59. Throughput
agents or buffers as analytical particularly for the main polypeptide
The amount of purified polypeptide
chromatography. to be collected. Ion-pairing agent or buffer
produced in a given time period. While
Ion pairing agents commonly used
low sample loads yield maximum
Gradient characteristics Protein Loading Capacity of RP-HPLC for analytical chromatography are
resolution, only small quantities are
To retain the resolution obtained on Materials of Different Particle Size less practical for process scale
purified per chromatographic run
an analytical column while increasing chromatography. Alternate ion-pairing
and throughput is low.
column diameter, the gradient shape 160 agents or buffers useful for process
Peak Width at Half Height (mm)

140 20–30 micron

must be maintained by keeping the chromatography include acetic acid— Purity
120 15–20 micron
ratio of the gradient volume to the which also converts the polypeptide to The purity of the polypeptide
column volume constant. For example, 80
the acetate form, useful in formulations expressed in percent of total weight
a 22 mm diameter column has about 60 —and phosphate. Acetate is presently of final purified product. Pure
23 times the volume of a 4.6 mm 40 10 micron used in the purification of several polypeptides are obtained by
diameter column of the same length 20 5 micron biotechnology derived polypeptide
avoiding overlap with adjacent
(22 divided by 4.6, squared). 0 200 400 600 800 1000
therapeutics61. peaks although this may limit the
A 1.0 mL/min gradient over 30 amount of sample that can be loaded
minutes on an analytical column has Gradient characteristics
onto the column.
a volume of 30 mL. To transfer the Figure 42. Although peak widths are much The comments in the laboratory scale
narrower with small particle materials at low purification section regarding
method to a 22 mm column, the sample loads, there is little difference in peak
gradient volume should be increased widths at high loads, where the column is scaling up elution gradients to larger
“overloaded”. Column materials: VYDAC® columns apply to process scale
23 times to 690 mL. The flow rate 214TP, 5 µm; VYDAC® 214TP, 10 µm; VYDAC®
can be increased 23 times while 214TP, 15–20 µm; VYDAC® 214TP, 20–30 µm purifications (see above). Very
maintaining the gradient time Eluent: 24–95 % ACN in 0.1% aqueous shallow gradients in the region
TFA over 30 min at 1.5 mL/min; Protein:
constant or the flow rate can be ribonuclease. where the polypeptide of interest
elutes are common.

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Yield Practical Loading Capacity In Figure 44, injections of 25, 100, There are many examples in the
The percent of polypeptide purified Preparative separations require 200, 500 and 1,000 micrograms of literature of practical purification
as a percent of the total amount of maximizing throughput by balancing ribonuclease and lysozyme illustrate of polypeptides at high loading
polypeptide present in the original resolution, yield and purity. Often the effect on resolution of increasing levels46–50. In one case 1.2 grams of
sample. Maximizing resolution improving yield comes at a cost of peak width resulting from increasing a synthetic peptide mixture were
enables recovery of most of the reduced purity or reduced throughput. sample loads. At 25 and 100 µg purified on a 5 x 30 cm column46.
loaded polypeptide while removing In practice this generally requires injections—in the region of optimum In a personal communication it was
impurities. If resolution is poor then “overloading” the column—that is, resolution—resolution between reported that 5 grams of synthetic
only the center of a peak is collected, injecting polypeptide samples greater ribonuclease and the small impurity peptide were purified on a 5 x 25 cm
reducing yield. than the sample capacity defined by preceding it remains constant (Figure column in two steps.
optimum resolution. As the sample 44A, B). Resolution begins to decrease
There are three measures of sample load is increased, polypeptide peak between ribonuclease and the impurity
capacity on a RP-HPLC column: widths increase (Figures 43 and 44), above 100 µg—the “overload” point.
■ the loading capacity with however peak shape remains reasonably The 200 µg load shows a definite
optimum resolution; symmetrical. This often allows the increase in peak width and consequent Effect of Sample Load on Protein
■ the practical sample loading loading of samples 10 to 50 times the loss of resolution (Figure 44C). At Peak Shape and Resolution
capacity; nominal sample capacity while still 500 mg there is considerable loss in
resolution (Figure 44D) and at 1,000 µg ribonuclease lysosyme
■ and, the maximum amount of retaining acceptable resolution.
polypeptide the column will bind. the impurity peak completely merges
with the ribonuclease peak (Figure 44E). impurity impurity

Sample Loading Capacity

Resolution between lysozyme and the
with Optimum Resolution Sample Loading Curve for preceding impurity peaks remains
Ribonuclease on Analytical Column constant to about 200 µg, after which
In chromatography the loading
limit of a column is normally 160 resolution is slowly lost. At 500 µg
defined as the maximum amount 140 (Figure 44D) the impurity peaks
of analyte that can be 120 appear only as shoulders on the
Peak Width (mm)

100 lysozyme peak and by 1,000 µg

chromatographed with no more
than a 10% increase in peak width. Practical loading range (Figure 44E) the impurity peaks have
60 (column is overloaded) completely merged with the lysozyme
Peak width and resolution remain peak. Resolution between the protein
20 Overload Point
constant up to the “overload” point 0
and impurity peak can be improved
which, for analytical (4.6 mm diameter) 0 400 800 1200 1600 by running a more shallow gradient.
columns, is about 100 to 200 µg for
most polypeptides (Figure 43). Figure 43. Peak width is constant with sample Since resolution between the two, well
loads up to 200 µg. Above 200 µg—the separated, major peaks—ribonuclease Figure 44. A. 25 µg each protein B. 100 µg
Loading samples greater than this “overload” point—the peak width gradually each protein C. 200 µg each protein D. 500 µg
amount results in broadened peaks increases. The practical loading region for and lysozyme—remains good even at each protein E. 1000 µg each protein Column:
ribonuclease is 200 to 5000 µg. Column: the 1,000 µg sample load and peak VYDAC® 214TP54 (C4, 5 µm, 4.6 x 250 mm)
and decreased resolution. VYDAC® 214TP54 (C4, 5 µm, 4.6 x 250 mm) Eluent: 25–50% ACN in 0.1% TFA over 25
Eluent: 24–95% ACN with 0.1% TFA over shape is not seriously degraded, very minutes at 1.5 mL/min. Sample: ribonuclease
30 minutes Sample: ribonuclease. high sample loads are possible for and lysozyme.
well separated peaks.
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Maximum Polypeptide Use shallow gradients Biological Activity and Small peptides and very stable proteins
Binding Capacity Resolution between closely eluting Reversed-Phase HPLC are less likely to lose biological
The maximum binding capacity of polypeptides may be improved by activity than large enzymes. Some
using a more shallow gradient slope. Biological activity of proteins specific points to keep in mind are:
a polypeptide on a reversed-phase depends on tertiary structure
column depends on the size and This is usually done by lengthening
the gradient time. Suggestion: Use and permanent disruption of Protein denaturation
characteristics of the polypeptide. tertiary structure eliminates
longer elution times and shallow Denaturation of proteins on
Small peptides have binding biological activity.
gradients to obtain maximum hydrophobic surfaces is kinetically
capacities of about 10 mg of peptide
resolution for closely eluting peaks. slow. Reducing the residence time of
per gram of separation material— RP-HPLC may disrupt protein the protein in the column generally
25 mg on a 4.6 x 250 mm column. tertiary structure because of the
Increase the column volume reduces the loss of biological activity.
Proteins have slightly higher binding hydrophobic solvents used for elution
Since sample capacity is a function
capacities between 10 and 20 mg or because of the interaction of the Solvent effects
of column volume, either column
of protein per gram of separation protein with the hydrophobic surface of Some solvents are less likely to
diameter or column length can be
material, depending on the ratio of the material. The amount of biological cause a loss of biological activity
increased for increased sample load. It
the area of the hydrophobic foot to activity lost depends on the stability of than others. Isopropanol is the best
is the volume of the column that is
the total molecular weight. the protein and on the elution conditions solvent for retaining biological
important, not the diameter or the length.
used. The loss of biological activity activity. Ethanol and methanol are
Although sample loads near the
Use large particle adsorbents can be minimized by proper slightly worse and acetonitrile
maximum binding capacity of a post-chromatographic treatment. causes the greatest loss of
When columns are “overloaded”,
column provide little resolution, biological activity.
particle size becomes less significant
they are useful for simple, fast
in obtaining resolution (Figure 42). HIV Protease Biological Activity
desalting of polypeptide samples. Stabilizing factors
Small particle materials give only
slightly better resolution than large Stabilizing factors, such as
Ways to Optimize particle materials under “overload” After lyophilization, dissolve residue at enzyme cofactors, added to the
Throughput and Resolution conditions and the higher cost, higher 5–15 mg/mL in 50 mM sodium acetate, pH 5.5, chromatographic eluent, may
containing 8 M urea, 1 mM EDTA and stabilize proteins and reduce the
back-pressure and practical difficulties 2.5 mM dithiothreitol.
Sample concentration of column preparation with small loss of biological activity.
Resolution between closely eluting particle materials make them impractical Refolding
polypeptides may be affected by for most preparative separations. Dilute with 9 volumes of 50 mM acetate, pH The most important factor in
sample concentration. Dilute samples 5.5, containing 1 mM EDTA and 2.5 mM maintaining or regaining biological
dithiothreitol, 10% glycerol, 5% ethylene
appear to spread out over the column Effective loading of the sample glycol and 0.2% Non-idet P-40 at 4˚ C.
activity is post-column sample
surface better than concentrated Load the sample in a solvent that will treatment. Dissolution of a collected
samples and this results in somewhat not interfere with adsorption of the Result protein in a stabilizing buffer often
better resolution. Recommendation: polypeptide. This generaly means Specific activity = 1.0 + -0.1 mmol substrate allows the protein to re-fold. An
hydrolyzed per minute per mg of protein—
Use dilute samples to improve keeping the organic content well example is HIV protease (Figure 45)56.
compared to specific activity of 1.2 for enzyme
resolution and sample loading capacity. below that required to elute the expressed in E. coli.
polypeptide from the column. Some
solvent in the sample, however, Figure 45. Procedure used to regain biological
activity of HIV protease after reversed-phase
improves sample loading. chromatography (Reference 56).

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Examples of Biological Use of Reversed-Phase Example of Large 6 Analysis of collected fractions for
Activity after RP-HPLC HPLC in the Purification Scale Purifications purity and yield;
of Commercial 7 Combining the optimum fractions
Trypsin Polypeptide Therapeutics Laboratory-scale purification for a final yield of 128 milligrams
Reversed phase chromatography has Perhaps the most compelling Several examples of the purification of GnRH antagonist at a purity of
been used to purify trypsin for use evidence that biological activity of synthetic peptides by RP-HPLC 99.7%.
in protein digestion57. is not inevitably lost during have appeared in the literature46–50. In
reversed-phase chromatography is one case46 128 mg of gonadotropin Removal of Virus Particles
Polio virus proteins the fact that several commercial releasing hormone (GnRH) antagonist During Reversed-Phase HPLC
Polio virus proteins purified by bio-therapeutics use reversed-phase was purified from 1.2 grams of Purification
reversed phase chromatography were chromatography in the purification synthesis mixture in two RP-HPLC
able to induce production of specific One of the benfits of incorporating
of the marketed product. purification steps (Figure 46). The a reversed-phase chromatography
antibodies in rabbits, indicating a ■ Erythropoetin may be procedure involved (see Reference 46
retention of biological activity23. separation step into a process to
purified using reversed-phase for details): produce large quantities of a
chromatography as an integral 1 Establishing elution conditions
Pollen allergens therapeutic protein is the removal
part of the purification process59. with triethylammonium phosphate or clearance of virus from the
The main protein allergen of
■ Leukine, a marketed polypeptide and acetonitrile on a five protein “soup”.
Parietaria judaica retained IgE-binding
therapeutic, uses reversed-phase micrometer, 4.6 x 250 mm, column;
activity even after RP-HPLC
HPLC as an integral part of its 2 Loading the synthetic peptide onto
purification because it eluted at
low acetonitrile concentration58. purification procedure60, 61. a 5 x 30 cm column packed with
15–20 µm adsorbent comparable to Purification of Synthetic Peptide
■ Human recombinant insulin
HIV protease purification uses reversed-phase the five micron material in
the column in Step One and GnRH
HIV protease regained most of chromatography in its production62. antagonist

Absorbance Units (280 nm)

its biological activity after elution with acetonitrile and
reversed-phase chromatography and triethylammonium phosphate;
While the conditions of
post chromatographic treatment to 3 Analysis of collected fractions for
reversed-phase chromatography
allow refolding (Figure 45)56. may cause some loss of tertiary purity and yield and combining
structure and biological activity, the best fractions for desalting
in most cases this loss of biological and final purification;
activity may be moderated or 4 Dilution and re-injection on the

eliminated by use of optimum same column;

0 Minutes 60
chromatographic conditions or by 5 Elution using acetonitrile and
post-chromatographic treatment. TFA to remove the non-volatile Figure 46. Purification of 128 mg of a synthetic
peptide, GnRH antagonist 1.2 grams of
phosphate salt and improve synthesis mixture were loaded onto a 5 x 30 cm
resolution further; column packed with VYDAC® 218TPB1520
(C18, 15–20 µm) and eluted with a gradient
of acetonitrile in water containing
triethylammonium phosphate.

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Viral Inactivation During
Reversed-Phase HPLC Purification

Viral inactivation by ethanol and chromatographic separation

R eversed-phase HPLC usually
reduces or eliminates viral
activity in protein preparations,
The data in the table below
illustrates that some viruses are
highly inactivated in ethanol
Log10 infectivity reduction by exposure to ethanol for 30 minutes (1st Row)
or a combination of ethanol and chromatographic separation (2nd Row).
making it a valuable step in (Xenotropic Murie Leukemia Virus
recombinant protein purification. and Pseudorabies Virus) while others XmuLV MVM Adeno 5 PRV
Viral inactivation occurs through (Minute Virus of Mice and Human Ethanol exposure >4.9±.13 .4±.2 0.1±.44 >4.6±.08
two mechanisms. First, exposure to Adenovirus type 5) are less strongly RP-HPLC in ethanol >5.9 2.9±.4 2.4±.44 >5.6±.32
ethanol inactivates many viruses. inactivated. The combination of
Second, viruses can be separated ethanol and chromatographic
from protein in the chromatographic separation, however, significantly XMuLV–Xenotropic Murine Leukemia Virus
step (see Figure 47). reduces the infectivity level of all MVM–Minute Virus of Mice
four viruses. Adeno5–Human adenovirus type 5
PRV–Pseudorabies Virus
Separation of Xenotropic Murine Leukemia Virus

Ethanol Gradient


100 XMuLV Virus

was below the
XMuLV Virus limit of detection
was detected in this peak
in this peak

0 50 100 150 200 mL

Figure 47. Separation of Xenotropic Murine Leukemia Virus (XMuLV) from target protein during
preparative HPLC. Column: VYDAC® C4, 20-30 mm Elution: Ethanol gradient
Data courtesy of Holly Harker and Marcus Luscher, Amgen, Boulder, Colorado

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Appendix A: Column Characteristics Appendix B: The Care and Maintenance
of Reversed-Phase Columns

Flow Rate
Maximum Practical
Sample Load
R eversed-phase HPLC columns,
if properly cared for, may give
good performance for over a thousand
Column Storage
RP-HPLC columns can be stored in
(mm) (1) (2) (3) organic solvent and water. For long
sample injections, depending on sample term storage the ion-pairing agent or
Capillary 0.075 0.25 µL/min 0.05 µg preparation and elution conditions. buffer should be rinsed from the
0.15 1 µL/min 0.2 µg Although the following ideas are column and the organic content
0.30 5 µL/min 1 µg specifically applicable to VYDAC® should be at least 50%.
0.50 10 µL/min 2 µg RP-HPLC columns, they also apply
to most other columns.
Chemical Stability
Microbore 1.0 25–50 µL/min 0.05–10 µg Reversed-phase HPLC columns are
Column Protection
stable in all common organic solvents
Column lifetime can be extended by including acetonitrile, ethanol,
Narrowbore 2.1 100–300 µL/min 0.2–50 µg filtering all solvents and samples isopropanol and dichloromethane.
and using an eluent filter and a guard When switching solvents it is
column. We recommend using an important to only use mutually
eluent filter between the solvent miscible solvents in sequence.
Analytical 4.6 0.5–1.5 mL/min 1–200 µg 10 mg delivery system and the injector to Silica-based RP-HPLC columns
trap debris from the solvents, pumps or are stable up to pH 6.5 to 7 and are
mixing chamber. We also recommend not harmed by common protein
Semi-preparative 10 2.5–7.5 mL/min 1,000 µg 50 mg using a guard column between the detergents such as sodium
injector and the column if samples dodecylsulfate (SDS).
contain insoluble components or
Preparative 22 10–30 mL/min 5 mg 200 mg compounds that strongly adsorb to
Pressure and
the material.
Temperature Limits
Column Conditioning RP-HPLC columns are generally
Process 50 50–100 mL/min 25 mg 1,000 mg stable to 60˚C and up to 5,000 psi
100 150–300 mL/min 125 mg 5,000 mg Because of the nature of the
(335 bar) back-pressure. Typical
reversed-phase surface, column
back-pressures for RP-HPLC
performance (resolution, retention)
columns are shown in Figure B-1.
may change slightly during the first few
injections of proteins. A column can
be conditioned by repeated injections
Figure A-1. of the protein until the column
1. Actual flow rates can be a factor of two higher or lower depending on the method.
characteristics remain constant
2. Sample Capacity is the quantity of polypeptide that can be loaded onto the column (requires injection of about 100 µg of
without reducing resolution.
protein) or by injection of 100 µg of
3. Maximum Practical Sample Load is approximately the maximum quantity of sample a commonly available protein, such as
that can be purified with reasonable yield and purity on the column. ribonuclease, followed by running an
acetonitrile/0.1% TFA gradient.

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RP-HPLC Column If the column back-pressure is high, Protein contamination or chloroform. When changing
Trouble-Shooting most HPLC columns can be If the loss in column performance from water to chloroform or
reversed and rinsed to try to flush appears to be due to adsorbed protein dichloromethane or back again it is
The performance of RP-HPLC
contaminants from the inlet frit. we recommend rinsing the column important to rinse the column with
columns may deteriorate for a
Begin the reverse rinse at a low with a mixture of one part 0.1 N a mutually miscible, intermediate
number of reasons including use of
flow rate—10 to 20% of normal— nitric acid and four parts isopropanol. solvent such as isopropanol or
improper eluents, such as high pH,
or 10–15 minutes and then increase Rinsing at a low flow rate—20% of acetone between the two less
contamination by strongly adsorbed
to the normal flow rate. normal—overnight is most effective. miscible solvents.
sample constituents, insoluble
materials from the solvent or sample Contaminated column Lipids or other very Spurious–"ghost"–peaks
or simply age or extensive use. Here Wash the column either with 10–20 hydrophobic contaminants Unexpected peaks sometimes appear
are some suggestions to restore the column volumes of a strong eluent If lipids or very hydrophobic small in HPLC chromatograms. These
performance of a RP-HPLC column. or run 2–3 ‘blank’ gradients (without molecules are causing the change in are usually caused by contaminants
sample injection) to remove less column performance, we recommend in the solvents used. Hydrophobic
High back-pressure
strongly adsorbed contaminants. rinsing the column with several contaminants in Solvent
Disconnect the column from the
injector and run the pumps to ensure column volumes of dichloromethane A-contaminants may be present in
that the back-pressure is due to the the water or the ion-pairing agent
column and not the HPLC system. or buffer—accumulate on the
column during equilibration and at
low solvent concentrations and elute
Typical Back-Pressures of RP-HPLC Columns Evidence of Solvent Contaminants as “ghost” peaks during the gradient.
as Source of Ghost Peaks This can be easily diagnosed by
making two gradient runs, the first
Column Size Flow Rate Typical Back-pressure
with a relatively long equilibration
(mm) (mL/min) (with 50:50 ACN:Water)
time–30 minutes—and the second
2.1 x 250 0.20 1000 –1800 psi
with a short equilibration time—
4.6 x 250 1.0 1000 –1800 psi
10 minutes (example, Figure B-2).
4.6 x 150 1.0 600 –1200 psi
The short equilibration will have
10 x 250 5.0 1000 –1800 psi
smaller peaks than the long
4.6 x 250 1.0 500 –1000 psi
equilibration if the “ghost peaks”
10 x 250 5.0 500 –1000 psi
are due to contaminants in the “A”
22 x 250 25.0 500 –1000 psi
Figure B-2. solvent because less contaminants
Figure B-1. will adsorb onto the column with the
short equilibration. To correct the
problem use higher purity or fresh
water or ion-pairing agent or buffer.

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Appendix C: The Effect of Surfactants
On Reversed-Phase Separations

P olypeptide samples sometimes

contain surfactants. To determine
the effect of surfactants on RP-HPLC
The separation on a C18 column of
the protein sample with SDS was
much worse (Figure C-1B) than the
Results on a C4 column were slightly
better than those obtained on the
C18 column (Figure C-2). The
small amounts of SDS affected the
digest separation and higher amounts
virtually destroyed resolution
polypeptide separations and on the separation of the same sample without presence of SDS in the protein (Figure C-3).
columns themselves, five proteins— SDS (Figure C-1A). Subsequent sample affected the chromatography
ribonuclease, insulin, lysozyme, chromatography of the sample without (Figure C-2B), however the effect Although surfactants usually degrade
myoglobin and ovalbumin—were SDS, however, showed no deterioration was less than on the C18 column RP-HPLC peptide separations, the use
chromatographed with and without (Figure C-1C), confirming that the (compare with Figure C-1). The SDS of octylglucoside, urea and guanidine
0.5% sodium dodecyl sulphate (SDS) SDS was removed in the gradient was removed in the gradient and did in the eluent have produced beneficial
in the sample (Figures C-1, C-2). and did not harm the column or not affect the column or subsequent results in some cases54, 55.
affect subsequent separations. separations (Figure C-2C).
Surfactants usually degrade RP-HPLC
Peptide separations are seriously polypeptide separations, however
Effect of Surfactants on Effect of Surfactants on affected by the presence of surfactant. they do not harm the column. If
C18 RP-HPLC of Polypeptides C4 RP-HPLC of Polypeptides Even trace amounts of SDS in a surfactants are present in the
peptide sample or protein digest sample, we recommend using a
A. Without SDS A. Without SDS
can reduce separation efficiency C4 reversed-phase column or
significantly12, 53. Peptide maps of a removing the surfactant prior
protein digest containing small to chromatography.
amounts of SDS showed that even

Effect of Surfactants on Peptide Map

B. With 0.5% SDS B. With 0.5% SDS

B. 0.001% SDS

C. After SDS C. After SDS

C. 0.01% SDS

D. 0.1% SDS

Figure C-1. Surfactants affect chromatography Figure C-2. Surfactant affects chromatography
(B) but do not harm column or subsequent (B) but does not harm column or subsequent
separations (C). Column: VYDAC® 218TP54. separations (C). Column: VYDAC® 214TP54 Figure C-3. The presence of even trace amounts of SDS causes a loss in resolution in a peptide
Eluent: 24–95% ACN in 0.1% TFA over Eluent: 24–95% ACN in 0.1% TFA over 30 min map. Column: VYDAC® 218TP52 (Narrowbore). Eluent: 2–80% ACN with 0.06% TFA over 120
30 min at 1.5 mL/min Sample: ribonuclease, at 1.5 mL/min Sample: ribonuclease, insulin, min at 0.25 mL/min. Sample: tryptic digest of carboxymethylated transferrin Data courtesy of K.
insulin, lysozyme, myoglobin and ovalbumin. lysozyme, myoglobin and ovalbumin. Stone and K. Williams. Ref. 12.

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Appendix D: Ion Exchange Chromatography
Orthogonal Analytical Techniques

R eversed-phase chromatography
separates polypeptides on the
basis of hydrophobicity; ion-exchange
reversed-phase and cation exchange
HPLC illustrates the complementary
selectivity of the two techniques
The Benefits of Ion-Exchange
The Benefits of Reversed-Phase
■ Relatively high sample loading ■ A high degree of selectivity based
chromatography separates on the (Figure D-1). On the cation exchange
basis of charge. These complementary column singly-charged oxytocin capacity compared to reversed- on differences in hydrophobicity
separation techniques offer synergistic elutes early, followed by the three phase. or molecular conformation.
capabilities in the analysis and doubly-charged peptides—neurotensin, ■ Resistance to strong reagents such ■ Use of volatile buffers or
purification of proteins and peptides angiotensin II and bradykinin. as 0.1 M NaOH, 0.1 M acid or ion-pairing agents.
and are often used together because Angiotensin I with four charges 6 M guanidine because of the ■ Freedom from interferences by
of the different separation elutes last. On reversed-phase the polymeric matrix. Relatively crude salt or buffers from ion exchange.
mechanisms. In series they offer peptides elute in the order of oxytocin, solutions can be loaded onto
better purification than can be bradykinin, angiotensin I, neurotensin ion-exchange columns because Ion-exchange chromatography is
achieved with either one alone; in and angiotensin II. The complementary adsorbed matrix components can normally used first, followed by
parallel they offer mutual confirmation selectivities provide two dimensional be removed with strong reagents. reversed-phase chromatography
of analytical results. Comparison of resolving power. ■ Addition of urea, acetonitrile or (Figure D-2). Crude samples can be
the separation of several peptides by non-ionic detergents to break-up loaded onto a polymer-based ion
complexes. exchange column without damaging
■ Optimization of elution selectivity the column; ion-exchange has a high
by adjustment of pH. loading capacity to accomodate
complex samples; and chaotropes
High Performance Reversed-Phase can be added to the sample to break
Comparison of High Performance Reversed-Phase and High Performance
Ion-Exchange Chromatography in the Separation of Peptides and Ion-Exchange Chromatography up protein complexes. The partially
Used in Series to Remove Impurities purified polypeptide, containing salts
in Lysozyme and buffers from the ion exchange
Reversed-Phase (218TP54) Cation Exchange (400VHP575) separation, can then be loaded onto a
5 reversed-phase column. The salts are
5 Cation
Exchange not retained and do not harm the
3 reversed-phase column. Purification
3 lysosome peak
from IEX onto RP based on hydrophobicity or
1 2 conformation then takes place
4 impurity on RP
1 4 and the collected sample elutes
in a volatile solution, ready for
impurity on RP Reversed-
final preparation.

0 25 min 0 40 min
Figure D-2. Strong Cation Exchange Column:
VYDAC® 400VHP575, Cation exchange, 5 µm,
7.5 x 50 mm Eluent: 10 mM phosphate, pH
Figure D-1. Reversed-Phase Column: VYDAC® 218TP54, C18, 5 µm, 4.6 x 250 mm Eluent: 6.5/25% ACN; gradient from 0–0.1 M NaCl in
15–30% ACN in 0.1% TFA over 30 minutes at 1.0 mL/min Strong Cation Exchange Column: 25 min at 1.0 mL/min Reversed-Phase Column:
VYDAC® 400VHP575, Cation exchange, 5 µm, 7.5 x 50 mm Eluent: 10 mM phosphate, VYDAC® 214TP54, C4, 5 µm, 4.6 x 250 mm
pH 2.7/25% ACN; gradient from 0-0.1 M NaCl in 20 min at 1.0 mL/min Sample: 1. oxytocin Eluent: 10–35% ACN in 0.1% TFA, 50 minutes
2. bradykinin 3. angiotensin II 4. neurotensin 5. angiotensin I. at 1.0 mL/min Sample: Lysozyme.
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Appendix E: The Effect of System Hardware
on Reversed-Phase Polypeptide Separations

R eversed-phase HPLC peptide

separations are sensitive to the
shape of the gradient and hence, to the
Figure E-2 shows the effect that the
gradient response delay has on
narrowbore columns run at low flow
Calculation of Desorbing
Solvent Concentration
the solvent concentration given by
the system when the polypeptide
elutes is higher than the actual
Because of internal volume in the
characteristics of the system hardware rates. The peptide separation on a solvent concentration that desorbs
flow system—tubing, mixing
being used. Pumps and gradient formers narrowbore HPLC column at 0.20 and elutes the polypeptide.
chamber, column void volume, etc—
can affect peptide separations in subtle mL/min (Figure E-2B) is compared
ways, especially at low flow rates. with the separation on an analytical
column at 1.0 mL/min (Figure E-2A) To calculate the solvent concentration that desorbs the polypeptide (CD):
Gradient Systems and using the same HPLC system and
Enter the retention time of the peak Example 33 min
Response Delay Time programmed gradient. The 10 minute
gradient response delay distorts the Subtract the retention time of the injection peak Example 2.5 min
To experimentally examine the actual Subtract the gradient response delay time Example 2 min
peptide separation (Figure E-2B).
gradient produced by an HPLC system,
Delaying sample injection and data And subtract any initial gradient hold time Example 5 min
replace the column with a short
collection ten minutes after starting
length of small diameter tubing and Equals corrected elution time (ETcorr) Example 23.5 min
the gradient cancels the effect of
run a 30 minute gradient at 1.0 ETcorr = Tretention - Tvoid - Tgradient Delay - THold
the gradient response delay and the
mL/min from water to 0.3% acetone
resulting narrowbore separation
(for absorbance) in water and monitor
(Figure E-2C) is similar to the The solvent concentration (CD) at the corrected elution time is:
at 254 nm. The UV profile represents
analytical separation (Figure E-2A). CD = CS + (ETcorr/Tg)(CE - CS); where
the gradient actually generated by the
CS = solvent concentration at start of gradient
system hardware (Figure E-1). The
CE = solvent concentration at end of gradient
gradient UV profile is used to:
Tg = time duration of gradient
■ Check on system reproducibility;
■ Determine system performance at
the extremes of the gradient; Gradient Hardware
Effect of System Hardware on Gradient Shape in Narrowbore HPLC
■ Calculate the gradient response System Evaluation
delay—the time from when the
controller or computer signals a
change in the gradient to when this
change actually reaches the column.
In the example (Figure E-1) the
gradient delay is about 3 minutes
(3 mL at 1 mL/min) calculated C
from when the run beguns to
where the profile begins to rise.
Figure E-1. The gradient generated by the Figure E-2. The system hardware gradient delay distorts the gradient shape at low flow rates and
Hardware systems that differ in system hardware is visualized by the profile of affects the peptide separation (B). Delaying sample injection to adjust for the gradient delay
gradient response delay times will a gradient increasing in acetone. Column: produces similar separation results (C) as obtained with an analytical column (A). Column: A.
Replaced by low-volume tubing. Gradient: VYDAC® 218TP54 (C18, 5 µm, 4.6 x 250 mm) B and C. VYDAC® 218TP52 (C18, 5 µm, 2.1 x
produce different gradient shapes, 0–0.3% acetone in water over 30 min at 1.0 250 mm) Eluent: 15–30% ACN in 30 min with 0.1% TFA. Flow rate: A. 1.0 mL/min B and
which may result in apparent mL/min. Detection: UV at 254 nm. C. 0.20 mL/min Peptides: 1. bradykinin 2. oxytocin 3. angiotensin II 4. neurotensin 5. angiotensin I
Note: In C, sample injection and data collection were delayed 10 min after initiating the gradient.
differences in peptide selectivity.
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Technical References

Basic Principles and Analytical Conditions Applications

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41 Evaluation of Genetic Stability of Recombinant Human Factor VIII by 49 Practical Aspects of Preparative Reversed-phase Chromatography of
Peptide Mapping and On-line Mass Spectrometric Analysis, M. Besman Synthetic Peptides, C.A. Hoeger, R. Galyean, R.A. McClintock and
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42 Application of capillary electrophoresis, high-performance liquid Conformation, Pages 753–764
chromatography, on-line electrospray mass spectrometry and 50 Process Purification of Polypeptides and Proteins by Reversed-phase
matrix-assisted laser desorption ionization-time of flight mass Column Chromatography: Misconceptions and Reality, P. Lu, C.D.
spectrometry to the characterization of single-chain plasminogen Carr, P. Chadwick, M. Li and K. Harrison BioPharm 14 (9), 28–35 (2001)
activator, A. Apffel, J. Chakel, S. Udiavar, W. Hancock, C. Souders,
E. Pungor, Jr., J. Chrom. A. 717, 41–60 (1995) Practical Aspects of Reversed-Phase HPLC
43 Direct analysis of protein complexes using mass spectrometry, A. Link, 51 Factors affecting the separation and loading capacity of proteins in
J. Eng, D. Schieltz, E. Carmack, G. Mize, D. Morris, B. Garvik and preparative gradient elution high-performance liquid chromatography,
J. Yates, III, Nature Biotech. 17, 676–682 (1999) Y-B. Yang, K. Harrison, D. Carr and G. Guiochon, J. Chrom. 590,
35–47 (1992)
Microbore, Narrowbore and Capillary Columns 52 Synthetic Peptide Purification by Application of Linear Solvent
44 Analysis of peptide mixtures by capillary high performance liquid Strength Gradient Theory, J.C. Ford and J.A. Smith, J. Chrom. 483,
chromatography: A practical guide to small-scale separations, 131–143 (1989)
M.T. Davis and T.D. Lee, Protein Science 1, 935–944 (1992) 53 Micropreparative Separation of Peptides Derived from Sodium
45 Microbore reversed-phase high-performance liquid chromatographic Dodecyl Sulfate-Solubilized Proteins, A. Bosserhoff, J. Wallach and
purification of peptides for combined chemical sequencing-laser-desorption R.W. Frank, J. Chrom. 473, 71–77 (1989)
mass spectrometric analysis, C. Elicone, M. Lui, S. Germanos, 54 Prevention of Aggregation of Synthetic Membrane Protein by Addition
H. Erdjument-Bromage and P. Tempst, J. Chrom. A, 676, 121–137 (1994) of Detergent, J.M. Tomich, L.W. Carson, K.J. Kanes, N.J. Vogelaar,
M.R. Emerling and J.H. Richards, Anal. Biochem. 174, 197–203 (1988)
Preparative Chromatography 55 Use of a Urea and Guanidine-HCl-Propanol Solvent System to Purify
46 Reversed-Phase High Performance Liquid Chromatography: a Growth Inhibitory Glycopeptide by High Performance Liquid
Preparative Purification of Synthetic Peptides, J. Rivier, R. McClintock, Chromatography, B.G. Sharifi, C.C. Bascom, V.K. Khurana and
R. Galyean, and H. Anderson, J. Chrom. 288, 303–328 (1984) T.C. Johnson, J. Chrom. 324, 173–180 (1985)
47 Preparative Reverse Phase High Performance Liquid Chromatography: 56 Substrate Analogue Inhibition and Active Site Titration of Purified
Effects of Buffer pH on the Purification of Synthetic Peptides, Recombinant HIV-1 Protease, A.G. Tomasselli, M.K. Olsen, J.O. Hui,
C. Hoeger, R. Galyean, J. Boublik, R. McClintock and J. Rivier, D.J. Staples, T.K. Sawyer, R.L. Heinrikson and C-S. C. Tomich,
Biochromatography 2, 134–142 (1987) Biochemistry 29, 264–269 (1990)
48 High Performance Preparative Scale Purification of Human Epidermal 57 A Simple and Rapid Purification of Commercial Trypsin and
Growth Factor, R.F. Burgoyne, M.F. Charette and T. Kierstead, Journal of Chymotrypsin by Reverse-Phase High-Performance Liquid
Analysis and Purification, October 1986, 48–53 Chromatography, K. Titani, T. Sasagawa, K. Resing and K.A. Walsh,
Anal. Biochem. 123, 408–412 (1982)
58 HPLC Purification of the Main Allergen of Parietaria judaica Pollen,
F. Polo, R. Ayuso and J. Carriera, Molecular Immunology 27(2),
151–157 (1990)

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Other Grace Vydac ADVANCES
59 Erythropoetin purification, Por-Hsiung and T. Strickland, U.S. Patent No. (Grace Vydac ADVANCES is a newsletter published by Grace Vydac.
04667016 For copies, contact Grace Vydac or visit our website at
60 Expression, Purification and Characterization of Recombinant Murine
Granulocyte-Macrophage Colony-Stimulating Factor and Bovine 63 Vydac ADVANCES, Spring 1997. Vydac Introduces 238TP, a New 300 Å
Interleukin-2 From Yeast, V. Price, D. Mochizuki, C.J. March, D. Monomeric C18 Reverse-Phase Column.
Cosman, M.C. Deeley, R. Klinke, W. Clevenger, S. Gillis, P. Baker and D. 64 Vydac ADVANCES, Spring 1997. What is 0.1% TFA? Changing the
Urdal, Gene 55, 287–293 (1987) modifier concentration provides another way to vary peptide selectivity.
61 Production of Recombinant Human Colony Stimulating Factors in Keeping it the same is important for reproducibility.
Yeast, S. Gillis, D. Urdal, W. Clevenger, R. Klinke, H. Sassenfeld, V. Price 65 Vydac ADVANCES, Winter 1998. Vydac 3 µm 300 Reversed-phase
and D. Cosman, Behring Institute Research Communications 83, Columns Speed Protein and Peptide Analyses.
1–7 (1988) 66 Vydac ADVANCES, Spring 1998. LC/MS and Microanalysis
62 Production Scale Purification of Biosynthetic Human Insulin By Applications Benefit from Vydac® Microbore Columns.
Reversed-Phase High-Performance Liquid Chromatography, 67 Vydac ADVANCES, Summer 1998. Optimum Peptide Purification.
E.P. Kroeff, R.A. Owens, E.L. Campbell, R.D. Johnson and H.I. Marks, Effects of Pore-Size, Buffers, pH, and Chemistry. Two Vydac Wide-Pore
J. Chrom. 461, 45–61 (1989) C18 Columns Provide Selectivity Alternatives.
68 Vydac ADVANCES, Summer 1998. The Concept of a Resolution Mixture.
Its Role in Developing and Validating Process Chromatographic Methods
69 Vydac ADVANCES, Spring 1999. Two New Reversed-Phase Columns
Permit Peptide Separations at Low TFA Concentrations
70 Vydac ADVANCES, Fall 1999. Peptide Separations on a C18 LC/MS
Column. Optimizing the Effects of Mobile-Phase Modifiers
71 Vydac ADVANCES, Fall 1999. Vydac Reversed-Phase Columns Aid in
Purification of Recombinant Alzheimer’s Proteins. University of Georgia
Group Produces Pure A1-42 in Milligram Quantities
72 Vydac ADVANCES, Spring 2000. Reversed-Phase Preparative and
Process Chromatography. A Powerful but Often Overlooked Method for
Protein Purification.
73 Vydac ADVANCES, Summer 2000. Peptide Separations. All 300 Å
Reversed-Phase Columns are Not the Same
74 Vydac ADVANCES, Winter 2001. Vydac’s Contribution to
Biopharmaceutical Development.
75 Grace Vydac ADVANCES, Winter 2001–2002. VYDAC® LC/MS
Pre-Packed Capillary Columns Benefit Peptide Analysis and Proteomics.
76 Grace Vydac ADVANCES, Winter 2001–2002. Practical Application
from the Protein Characterization and Proteomics Laboratory at University
of Cincinnati College of Medicine.

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Hesperia, California, USA
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About the author Phone: (800) 247-0924 or (760) 244-6107
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David Carr, a graduate of U.C. Berkeley, first became involved in HPLC
Columbia, Maryland, USA
in 1971, when the technique was in its infancy. As the technical marketing Executive offices, sales and marketing
management, and separations research
manager of Vydac from 1984–1996, David was involved in the use of
W. R. Grace & Co.– Conn.
reversed-phase HPLC for protein and peptide separations for both analytical 7500 Grace Drive
and preparative purposes. Working with companies such as Genentech, Columbia, MD 21044 USA
Phone: (410) 531-4000
Amgen, and Immunex, David assisted in developing protein and peptide Toll Free: (800) 638-6014
Fax: (410) 531-4273
separation methods for quality control as well as consulting on large-scale
preparative separations. Since 1996 David has developed and instructed Baltimore, Maryland, USA
ISO manufacturing and shipping
courses in analytical biotechnology and HPLC. His short course, Fundamentals
W. R. Grace & Co.– Conn.
in Analytical Biotechnology, is very popular among biotechnology companies 5500 Chemical Road
Baltimore, MD 21226 USA
(details may be found at David is the author of Phone: (410) 355-4900
Fax: (410) 354-8945
the first two editions of The Handbook of the Analysis and Purification
of Peptides and Proteins by Reversed-Phase HPLC as well as this, the Worms, Germany
Separations research and ISO manufacturing
Third Edition.
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In der Hollerhecke 1
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The information contained herein is based on our testing and experience and is offered for the user’s consideration, investigation
and verification. Since operating and use conditions vary and since we do not control such condiitions, we must DISCLAIM
ANY WARRANTY, EXPRESSED OR IMPLIED, with regard to results to be obtained from the use of this product. Test methods are
available on request. © 2002 W.R. Grace & Co.-Conn. All rights reserved. VYDAC is a registered trademark of The Separations
Group, a wholly-owned subsidiary of W. R. Grace & Co.–Conn.
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