Simplifying 2D -LC, 2D-LC/MS Automated Workflows
Fast and Efficient Multi-Dimension LC & LC/MS method development.
February 2, 20161
Two Dimensional UHPLC
The selective transfer of a fraction (or fractions) from one chromatographic column to a secondary chromatographic
column for further separation
Why Two Dimensional LC?
� Further resolution of a complex mixture that cannot be separated on a single mode/column
� Sample cleanup by removing matrix or interfering compounds
� Increase sample throughput (two separations going on at once)
� Trace enrichment of major compounds of interest (column focusing)
� Increased peak capacity
� Second dimension mobile phase amenable to mass spectrometry
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Resolution - a measure of the ability to separate two components A Function of Selectivity, Column Efficiency, or Re tention
February 2, 2016
Diploma Training Fundamentals. Agilent Internal
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Re
solu
tion
Increase N Increase Alpha
Increase k'
Plates: 5000 10000 15000 20000
25000Alpha: 1.10 1.35 1.60 1.85 2.1
k: 2.0 4.5 7.0 9.5 12.0
Selectivity Affects Resolution Most• Change bonded phase
• Change mobile phase
Rs = N½/4 • (α-1)/α • k/(k+1)
α
Nk
Column Modes CombinationsMode Combination* Application Advantage Disadvantage Column Examples
IEC & RP PROTEOMICS Orthogonality Low Peak Capacity AGILENT BIO-IEX;
ZORBAX STABLEBOND,
ECLIPSE, EXTEND,
BONUS RP, POROSHELL
SEC & RP POLYMERS Orthogonality Low Peak Capacity AGILENT PL-GEL
(VARIOUS
BEDS);ZORBAX
STABLEBOND, ECLIPSE,
EXTEND, BONUS RP,
POROSHELL
NP & RP PHARMACEUTICALS
METABOLOMICS
Orthogonality Solvent compatibility, limited
application
ZORBAX CYANO,
AMINO, SIL;ZORBAX
STABLEBOND, ECLIPSE,
EXTEND, BONUS RP,
POROSHELL
RP & RP PHARMACEUTICALS
METABOLOMICS
Miscible Solvents, broadest
applications,fast speed, gradient
on both dimensions, highest peak
capacity
Strongly depends on column
choice of mobile phase choice ZORBAX STABLEBOND,
ECLIPSE, EXTEND,
BONUS RP, POROSHELL
AFFINITY & RP PROTEOMICS Orthogonality Low Peak Capacity, Off-line AGILENT MULTIPLE
AFFINITY REMOVAL
COLUMNS
SEC & NP POLYMERS Orthogonality Low Peak Capacity AGILENT PL-GEL
(VARIOUS
BEDS);ZORBAX CYANO,
AMINO, SIL
SEC & IEC PROTEOMICS Orthogonality Low Peak Capacity AGILENT BIO-
SEC;AGILENT BIO-IEX
* Stoll et al, Jchrom A, 1168 (2007)
3-43
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Road Blocks to Two Dimensional Chromatography
� Typically first dimension gradient is a long, slow gradient followed by rapid, repeated gradients on the second dimension so a very low delay volume pump is needed capable of ballistic gradients
� Difficult to coordinate timing between first and second dimension gradient
� Difficult to coordinate valve timing between first and second dimension
� Difficult to coordinate heart-cutting first dimension detector with trapping valve
� Any changes to one time table necessitates changes to all the other tables
Complex Gradient and Valve Switch Tables
Capillary pump 2:Gradient across
analytical column
Time % B Time % B0 3 175 35 3 201.1 65
26.1 65 201.2 326.2 3 210 335 3 236.1 65
61.1 65 236.2 361.2 3 245 370 3 271.1 65
96.1 65 271.2 396.2 3 280 3105 3 306.1 65
131.1 65 306.2 3131.2 3 315 3140 3 340 65
166.1 65 340.1 90166.2 3 345 90
Time Position0 column 2
26.1 column 135 column 2
61.1 column 170 column 2
96.1 column 1105 column 2
131.1 column 1140 column 2
166.1 column 1175 column 2
201.1 column 1210 column 2
236.1 column 1245 column 2
271.1 column 1280 column 2
306.1 column 1315 column 2345 column 1
Time Position0 Pos 15 Pos 230 Pos 165 Pos 2100 Pos 1135 Pos 2170 Pos 1205 Pos 2240 Pos 1275 Pos 2310 Pos 1
6-port valve:timetable
10-port valve:timetable
Time % B0 06 0
135 10200 20230 30280 50295 100320 100
320.1 0350 0
Capillary pump 1:Gradient across SCX
column
Principles of Two Dimensional HPLC
� Long efficient first column retains and separate sample components in one chromatography mode (first dimension)
� The eluent flows through valve with injection loops (Comprehensive or Heart Cutting modes)
� The loop content is automatically introduced into a 2nd, fast column (UHPLC) for an orthogonal separation mode
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Two Approaches to 2D HPLC
�Comprehensive : All of the sample from the first column is ‘trapped & released’ on to the second column in sequential fractions throughout the first dimension run
�Heart-cutting: Detector in the first dimension detects peaks to be trapped and released on to the second dimension column
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2D-LC SetupAdding 2nd dimension
2D column
• Purpose of 2D-LC: To increase the total separation power.
• Aliquots of effluent of the 1D-column are handed to the 2nd dimension for 2D analysis.
• Ideal case: Two columns with orthogonal separation mechanism, where separation power of 1D and 2D multiplies.
• Operation modes: Comprehensive 2D-LC and Heart-Cutting 2D-LC
1D column
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1D column
2D column
1. Peak sampling from 1D.
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Dual Loop Workflow
The Sampling Device / Interface
1D column
2D column
1. Peak sampling from 1D.
Dual loop workflow
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The Sampling Device / Interface
1D column
2D column
1. Peak sampling from 1D.
Dual loop workflow
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The Sampling Device / Interface
1D column
2D column
1. Peak sampling from 1D.
2. Valve switch.
Dual loop workflow
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The Sampling Device / InterfaceDual loop workflow
1. Peak sampling from 1D.
2. Valve switch.
3. 2D-LC.
4. Repeat 1.- 3.
2D column
1D column
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The Sampling Device / Interface
1. Peak sampling from 1D.
2. Valve switch.
3. 2D-LC.
4. Repeat 1.- 3.
2D column
1D column
Dual loop workflow
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The Sampling Device / Interface
1. Peak sampling from 1D.
2. Valve switch.
3. 2D-LC.
4. Repeat 1.- 3.
2D column
1D column
Dual loop workflow
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The Sampling Device / Interface
1. Peak sampling from 1D.
2. Valve switch.
3. 2D-LC.
4. Repeat 1.- 3.
2D column
1D column
Dual loop workflow
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The Sampling Device / Interface
1. Peak sampling from 1D.
2. Valve switch.
3. 2D-LC.
4. Repeat 1.- 3.
2D column
1D column
Dual loop workflow
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The Sampling Device / Interface
1. Peak sampling from 1D.
2. Valve switch.
3. 2D-LC.
4. Repeat 1.- 3.
2D column
1D column
Dual loop workflow
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Comprehensive 2D -LCPrinciple of Operation • Entire effluent of 1D-separation
and thus every peak gets fully sampled and passed to 2D.
• Frequency: ≥ 2 snipets (cuts) per peak to avoid de-separation.
• Requires fast 2D-gradients (sec), short columns (≤ 5 cm) and high flow rates (≥ 2 mL/min).
• Used for Complex / Unknown samples.
2D-chart
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Heart-Cutting 2D -LCPrinciple of Operation
• Fractions of specific 1D-peaks are sampled and subjected to 2D.
• Decouples 1D / 2D time scale.
• Allows for longer 2D-gradients and longer 2D-columns with higher separation efficiency.
• Better data quality for those peaks of interest.
• Used for Targeted analysis of known samples / increase confidence.
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Sampling Device / Interface
1D column
2D column
1. Peak sampling from 1D.
Dual loop workflow with Agilent 2 pos / 4 port Duo Valve *
* Duo Valve specifically designed for 2D-LC Application. Two identical flow paths.
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Heart-Cutting Workflow with Dual LoopFirst Cut Sent to 2D
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Limit of Heart-Cutting Workflow with Dual LoopMissed Next Two Peaks
First cut is being analyzedNext two peaks are missed
Limit of Heart-Cutting Workflow with Dual LoopLimited Volume
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New Multiple Heart-Cutting (MHC) Solution
From dual loop to MHC
Deck with 6 loops
One of the loops replaced with selector valve fitted with 6 loops Parking deck cluster offering 7 sampling positions.
Deck-B
Deck-A
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Both loops replaced with two selector valves fitted with 6 loops each Parking deck cluster offering 12 sampling positions.
Different view for better illustration
=Deck-B
Deck-A
New Multiple Heart-Cutting (MHC) Solution
Loop-1
Loop-6
Loop-1
Loop-6
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February 2, 2016Confidentiality Label
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1290 Infinity II 2D -LC Solution with Multiple Heart-CuttingHigh Resolution Sampling
1D
Where to take the cut?
2D
February 2, 2016Confidentiality Label
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1290 Infinity II 2D -LC Solution with Multiple Heart-CuttingThe easy Interface
variables:snip timenumber of snips
February 2, 2016Confidentiality Label
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1290 Infinity II 2D -LC Solution with Multiple Heart-CuttingOptimised Peak Parking
February 2, 2016Confidentiality Label
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1290 Infinity II 2D -LC Solution with Multiple Heart-CuttingOptimised Peak Parking
ChemStation Dashboard: All modules in one dashboardcan be relabled individually, e.g. „BinPump-1st-Dim“
Agilent 2D -LC Add -On Software
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2D-LC System Configuration“One screen for the entire system”
Define 1D / 2D pump
Define detector in the second dimension
Define peak detector (optional)
Select the valve(s) to be used for 2D-LC
injection
Select a possible valve / loop
configuration
Graphical representation of the selected valve / loop configuration:• Flow path 1D & 2D• Animated valve switching
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Select the 2D-LC mode: comprehensive
/ heart-cutting
Define the gradient of the 2nd dimension
Define repetition of 2nd dimension
gradient (Modulation time)
Show rollout of gradient in the 2nd dim
over the runtime of the 1st dimension
Define time window(s) where the
selected 2DLC mode is active
Method User Interface 2D-LC specific parameters of the 2D-pump
Graphical editing of gradient shift Access to standard
method UI of the pump
Operation values, warnings
Close-up of 2D-gradient
Solvent & Flow-Settings
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February 2, 2016Confidentiality Label
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1290 Infinity II 2D -LC Solution
Heart-cutting 2D-LC
Sam
ple
com
plex
ity
Multiple heart-cutting 2D-LC
Comprehensive 2D-LC
Comprehensive 2D-LC/(IMS)-MS/MS
API impurity profiling
Complex formulations
Bio-pharmaceuticals
Natural products,biological samples....
Peptide analysis after tryptic digestion and intact protein analysis using comprehensive 2D-LC on the same columns in th e first and
second dimension.
Comprehensive 2D -LCAnalysis of E.coli Tryptic Digest and Intact Protein
• In the field of protein analysis, sample complexity is enormous. It is not uncommon to be confronted with samples containing hundreds of proteins, and following digestion, thousands of peptides.
• Combination of SCX with RPLC for the analysis of E.coli tryptic digest and intact protein.
• Both intact protein and peptide analyses were done with the same instrument and column configuration using DAD and Q-TOF LC/MS.
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Data from Agilent Application Note 5991-5179EN
Analysis of E.coli Tryptic Digest and Intact ProteinPeak Capacity for Tryptic Digest
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Gradient time [min]
Elutionwindow[min]
Peak capacity
70 – 75 10 – 63 1927
140 – 150 10 – 112 3138
210 – 225 10 – 158 3750
Separation by IEX (Bio SCX, NP 10)
Sep
arat
ion
byR
P (
300S
B-C
18)
Analysis of E.coli Tryptic Digest and Intact ProteinPeptide Analysis
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Identity of the spots was determined by searching the MS/MS spectra against the E. coli protein database (SWISS-PROT) using Agilent MassHunterSpectrum Mill software.
Analysis of E.coli Tryptic Digest and Intact ProteinPeptide Analysis
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Analysis of E.coli Tryptic Digest and Intact ProteinPeptide Analysis
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MS/MS spectra of some selectedPeptides.
Analysis of E.coli Tryptic Digest and Intact ProteinProtein Analysis
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Separation by IEX (Bio SCX, NP 10)
Sep
arat
ion
byR
P (
300S
B-C
18)
Analysis of E.coli Tryptic Digest and Intact ProteinProtein Analysis
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Raw and deconvolutedspectra of spots 2 and 6.
Analysis of complex glycan pattern of EPO after enz ymatical release and labelling of glycans bei 2D-LC with Fluorescence Detection.
Comprehensive 2D -LCAnalysis of Complex N -Glycans in Biopharmaceuticals
• Erythropoietin (EPO) is a 30,400 Da glycoprotein hormone that regulates the production of red blood cells (erythropoiesis).
• The molecule consists of a 165 amino acid single polypeptide chain and a complex carbohydrate addition.
• The glycosylation of EPO is highly variable because it contains multiple glycosylation sites, each of which can have a wide variety of glycan structures. This results in a huge complexity of glycan structures that is referred to as microheterogeneity.
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Data from Agilent Application Note 5991-5349EN
Analysis of Complex N -Glycans in BiopharmaceuticalsEPO Structure
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The glycosylation portion of EPO consists of three N-linked glycosylation sites atAsn 24, 38, and 83, and one O-linked glycosylation site at Ser 1261.
Each of the three N-linked glycans is likely to contain up to four sialic acids. Theamount of NeuAcs in EPO has a huge influence on the molecule’s net charge,which is used to classify EPO isoforms.EPO structure with four examples of
N-glycans typically occurring in EPO
Analysis of Complex N -Glycans in BiopharmaceuticalsComprehensive 2D -LC Analysis
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Comprehensive HILIC/WAX 2D-LC separation of EPO, showing highly orthogonal separation. The IEX chromatography in the second dimension reveals the charge pattern of the glycans.
Separation by Glycan Mapping column
Sep
arat
ion
byIE
X (
Bio
WA
X)
Peptide mapping for identity and purity assessment of Trastuzumabby comprehensive 2D-LC using SCX/RP and HILIC/RP
combinations.
Comprehensive 2D -LCAnalysis of Monoclonal Antibody Digests
• Trastuzumab, marketed as Herceptin, is a 150 kDa large molecule used in the treatment of HER2 positive metastatic breast cancer.
• Following trypsinolysis, over 100 peptides with varying physicochemical properties present in a wide dynamic concentration range are expected.
• This Application reports on the combination of strong cation exchange (SCX) and RPLC and on HILIC and RPLC, two combinations shown to provide good orthogonality towards the analysis of peptides.
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Data from Agilent Application Notes 5991-2880EN and 5991-4503EN
Analysis of Monoclonal Antibody DigestsStructure and amino acid sequence
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Analysis of Monoclonal Antibody DigestsSCX or HILIC in the First Dimension Provide Differe nt Selectivity
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Analysis of Monoclonal Antibody DigestsAnalysis of Nonstressed and Oxidatively Stressed Trastuzumab
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Analysis of Monoclonal Antibody DigestsAnalysis of Nonstressed and Oxidatively Stressed Trastuzumab
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Thank You!!!!
Questions?
June 2015
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