biomolecule characterization and analyses

114 Pages·2016·16.88 MB·English

BIOMOLECULE CHARACT ERIZATION AND ANALYSES
[ INNOVATIVE HPLC, UHPLC, and UPLC CHEMISTRY CONSUMABLES FOR BIOSEPARATIONS ]
BIOMOLECULE CHARACTERIZATION AND ANALYSES
Innovative Technologies from the Leader in Separation Science and Analytical Biochemistry
Advances in the areas of genomics, proteomics, metabolomics, and molecular and system biology continue to revolutionize the
diagnosis and treatment of diseases and increase our fundamental understanding of biological processes.
As a leading analytical supplier of instrumentation, software, service and support, and chemistry products, Waters® is
uniquely positioned to provide researchers the tools, technologies, and integrated solutions desired to tackle the formidable
challenges involving various biomolecules. Beginning with a keen understanding of today’s biomolecule-related challenges,
Waters scientists and engineers continuously seek purposeful innovations that help deliver impactful solutions in applications
ranging from proteomics and biomarker discovery through the commercialization of advanced biopharmaceuticals. We continue
to develop new, innovative columns and sample preparation consumables that support the HPLC, UHPLC, UPLC,® and LC-MS
analyses of peptides, oligonucleotides, proteins, amino acids, and glycans.
Waters comprehensive chemistry and consumables family includes:
ƒ Peptide columns for nano, capillary, analytical, and preparative peptide applications
ƒ Protein size-exclusion, ion-exchange, hydrophobic-interaction, hydrophilic-interaction, and reversed-phase columns for
analytical HPLC, UHPLC, UPLC, and lab-scale purification applications
ƒ AccQ•Tag® Ultra chemistry specific for Waters UPLC Amino Acid Analysis Solution, as well as Pico•Tag® and AccQ•Tag
for HPLC-based amino acid analyses
ƒ Oligonucleotide columns for synthetic oligonucleotide and DNA/RNA fragment isolations and analyses
ƒ New GlycoWorks™ RapiFluor-MS™ labeling and sample preparation kits and Waters Glycan Columns for the analysis
of released glycans
ƒ Analytical Standards and Reagents consumables and kits for MS and LC-MS applications of peptides, proteins,
and other biomolecules
ƒ New ACQUITY UPLC® Glycoprotein BEH Amide, 300Å Column for the analyses of intact glycoproteins, glycoprotein
fragments, and glycopeptides
In addition, our ACQUITY UPLC Protein BEH SEC, 125Å, 200Å, and 450Å guards, columns, and quality controlled protein/peptide
standards, as well as our new ACQUITY UPLC Glycoprotein BEH Amide, 300Å offering, were developed for use on ACQUITY UPLC
Systems to help obtain accurate, precise, and highly resolving quantitative analysis of therapeutic proteins such as mAbs.
DESIGNED AND QC TESTED WITH RELEVANT BIOMOLECULES
TO HELP ENSURE COLUMN-TO-COLUMN CONSISTENCY.
Bioseparations Columns Analytical Standards and Reagents
www.waters.com/biosep asr.waters.com
TABLE OF CONTENTS
Factors to Consider when Investing and Using an HPLC, UHPLC, or UPLC Column for Bioseparations .....................................3
Part 1: Column Selection and Installation .....................................................................................................................................................................3
Part 2: Bioseparation Method Development ...................................................................................................................................................................6
Amino Acid Analysis Solutions .........................................................................................................................9
AccQ•Tag Ultra Chemistry and the UPLC Amino Acid Analysis Solution .....................................................................................10
Accurate Amino Acid Analyses from Varied Sample Matrices .......................................................................................................10
AccQ•Tag Amino Acid Analysis Using HPLC ..................................................................................................................................15
Pico•Tag HPLC Method ......................................................................................................................................................................16
Reference Materials for Amino Acid Analysis Solutions ................................................................................................................18
Glycan and Glycoprotein Separations .............................................................................................................19
Bringing Together Complementary Techniques to Streamline Glycan Analysis ............................................................................19
Intact Glycoprotein Analyses ............................................................................................................................................................20
Glycoprotein Subunit Analysis ..........................................................................................................................................................22
Glycopeptide Analyses ......................................................................................................................................................................24
Benchmarking, Method Development, and Troubleshooting: Glycoprotein Performance Test Standard ......................................26
Released Glycan Analysis .................................................................................................................................................................27
Sample Preparation ............................................................................................................................................................................28
Benchmarking, Method Development, and Troubleshooting: Glycan Performance Test Standards
and Dextran Calibration Ladders .......................................................................................................................................................30
Monosaccharide and Sialic Acid Analyses from Glycoproteins ......................................................................................................31
Reference Materials for Glycan and Glycoprotein Separations.......................................................................................................34
Oligonucleotide Separations ..........................................................................................................................35
Columns for Large DNA/RNA Species ..............................................................................................................................................38
Gen-Pak FAX Anion-Exchange Columns ...........................................................................................................................................38
Benchmarking, Method Development, and Troubleshooting: MassPREP Oligonucleotide Standard ............................................39
Oasis µElution Plates .........................................................................................................................................................................39
Reference Materials for Oligonucleotide Separations .....................................................................................................................40
Peptide Separations .......................................................................................................................................41
Peptide BEH C , 130Å and 300Å, and Peptide CSH C , 130Å Columns ..................................................................................41
18 18
Peptide CSH C , 130Å Columns .....................................................................................................................................................42
18
Benchmarking, Method Development, and Troubleshooting: Cytochrome c Digestion Standard .................................................48
Cation-Exchange Peptide and Polypeptide Separations ..................................................................................................................50
Therapeutic Peptide Method Development Kit .................................................................................................................................50
BioSuite HPLC and UHPLC Peptide Analysis Columns ....................................................................................................................51
BioSuite Cation-Exchange HPLC Columns ........................................................................................................................................52
Benchmarking, Method Development, and Troubleshooting: MassPREP Peptide Standard ..........................................................53
Delta-Pak HPLC and UHPLC Columns ...............................................................................................................................................54
Symmetry HPLC and UHPLC Columns ..............................................................................................................................................55
Additional Peptide Consumables ......................................................................................................................................................56
Reference Materials for Peptide Separations ...................................................................................................................................60
www.waters.com/biosep 1
TABLE OF CONTENTS (CONTINUED)
Protein Separations ........................................................................................................................................61
ACQUITY UPLC SEC System Solution...............................................................................................................................................61
Waters Insulin HMWP HPLC and UHPLC Columns ...........................................................................................................................63
XBridge Protein BEH SEC, 125Å, 200Å, and 450Å Columns and Protein Standard Test Mixtures ...........................................67
Benchmarking, Method Development, and Troubleshooting: BEH SEC Protein Standard Mix ..................................................67
Protein BEH C , 300Å Columns .......................................................................................................................................................69
4
Benchmarking, Method Development, and Troubleshooting: MassPREP Protein Standard Mix ....................................................72
ACQUITY UPLC Glycoprotein BEH Amide, 300Å Column ............................................................................................................73
Lifetime Testing of ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 µm Columns for Profiling
IgG Subunit Glycoforms .....................................................................................................................................................................74
Protein-Pak Hi Res Ion-Exchange (IEX) Columns for ACQUITY UPLC Applications .......................................................................76
Benchmarking, Method Development, and Troubleshooting: Ion Exchange Standards .................................................................77
Protein-Pak Hi Res HIC Column and HIC Protein Standard ..............................................................................................................79
BioSuite Ion-Exchange HPLC Columns ..............................................................................................................................................80
Protein-Pak PW Series Columns ........................................................................................................................................................81
Protein-Pak High Resolution (HR) Ion-Exchange Glass Columns .....................................................................................................82
Advanced Purification (AP) Glass Columns ......................................................................................................................................83
Accell Plus Ion-Exchange Packings ...................................................................................................................................................85
Accell Plus Sep-Pak Cartridges .........................................................................................................................................................85
Accell Plus PrepPak Cartridges (47 x 300 mm) ..............................................................................................................................85
Accell Plus Ion-Exchange Bulk Packings ..........................................................................................................................................85
Ion-Exchange Sample Preparation with Sep-Pak Cartridges ............................................................................................................85
BioSuite Size-Exclusion (SEC) HPLC Columns ..................................................................................................................................86
Protein-Pak and Shodex Size-Exclusion (SEC) HPLC Columns .........................................................................................................88
Symmetry300 C HPLC and UHPLC Columns .................................................................................................................................89
4
Protein-Pak Affinity Columns ............................................................................................................................................................90
BioSuite pC and pPhenyl Reversed-Phase Chromatography (RPC) HPLC Columns ....................................................................91
18
BioSuite Hydrophobic-Interaction Chromatography (HIC) HPLC Columns ......................................................................................92
Shodex and Protein-Pak Phenyl HIC HPLC Columns ........................................................................................................................93
Reference Materials for Protein Separations ....................................................................................................................................94
MS and LC-MS Consumables for Biomolecules ................................................................................................95
Nano- and Micro-Flow LC-MS ............................................................................................................................................................95
ionKey/MS ..........................................................................................................................................................................................96
ACQUITY UPLC M-Class Nano and micro Columns and Trap Columns ..........................................................................................99
Particle Technology .........................................................................................................................................................................102
ACQUITY UPLC M-Class with HDX Technology ............................................................................................................................104
MassPREP On-Line Desalting Devices ............................................................................................................................................105
Standards and Kits ..........................................................................................................................................................................107
TruView LCMS Certified Vials ..........................................................................................................................................................109
Waters Certified Containers .............................................................................................................................................................109
pH Buffers .........................................................................................................................................................................................109
Reference Materials for MS and LC-MS Consumables for Biomolecules .....................................................................................110
2
FACTORS TO CONSIDER WHEN INVESTING AND USING AN
HPLC, UHPLC, OR UPLC COLUMN FOR BIOSEPARATIONS
Many factors can affect the quality of data obtained from LC-based separations of peptides, proteins, and other biomolecules. The following pages list
just a few of the important factors to consider when selecting an appropriate HPLC, UHPLC, or UPLC column for an analytical or lab-scale applications.
Once an appropriate column is selected, time must be invested in developing a satisfactory separation, so we have also included a few useful method
development “tips and tricks”. We hope that these few examples will help chromatographers select a column and develop a method that matches their
specific instrumentation and application needs.
Part 1: Column Selection and Installation
Effect of Particle Composition on SEC Peptide Separations
ƒ Particle composition (e.g., silica, polymer, hybrid) influences desired LC separations
ƒ These “secondary interactions” can be beneficial or detrimental
ƒ Particle composition can also influence column life (e.g., silica-based at pH >7)
2.5 Rs=3.14 In the size-exclusion chromatographic (SEC) separation
2.0 Column: Silica SEC, 125Å, 5 µm, 3 4 shown, a series of synthetic peptides are separated on
7.8 x 300 mm
1.5 Flow rate: 0.84 mL/min 5 apa crotilculmesn (tcoopn)t avisn. inWga t1e0rs0 d%io sl-ilciocaat-ebda,s ebdri,d dgieodl--coated
1.0 1 2 ethylene hybrid (BEH Technology™) particles that have
0.5
less-undesired-free silanols. Consequently, and as
0.0
shown in this example, use of SEC columns that contain
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 min
e (214 nm) 122...505 C7F.loo8lw uxm r3an0t:e0 X: mB0.mr8id4g me LP/rmotine in BEH SEC, 125Å, 3.5 µRms=, 2.63 Rs=3.90 BsaoefpE crHtooh ntepidnsaaeirn rtpyi cpe liepeoatsnik dirscee ssainu nmtldetso rl areiencs t srice ooplnmieasa pbbklaee rtt.aa wFtiileovienre nglmy tm ohlreeaes k usii nbnuifgqno udrqmieutsiaaninrt eiatoidntn ad,t ion
anc 1.0 reference application note 720005369EN.
b
or 0.5
Abs 0.0
UV 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 min
Column: Two XBridge Protein BEH SEC, 125Å, 3.5 µm, Rs=4.24
2.5 7.8 x 600 mm
2.0 Flow rate: 1.68 mL/min Rs=3.11
1.5
1.0
0.5
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 min
Effect of Column Length on Reversed-Phase Protein Separations
ƒ Use of longer LC columns can translate into improved component resolution
ƒ Analysis time increases as column length increases
ƒ Separated peak volume increases as column length increases
2.1 x 50 mm 0.296 Increasing the length of the column will increase the resolving power
for a separation. This is shown with the separation of a protein
0.60
0.222 mixture. The additional small peaks surrounding the Phosphorylase b
AU can be seen more readily on the longer column, as seen in the inset,
0.148
0.40 but it comes at the cost of a 3-fold increase in run time and ~40%
AU 0.074 loss of sensitivity. Depending on the application objective, this may
be a useful parameter to improve resolution. For more information,
0.20 0.28815.00 15.60 16.20 16.80 17.40 18.00 min reference application note 720003875EN.
0.216
0.00 AU
0.144
0.60 2.1 x 150 mm 0.072
50.40 51.00 51.60 52.20 52.80 min
0.40
AU
0.20
0.00
0 11 22 33 44 55 min
www.waters.com/biosep 3
Effect of Particle Size on SEC Protein Separations
ƒ Well-packed columns containing small particles can improve a separation
ƒ System back pressure will increase as particle size decreases
ƒ Consequently, LC instrumentation can limit potential column use
00..1168 A 00..001280 C ImgoGnomer A(p /cno:m 1p8a6ri0so0n6 8of4 s2e)p aanradt iIonntasc ot fm WAabte Mrsa BssE HC4he5c0k SStEaCn dPraortde i(np /Snt:a 1n8da6r0d 0M6i5x 52,
0.14 0.016 diluted to 1 mg/mL) on 450Å, silica-based 8 μm (Frames A and C) and
0.12 0.014
00..0180 2 3 4 00..001102 IgG dimer Buffer 4sa5m0eÅ d, iBmEeHn s3i.o5n sμ m(7 .(8Fr ax m3e0s0 B m amn)d aDn)d S sEeCp acoralutimonnss .w Beorteh pceorlfuomrmnse dw weriet ht he
m)0.06 1b 5 m)00..000068 the same flow rate (0.84 mL/min) and with the same sample loads. Peak
Absorbance (280 n000000......000111024468 B 1a Absorbance (214 n0000....000020000024 D ImgoGnomer iMmCad pDyeapnonart)godi,lx t o1Diiemb bsti)na hftt eoh(e 1rylm yr4co oh 1glKre5olDocm0uab l)auKa,lt diraon angw (dr3e6a iu0m6gr0h9ast c sAKKi lDo D a(faa1 n,t) 1d,ha 2e2nB ) d I Dga Ig≥Gare)G4 .m: 5 F(11o0oanr5 ) Ko t0tmDh heKaye ,Drrc o,rha egdr)s,oli ompm3be)eauc rBtlt,ioi nSvagAe ndrla dyi(mm 6. mF7esou r i rlKn(t 1miD mf.aro3a)er, mr e4 ae)rs e
UV 0.12 UV 0.015 IgG dimer Buffer information, reference application note 720005202EN.
0.10
0.08 0.010
0.06 IgG
0.04 0.005 multimer
0.02
0.000
0.00
6 8 10 12 14 16 min 9 10 11 12 13 14 min
Choosing an Analytical Column that Best Matches LC-based Instrumentation
ƒ Standard methods can be performed to measure system dispersion (band broadening)
ƒ Different LC systems have different dispersion values (e.g., band broadening)
ƒ Optimal chromatographic separations are obtained when the appropriate column (e.g, column I.D., particle size) and separation conditions
(e.g., flow rate, temperature, gradient) are selected based on LC system design
HPLC UHPLC UPLC
Dispersion > 30 µL Dispersion 12–30 µL Dispersion < 12 µL
Columns: 3.0–4.6 mm I.D.; Columns: 2.1–4.6 mm I.D.; Columns: 1.0–4.6 mm I.D.;
3–10 µm particles 1.7–5 µm particles 1.6–5 µm particles
Recommended column: Recommended column: Recommended column:
4.6 mm I.D., 5 µm particles 3.0 mm I.D., 2.5 µm particles 2.1 mm I.D., 1.7 µm particles
Typical operating pressure: < 6,000 PSI Typical operating pressure: < 10,000 PSI Typical operating pressure: < 15,000 PSI
Dispersion – n. Broadening of an analyte band due to both on-column effects (diffusion and mass transfer kinetics which are both dependent on particle size and linear velocity) and
system effects (tubing internal diameter [I.D.] and length, connections, detector flow cell volumes, etc.) True separation performance is governed by the system dispersion paired with a
flow rate range that yields the highest possible efficiency for a given analytical column. Due to these dispersion levels, we can appropriately match the right type of column size (volume)
with the system dispersion. UPLC, having a very low dispersion volume, provides the greatest flexibility in terms of the columns that can be run on the system.
Effect of LC System Dispersion on SEC Monoclonal Protein Separations
ƒ LC Systems with lower system volumes better maintain column generated separations
ƒ Isocratic-based SEC separations are most sensitive to deleterious band broadening effects
0.30 The extra system volume of the traditional HPLC System
USP Res = 1.37 Alliance® HPLC (top) caused the column separated peaks to “partially
Dispersion > 30 µL remix” resulting in a USP resolution factor of 1.37 vs.
0.20 the superior 2.37 value obtained when the separation
U was performed on an ACQUITY UPLC System. This
A
slide shows how the LC system’s “band broadening”
0.10 can adversely affect the quality of the mAb separation
generated with the same XBridge® Protein BEH SEC,
200Å, 2.5 μm Column, SEC eluent, and sample.
0.00
0.25 USP Res = 2.37 ACQUITY UPLC
H-Class Bio
0.20
Dispersion < 12 µL
U0.15
A
0.10
0.05
0.00
2 4 6 8 10 12 14 16 18 20 22 24 26 28 min
4
Importance of Making Good Connections from Column to LC System
ƒ Poor column-to-instrument connections can degrade a chromatographic separation
ƒ Perceived column leaking can also be caused by a poor connection
UPLC HPLC Resulting Peak Shape
Tubing flush with end-fitting and good seal
Proper
Connection
Column tubing not seated properly - creates a void
Unswept volume causing band broadening
Improper
Connection
Importance of Batch-to-Batch and Column-to-Column Reproducibility
ƒ Column reproducibility is a key attribute when selecting a column
ƒ QC testing with relevant biological standards can help ensure consistency
XBridge Protein BEH SEC, 200Å XBridge Protein BEH SEC, 450Å
0.05 Ba1tach 11b, Column2 1 3 4 5 00..0150 Batch 1, Column 1 1b 2 3 4 5
1a
0.00 0.00
Batch 1, Column 2 0.10 Batch 1, Column 2
0.05 0.05
0.00 0.00
m) Batch 2, Column 1 Batch 2, Column 1
Absorbance (280 n 000...000055 Batch 2, Column 2 0000....01005005 Batch 2, Column 2
UV 0.00 0.00
Batch 3, Column 1 0.10 Batch 3, Column 1
0.05 0.05
0.00 0.00
Batch 3, Column 2 0.10 Batch 3, Column 2
0.05 0.05
0.00 0.00
6 8 10 12 14 min. 7 8 9 10 11 12 13 14 15 min.
Shown are overlays of the separations of Waters BEH200 SEC Molecular Weight Standard (p/n: 186006518) on 200Å and 450Å BEH 3.5 μm SEC columns. Two columns
(300 mm length x 7.8 mm I.D.) were packed from 3 individual manufactured batches of particles to evaluate both batch-to-batch and column-to-column reproducibility.
Peak identities are: 1a) thyroglobulin dimer (1.34 MDa), 1b) thyroglobulin (669 KDa), 2) IgG (150 KDa), 3) BSA (67 KDa), 4) myoglobin (14 KDa), and 5) uracil (112 Da).
Separations were performed on an ACQUITY UPLC H-Class BIO System.
www.waters.com/biosep 5
Part 2: Bioseparation Method Development
Eluent Effect on SEC Peptide Separations
ƒ Non-desired, secondary interactions can compromise LC separations
ƒ Use of an appropriate LC eluent can minimize secondary interactions
Method development experiments evaluated the effect of
Analyte MW
mobile-phase pH and salt concentration. The results showed
1 Ubiquitin 8,565
minimal effect of salt concentrations (150–350 mM) and
25 mM sodium 1 2 Aprotonin 6,511 mobile-phase pH (6.2–7.4) on retention time (data not shown).
phosphate pH 6.2, 3 Angiotensin I 1,296 All of the aqueous mobile phases resulted in later than expected
250 mM sodium chloride
4 Bradykinin 1,060 elution for most small peptides and proteins (<17,000 Da)
2
5 Bradykinin fragment 1-7 757 as well as elution order that did not correspond to published
molecular weight values. For example, bradykinin fragment 1–7
5
2a 4 3 (MW 757) eluted before greater molecular weight peptides such
as angiotensin I (MW 1296) and bradykinin (MW 1,060). These
results also suggest the non-ideal interactions of the tested
min
peptides with the media is not solely due to an “ion-exchange”
mechanism since increasing salt concentration had no
significant impact on retention time. For more information,
1
30% acetonitrile, 2 reference application note 720004412EN.
0.1% TFA
3
4 5
2a
min
Effect of pH on Ion-Exchange Protein Separations
ƒ pH has a significant influence on IEX separations
ƒ Optimal pH for a separation is sample dependent
1. Ribonuclease A To illustrate the effect of buffer pH, a mixture of proteins
3 was separated using weak cation-exchange chromatography
2. Cytochrome c 1 2 at various pH values. At a pH of 6, different selectivity
0.020 was observed for the most basic proteins vs. the separation
3. Aprotinin pH 6.0
AU 0.010 at pH 6.2 and greater. At pH 6, ribonuclease A elutes
before cytochrome c; this elution order is reversed when the
0.000 separation was performed at pH 6.2 or greater, as shown
3 in the figure. Sample: bovine, α-chymotryspinogen, bovine
1 ribonucelase A, equine cyctochrome c. Column: Protein-Pak™
0.020 2 Hi Res CM 7 μm, 4.6 x 100 mm. Conditions: 20 mM buffer
AU 0.010 pH 6.2 (MES or sodium phosphate) pH 6 to 7.2, 1 mL/min, 0 to 0.2
M NaCl in 34 min at 30 °C.
0.000
3
1
0.020 2
pH 6.4
AU 0.010
0.000
1 3
0.020 2
pH 7.2
AU 0.010
0.000
10 15 20 25 30 35 min
6
Temperature Effect on Reversed-Phase Protein Separations
ƒ Use of “room temperature” is NOT always the ideal separation temperature
ƒ Use of a column heater is strongly recommended for reproducible analyses
The intact IgG sample gave no observable peak at 40 °C,
0.75 but recovery for the IgG sample improves with increasing
0.50 temperature. There is not a measurable increase in recovery
AU 40 °C or improvement in peak shape above 80 °C. Column
0.25 temperature has a large effect on reversed-phase separation
0.00 of molecules. Changes in recovery and selectivity are
not uncommon with small molecule separations. While
0.75 increasing the temperature for proteins can significantly
improve recovery, particularly for intact monoclonal
U 0.50 60 °C antibodies, it doesn’t generally affect the selectivity of
A
0.25 the separation. However, not all proteins require higher
temperatures for improved recovery. In fact, some protein
0.00 separations have more desirable results with lower
separation temperatures. Therefore, it is recommended
0.75
that an evaluation of temperature be included in any
0.50 method development strategy for new samples. For more
AU 90 °C information, reference application note 720003875EN.
0.25
0.00
8.40 9.60 10.80 12.00 13.20 min
Effect of Flow Rate on Reversed-Phase Protein Separations
ƒ Use of lower flows can translate into improved component resolution
ƒ Analysis time will increase as flow rate increases
ƒ Sample complexity can influence selected separation flow rate
Decreasing the flow rate provides increased resolution without
a compromise in the sensitivity, as seen in this separation of
0.75 0.30
75 µL/min the protein mixture at 40 °C. The improved separation of the
0.20
AU Phosphorylase b sub-units can be seen (inset) at the lower flow
0.10 rate. The run time of the analysis is increased proportionally
0.50
U 0.00 to preserve the same gradient slope in both separations. Flow
A 42.00 43.20 44.40 45.60 46.80 min rate is seldom treated as an important parameter in method
0.25 development except as an indirect modification of gradient slope.
The impact of this variable is, however, more significant for
larger molecules. For more information, reference application
0.00 note 720003875EN.
0.75 200 µL/min
0.30
0.20
0.50 AU
U 0.10
A
0.00
0.25 15.60 16.00 16.40 16.80 17.20 min
0.00
0.00 9.60 19.20 28.80 38.40 48.00 min
www.waters.com/biosep 7
Effect of Gradient Duration of a Reversed-Phase Peptide Separation
ƒ Use of longer gradient time can translate into improved component resolution
ƒ Analysis time will increase using longer gradients as will peak volume
ƒ Sample complexity can influence selected gradient duration
Protein-Pak Hi Res Q, 4.6 x 100 mm 8 10 cv gradient The use of salt gradients can also be used to analyze
variants of a single protein. In this example, chicken
albumin was analyzed by anion-exchange chromatography.
3 4 7
6 9 Three different gradient slopes were employed to analyze
AU0.02 1 2 5 10 the variants of albumin formed by post transitional
modifications, such as methylation, phosphorylation, and
glycolyslation. As can be observed in the chromatograms,
0.00
the gradient slope can affect the number of variants
6 5 cv gradient detected, with shallower gradients reducing sensitivity but
allowing for resolution of additional variants. Note: All
AU0.02 3 4 5 7 gradients were performed at same flow rate from 0 to 0.5M
1 2 NaCl at same buffer pH.
0.00
6
AU0.02 3 4 5 7 3 cv gradient
1 2
0.00
4 6 8 10 12 14 16 18 20 22 min
Longer gradient: increased resolution, lower sensitivity.
Effect of Gradient Slope on an IEX Protein Separation
ƒ Gradients of differing salt concentration affect in IEX protein separations
ƒ Selected start and final salt concentration based on sample composition
ƒ Analysis time and component resolution increases using increasingly shallow gradients
Gradients of differing salt concentration are frequently used in
Protein-Pak Hi Res CM, 4.6 x 100 mm Ovalbumin 1 IEX protein separations to optimize retention time, component
48 1 Elutes in high salt wash step Myoglobin 2 resolution, and overall analysis time. In this example, a
5 Ribonuclease A 3 protein mix was analyzed using three different salt gradients
36 2 but keeping the run time identical. The higher salt gradient
%24 0-0.15M NaCl 3 Cytochrome c 4 shown on the bottom of the chromatogram increases the salt
4 Lysozyme 5 concentration from 0-0.5M NaCl, while the top chromatogram
12
has a gradient which ends at 0.15M NaCl, with the middle
0 chromatogram ending at 0.3M NaCl — note effect gradient
3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80 min
1 2 has on separation. Flow rates same for all separations.
28
4 5
21 0-0.3M NaCl
% 14
3
7
0
Unbound 3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80 min
proteins 2
48 1 4 5
36
% 24 3 0-0.5M NaCl
12
0
0.00 3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80 min
Higher salt gradients result in earlier elution of bound proteins.
High salt wash may be needed in shallower gradients to elute tightly bound proteins.
8