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4/4/2013
1
BioHPLC columns
Paul DinsmoorBiocolumn Technical Specialist
April 23-25, 2013
Size Exclusion BioHPLC Columns
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NEW Size Exclusion Columns
• 5m Particle
• 100Å, 150Å, 300Å, 500Å, 1000Å, 2000Å pore sizes
• High stability and long lifetime
• Great reproducibility
• Unique, 3m particle
• 100Å, 150Å, 300Å pore sizes
• Highest resolution
• Highest efficiency
• Faster SEC separations
Improved Efficiency With Smaller Particles
A il t Bi SEC 3 300Å
Column Pressure
Bio SEC-3(7.8x300mm)
93 bar
Bio SEC-5(7.8x300mm)
45 barPeak Protein
SEC-3, 300Å (7.8x300mm)
SEC-5, 300Å (7.8x300mm)
1 Th l b li 2460 1120
Column: Bio SEC-3 300Å and Bio SEC-5 300Å
Buffer: 150 mM Phosphate buffer, pH 7
Flow rate: 1.0 mL/min for 7.8x300 mm
Temperature: Ambient (~23° C)Min
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Agilent Bio SEC-3, 300Å,7.8x300mm
Agilent Bio SEC-5, 300Å,7.8x300mm
1 Thyroglobulin 2460 1120
2 BSA Dimer 5100 2720
3 BSA 13090 6590
4 Ribonuclease A 22000 11160
5 Uracil 38500 27860
Temperature: Ambient ( 23 C)
Detection: UV 214nm
Injection: 10 µL (3 L for 4.6x300 mm)
Sample: 1) Thyroglobulin (1.0 mg/mL), 670 kD; 2) BSA
dimer, 132 kD; 3) BSA (1.0 mg/mL), 66 kD; 4)
Ribonuclease A (1.0 mg/mL), 13.7 kD, and 5) Uracil
(2.5 g/mL), 120D.
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Column: Agilent Bio SEC-3, 7.8 x150mm Sample: mAb (2mg/ml)Injection: 5ulFl t 1 0 1 5 d 2 l/ i (56 b 75 b 105 b )
2.0 1.5 1.0ml/m ml/min ml/min
Fast separation of dimer from monomer using theAgilent Bio SEC-3 300 Å 7.8 x 150 mm column
Flow rate: 1.0, 1.5 and 2ml/min (56 bar , 75 bar, 105 bar)Eluent: 150mM sodium phosphate Detection: 220nm
Flow Rate ResolutionMonomer/Dimer
Monomer Efficiency
PercentageDimer
1.0ml/min 1.53 3,510 0.64
1.5ml/min 1.43 2,502 0. 47
monomer
dimer
2.0ml/min 1.13 1,917 0.64
Fast SEC – TB5990-8613EN
Peptides ProteinsGlobular proteins
Column Choice: Resolving Ranges
Thyr
oglo
bulin
IgG
Ovalb
umin
BSA
Myo
globin
Insu
lin
Agilent Bio SEC 100Å
Agilent Bio SEC 150Å
Agilent Bio SEC 300Å
ProSEC 300S
Agilent Bio SEC 500Å
Agilent Bio SEC 1000Å
Agilent Bio SEC 2000Å
100D 1kD 10kD 100kD 1,000kD 10,000kD
Zorbax GF-250
Zorbax GF-450
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Ion ExchangeBioHPLC Columns
NEW Ion Exchange Columns
• Non-porous PS/DVB particles (polystyrene divinylbenzene)
• Uniform polymeric coating with SCX, WCX, SAX, WAX layers, designed for protein and peptide separations
• Available in 10 µm, 5 µm, 3 µm, 1.7 µm particle sizes
• High surface area
• High capacity
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Agilent Bio IEX ColumnsComparing Separations on Each Particle Size
Column: Bio WCX-NP, 4.6x50mmBuffer A: 20 mM PBS
1.7 m
3 m
5 m
Buffer A: 20 mM PBSBuffer B: A+1.0 M NaClGradient: 0-100%B (20 min)Flow rate: 1.0 mL/min for NP10, NP5, NP3
0.75 mL/min for NP1.7Sample1) Ribonuclease A2) Cytochrome C3) LysozymeConcentration: 1.0 mg/mLDetector: 280 nm
Average N ~80,000 for WCX-NP1.7
Peak N
Min0 2 4 6 8 10 12 14 16 18 20 22
5 m
10 mPeak N
Ion Exchange ChromatographyCharge Isoform Analysis of Monoclonal Antibodies
Columns: Agilent Bio MAb, NP10, 4.6x250 mm
A
B
C
D
E
Mobile phase: A, 10 mM phosphate, pH 7.5B, A + 0.1M NaCl
Gradient: A) 15-75%B in 30 minB) 15-65%B in 30 minC) 15-55%B in 30 minD) 15-47.5%B in 30 minE) 15-40%B in 30 min
Flow rate: 0.8 mL/minSample: Monoclonal AntibodyInjection: 10 L (1.5 mg/mL)Temperature: 25 oC Detection: UV 214 nm
Min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
E
Optimization of method conditions for the isoform characterization of a monoclonal antibody. Changes in the buffer conditions, pH and gradient
conditions sharpen peaks and increase resolution of acidic and basic isoforms.
Detection: UV 214 nm
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Reverse PhaseBioHPLC Columns
NEW! Zorbax 300 SB RRHD for Proteins and Peptides!
• Stablebond 300 silica
• C-18, C-8, C-3, and diphenyl bonded phase
• 1.8 um particle size
• 1200 Bar pressure limit for uHPLC
• 2.1 x 50 mm and 2.1 x 100 mm
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UHPLC Columns Increase Resolution and Increase Speed
ColumnLength(mm)
Resolving Power
N(5 µm)
Resolving Power
N(3.5 µm)
ResolvingPower
N(1.8µm)
Analysis Time*
150 12,500 21,000 32,500
100 8,500 14,000 24,000
75 6000 10,500 17,000
50 4,200 7,000 12,000
30 N.A. 4,200 6,500
AnalysisTime
PeakVolume
-33%
-50%
-67%
-80% Solvent15 N.A. 2,100 2,500
%
-90%
SolventUsage
* Reduction in analysis time compared to 150 mm column
300SB-C18 RRHD Protein DigestTB# 5990-7989EN
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Column: Zorbax 1.8um 300SB-C18 RRHD, 2.1x100mmA : .1% TFA in water, B: .085% TFA in ACNGradient: 20%B 1min, 2-45%B 8.8min, 45-95%B .2min, 95%B 2min, 98-2%B .2min, 20%B 1.8minFlow rate: .5 ml/min, Temp: 50C, Pressure: ~640barSample: Protein digest, 5ul inj vol, 1 mg/ml
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Fast Separation of Reduced and Alkylated MAb
mAU
100
120
4.0
10
Heavy chain 1 Heavy chain 2
Conditions optimized for the ZORBAX RRHD 300SB-C3 Column
CHO Cell Derived MAb
min1 2 3 4 50
20
40
60
80
1000
.45
40
.50
10
.56
9
0.7
66
3.8
97
Light chain
Columns: ZORBAX RRHD 300SB-C3, 2.1 x 100 mm, 1.8 µmSample: Reduced MAb (IgG1) (1 0 mg/mL)- BioCreative IgG1
Time (min)
%B
15
Sample: Reduced MAb (IgG1) (1.0 mg/mL) BioCreative IgG1Sample injection: 2 µLMobile phase A: 0.1% TFA in waterMobile phase B: 80% n-propyl alcohol, 10% ACN, 9.9% water and 0.1% TFATemperature: 74 °CFlow rate: 0.5 mL/minDetection: UV, 280
0 1
2 20
5 50
6 90
6.1 1
TB# 5990-9667EN
Comparison C3 and Diphenyl Phases
mAU
6
8
10
12 1.33
7
1.58
7
1.66
6
1.75
4
1.81
1
ZORBAX RRHD 300SB-C3, 1.8 µm
Ribonuclease A, Cytochrome C and Lysozyme (3 mg/mL)
Time (min)
%B
min1 1.2 1.4 1.6 1.8 2 2.2 2.4
2
4
6
mAU
4
6
8
10
12 1.34
5
1.56
4
1.64
6
1.78
7
1.8
46
ZORBAX RRHD 300-Diphenyl, 1.8 µm
0 10
2.5 70
Arrows indicate better separation resolution of ZORBAX RRHD 300-Diphenyl
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min1 1.2 1.4 1.6 1.8 2 2.2 2.4
2
TB# 5990-9668EN
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AdvanceBio Peptide Mapping Column for HPLC and UHPLC:
• 2.7um Superficially Porous• 120A pore size p• 600 bar pressure limit • 2um frit to reduce clogging
Greater analytical confidence: Each batch is tested with a rigorous peptide mix to ensure suitability and reproducibility
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Save Time: 2 to 3 times faster than fully porous particles
Increased Flexibility: Highly compatible with TFA and formic acid mobile phases for efficient LC/MS analysis
Decrease the diffusion time for macromolecules and limit the diffusion path!
Superficially Porous Particle Technology
The particle has a solid core (1.7um) and porous outer layer with a 0.5um diffusion path
Reduces secondary interactions & enables selective separation for a wide range of peptides.
.
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Chromatographic ComparisonBSA tryptic digest
mAU
40
50
60
70
2.7um AdvanceBio Peptide Mapping, 2.1 x 150mm, gradient adjusted for length
104 peaks319 Bar
Greater Rs, increased sensitivity
min0 2.5 5 7.5 10 12.5 15 17.5 200
10
20
30
40
mAU
30
40
Vendor B, 1.7um, C18, 2.1 x 100mm, gradient adjusted for length
97 peaks476 Bar
Less Rs, decreased sensitivity
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min0 2 4 6 8 10 12
0
10
20
Conditions: water/ACN (TFA), 40C, .3 mL/min
Peptide Mapping for LC/MS
mAU
250
300
Agilent LC/MS 100% sequence coverage
Digested EPO ProteinGlycopeptide mapping
TB# 5991-1813EN
min0 5 10 15 20 25 300
50
100
150
200
Digested EPO (recombinant humanized) separated on a 2.1 x 250mm AdvanceBio Peptide Mapping ColumnMobile phase: A-water (0.1%FA), B- ACN (0.08%FA), temp: 55C, Flow: 0.5mL/min
E ll t ti bilit f ti tid f hi hl d i d t i i
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Excellent separation capability for generating peptide maps of highly desired proteins, i.e., glycopeptide mapping during fast analysis times
Provides compatibility with TFA and Formic acid mobile phases for LC/MS analyses
Pressure maintained below 600bar for 250mm column length
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Poroshell 300
300 A pore size
Stablebond chemistry
available in C-3, C-8,C-18, and C-18 Extend
5 um particle size
Porous Shell
SOLID CORE
5 m
High Flow Rates with 2.1 mm ID Poroshell for High Resolution and Fast Separations
34
5
Columns: Poroshell 300SB-C18 Sample:
TB# 5989-9899EN
12
67
8
2.1 x 75 mm, 5 mMP: A: 0.1% TFA
B: 0.07% TFA in ACNGradient: 5 – 100% B in 1.0 min.Flow Rate: 3.0 mL/min.Temperature: 70°CPressure: 250 barDetection: UV 215 nm
Sample:1. Angiotensin II2. Neurotensin3. Rnase4. Insulin5. Lysozyme6. Myoglobin7.Carbonic Anhydrase8.Ovalbumin
0 0.5 1.0Time (min)
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Reversed PhaseHeavy and light chain analytical characterization
Use of 1200 LC, Poroshell 300SB columns and UV detection to characterize antibodies
23
TB# 5989-0604EN
More Poroshell Bonded Phases Provide Selectivity Options to Enhance Resolution:
mAU
300
400
Poroshell SB-C18, 2.1 x 75 mm
15, 6
7
6
1.5
23
4
5, 6
7
4
5, 6
7
Poroshell 300SB-C182.1 x 75 mm
min0 0.5 1 1.5 2 2.5
0
100
200
2 3 4
8
9 0.9
76
1.0
77
1.2
89
1.3
95
1.4
63
1.7
41
1.8
60
mAU
300
400
51
23
4
7Poroshell SB-C3, 2.1 x 75 mm
1.4
97
5
4 6
7
5
4 6
7
1.51.5
Samples:
1. Angiotensin II
2. Neurotensin
3. RNase A
4. Insulin B Chain
5. Insulin
6. Cytochrome C
7. Lysozyme
8. Myoglobin
9. Carbonic
Anhydrase
Poroshell 300SB-C32.1 x 75 mm
min0 0.5 1 1.5 2 2.5
0
100
200 2 4
6 8
9
0.9
01
1.0
36
1.2
64
1.3
54 1.4
30
1.4
49
1.7
39
1.8
37
Column: Agilent Poroshell (2.1 x 75 mm); Temp.: 70 0C; Flow: 0.5 mL/min; Det: UV 215 nmMobile Phase: A= 0.1% TFA/H2O, B= 0.07% TFA/ACN; Gradient: 5-100% B in 3.0 min
1.51.5
Anhydrase
•Changing from SB-C18 to SB-C3, within the Poroshell family results in resolution of peaks 5 and 6, still in 3 min!
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PLRP-S
Polystyrene divinylbenzene bead
A il bl i 100 300 1000 d 4000A iAvailable in 100, 300, 1000, and 4000A pore sizes
Particle sizes 3, 5, 8, 10, and higher
Various geometries from Capillary to preparative
PLRP-S Continued:
Features
pH 1-14
Extreme buffer concentrations
Benefits
Acid and base cleanup
Typically < 8M
High temperature stability
Durable and Resilient
Inherently hydrophobic so does not require a bonded alkyl chain to confer hydrophobicity
yp y
200°C
Long Lifetimes
Avoids typical silica problems of silanol group
Analysis of very large biomolecules or high speed separations
1000A & 4000A pores
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25-bp Ladder Double Stranded DNA Ladder1
12% PAGE
25
50
100
1 1
HPLC
PLRP-S 100ÅColumn: PLRP-S 150x2.1mm ID
11
25
25
50
100
100
50
1 1
PLRP-S 300Å
PLRP-S 1000Å
Eluent A: 100mM TEAAEluent B: 100mM TEAA, 50% ACNGradient: 12.5%-50%B in 150 minsFlow Rate: 200µl/min
Pore Size Resolving Range
100Å 50-70 bp
300Å Up to 250-300 bp
1000Å Up to 400-450 bp
25
50
100
11
time
PLRP-S 4000Å
1000Å Up to 400-450 bp
4000Å >500 bp
In Summary
Agilent has many options for the analysis of bio-molecules and will continue to develop new products to address this growing market.
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NEED ASSISTANCE?
LC Column help desk1-800-227-9770
• orders• orders• customer service(option 1,1)
• technical support• applications assistance(option 3,3,2)(option 3,3,2)
29
BioHPLC Columns on the Agilent Website
To learn more and order online visit www.agilent.com/chem/BioHPLC
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Product Literature
New BioHPLC ColumnSelection Guide
#5990-9384
32
New AdvanceBio Peptide Column Brochure
#5990-8124EN
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New BioHPLC SEC New Zorbax SB 300Aand IEX Brochure 1.8um RRHD Brochure
#5990-5195 #5990-8124EN
Biomolecule PurificationBrochure
#5990-8335
New Protein Identification And Impurity Profiling
#5991-0625EN
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35
New LC Handbook#5990-7595
New Primer: Recombinant Protein Characterization#5990-8561
Part Numbers
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Agilent Bio SEC-5Ordering Information
Agilent Bio SEC-3Ordering Information
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Agilent Bio IEX Ordering Information
AdvanceBio Peptide Mapping ColumnsOrdering Info
Dimension AdvancedBio Peptide Mapping ColumnPart #’sPart # s
4.6X150mm 653950-902
3.0X150mm 653950-302
2.1X150mm 653750-902
2.1 X 250mm 651750-902
2.1X100mm 655750-302
4.6 mm Guard column 850750-911
3.0 mm Guard column 853750-911
2 1 mm Guard Column 851725-911
40
2.1 mm Guard Column 851725 911
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Poroshell 300 Ordering Information
41
Zorbax 1.8um 300SB RRHDOrdering Info
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Zorbax 300SBOrdering Info
43
PLRP-SOrdering Info
44
