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Dr. Frank Steiner Manager HPLC Solutions Marketing
Co-Authors: Remco Swart, Evert-Jan Sneekes, Michael Heidorn, Maria Grübner, Andreas Dunkel, Thomas Hofmann, Juri Leonhardt, Thorsten Teutenberg
What is the True Value of Peak Capacity in 2D-LC and 1D-UHPLC when it comes to Highly Complex Real Life Samples?
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Outline
• Fundamentals on peak capacity and pre-requisites • 1D-LC in complex samples fingerprinting and proteomics
• 2D-LC in proteomics, food metabolomics and environmental
• Considerations on the value of the peak capacity concept
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Peak Capacity – History and Definition
• Peak capacity is the maximum number of resolvable peaks by a given chromatographic method
• The theoretical number considers Rs=1.0 (4σ resolution) • Best if equidistant peaks of equivalent width ( Gradient)
C.S. Horvath, S.R. Lipsky, Anal. Chem., 1967, 39(14), pp 1893–1893
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Peak Capacity – Optimization Strategy
Peak capacity: nC = 1 + tG/w Peak width: w ~ √N or √N* (gradient)
LC Variables affecting N or N*: • Stationary phase (dp…) • Temperature (T) • Column length (L)
Flow Rate: 2.2 µL/min Gradient: 0–35% CH3CN, 0.05% TFA
Influence of Column Length and Gradient (PS-DVB Monolithic Column)
0
50
100
150
200
250
300
0 20 40 60 80 Gradient (time)
Peak
Cap
acity
15 cm Monolith 5 cm Monolith
14
1 +⋅⋅
=+≈M
GGP t
NtWtc
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Peak Capacity – What can be achieved in UHPLC?
• Assuming UHPLC can reach N = 100,000 – 150,000 per column (H = 4 µm, L = 40 – 60 cm) nc = 600 – 800
• Giddings: …in order to resolve 98% of the components, the peak capacity must exceed the number of components by a factor of 100.
J.C. Giddings, J. Chromatogr. A, 1995, 703, p 3
G
MC
VVBc
BcNn⋅⋅∆+
⋅∆⋅⋅+=14
11Rule of thumb for wide range gradient (0-100%) and VG/VM > 30
NcP ⋅≈ 2
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Target: Peak capacity (nc) in 2D-LC = 1nc x 2nc
2D-LC to Go Beyond these Limitation
Pre-requisites:
• Combination of two orthogonal separation mechanisms • Independent retention and selectivity criteria in both dimensions
• Use of entire retention space in both dimensions • Peaks must go in both dimensions from low to high retention
• Optimized sampling rate from 1st dimension • Murphy-Schure-Foley rule of oversampling: 4 fractions per 8σ volume
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1D SCX separation
2 D R
P se
para
tion
6.0 10.0 15.0 19.025.0
40.0
50.0 PMD 10pmolmin
min
min
min
Are All Three Criteria Fulfilled in this Example?
1nc = 30, 10 fractions
• Orthogonality: Random pattern fulfilled • Retention space coverage: Upper right empty partially fulfilled • Right oversampling: 60 fractions required not fulfilled
9.0 12.0 14.0 16.0 18.0 21.025.0
30.0
35.0
40.0
45.0
50.0 E-coli pH 9.6 2D retention mapmin
min
min
min
Example for poor Orthogonality (RPxRP)
“Gel plots” above can be constructed from 2D-LC runs by Thermo Scientific™ Dionex™ Chromeleon™ Chromatography software
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Outline
• Fundamentals on peak capacity and pre-requisites • 1D-LC in complex samples fingerprinting and proteomics
• 2D-LC in proteomics, food metabolomics and environmental
• Considerations on the value of the peak capacity concept
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Untargeted Exploratory Assays for TCM
• nC should increase with √L √1.6 = 1.26 fulfilled • Is there a measurable value of the nC increase?
Conditions: • Mobile Phase A: H2O w/ 0.1% Formic Acid; Mobile Phase B: Acetonitrile w/ 0.1% Formic Acid • Flow: 670 µL/min • Column: Thermo Scientific™ Acclaim™ RSLC 120 C18, 2.2 µm, 2.1 x 250 mm • Injection Volume: 5 µL of TCM preparation • UV Detection: 280 nm • Column Oven: 45 °C , still air; pre-heater: 45 °C
L = 250 mm, tG = 18 min L = 400 mm, tG = 29 min
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Some Numbers on the Full Chromatograms
Criterion nC = 590 method nC = 720 method Total peak number 125 206 Number of peaks with RS > 0.8 118 187 Number of peaks with RS > 1.0 114 179 Number of peaks with RS > 1.2 108 168 Number of peaks with RS > 1.5 101 163
There is significant value in the increase of nc for this fingerprinting experiment
L = 250 mm, tG = 18 min L = 400 mm, tG = 29 min
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Real Life Example in nanoUHPLC-MS-MS
• Protein ID of HeLa Cell Line digest using Orbitrap Velos MS
• Investigation on the benefit of nano UHPLC in protein ID
• Performed at Research Institute for Molecular Pathology • Group of Karl Mechtler (Vienna, Austria)
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Results of Long Column nanoUHPLC-MS/MS
Thomas Köcher, Remco Swart, Karl Mechtler Anal. Chem., 2011, 83(7), pp 2699–2704, DOI: 10.1021/ac103243t
• Column: Thermo Scientific™ Acclaim™ PepMap™ RSLC, 75 µm ID × 25/50 cm, 2 µm C18
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Peptide Identification Rate vs. Peak Capacity
• Good correlation between identified peptides and nc
• Tremendous number peptides / nc proofs powerful MS
25 cm column 50 cm column
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Outline
• Fundamentals on peak capacity and pre-requisites • 1D-LC in complex samples fingerprinting and proteomics
• 2D-LC in proteomics, food metabolomics and environmental
• Considerations on the value of the peak capacity concept
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Concept of Automated 2D-LC Analysis
Injection • Zero sample loss • Sample cooling
Fractionation • cooled well plates • µL volumes
Derivatization (optionally) • Digestion • Heating
Reinjection • MultiDimensional LC • Affinity
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SCX x RP vs. RP at pH 9.6 x PR at pH 1.9
1D SCX separation
2 D R
P se
para
tion
1D RP separation, pH = 9.6 2 D
RP
sepa
ratio
n, pH
= 1.
9 9.0 12.5 15.0 17.5 21.0
25.0
30.0
35.0
40.0
45.0 PMD pH 9.6 2D retention mapmin
min
min
min6.0 8.0 10.0 12.0 14.0 17.0
25.0
30.0
35.0
40.0
45.0
50.0 PMD 10pmol 2D retention mapmin
min
min
min
5-Protein Mixture Digest
• 10 fractions determine peak capacity in 1D • Column format and eluting conditions in 2D equivalent
equal nc
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3.0 10.0 15.0 19.025.0
30.0
35.0
40.0
45.0
50.0 E-coli SCX 2D retention mapmin
min
min
min
9.0 12.0 14.0 16.0 18.0 21.025.0
30.0
35.0
40.0
45.0
50.0 E-coli pH 9.6 2D retention mapmin
min
min
min 25 30 35 40 min 45
25 30 35 40 min 45
RP x RP
2D-RP x RP vs. 2D-SCX x RP for Complex Sample E. coli Tryptic Digest
SCX x RP
441 Proteins identified
352 Proteins identified
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16% 33% 51% RP x RP SCX x RP
Proteins Identified by SCX x RP and RP x RP
• 2D-LC with good retentions space coverage is very powerful • Both methods are complementary Selectivity matters!!!
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2D-LC RP-HILIC in Food Metabolomics
• Automation of a comprehensive offline 2D-LC system • Coupling of RP and HILIC → change of mobile phase / fraction solvent • Aspired workflow:
separation in 1st dimension (preferably RP)
monitoring by UV detection
fractionation
preparation
take up detection (CAD / MS)
separation in 2nd dimension (preferably HILIC)
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Wide Range of Methods Explored
Thermo Scientific™ Acclaim™ Trinity™ P1 x Thermo Scientific™ Accucore™ 150-Amide-HILIC
Thermo Scientific™ Hypersil GOLD™ PFP x Acclaim HILIC
• 18 RP, 14 HILIC columns run with test mixture (consistent conditions) • Model analyte mixture
• Representative for properties of real-life metabolomic samples • Broad polarity range coverage • Characteristic substance classes
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Orthogonality and Surface Coverage Calculations artificial data sets
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Peak Capacities of Single Methods and Combinations
0 50
100 150 200 250 300
nC of each characterized column under relevant conditions
1nc x 2nc (not corrected) 1nc x 2nc x surface coverage (binning)
• Surface coverage is crucial for realistic estimate of accessible nc
• Surface coverage consideration reduces nc in practical methods to < 50%
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1D and 2D Real Life Sample Analysis - Experiments
urine fractionation (30x, pooled) evaporation to 1/3
direct re-injection
dilution 1:1 (ACN)
evaporation to dryness, take up in 1/3 solvent (ACN/Water, 1:1)
5 µl
10 µl
5 µl
5 µl TOF-MS Full Scan
RP
Trinity P1
HILIC
Accucore Amide
HILIC
Accucore Amide
5 µl
• Urine Sample from food metabolomics case study • 1D-LC and differently prepared fractions from 1D injected into 2D • Peak counting in 2D runs with TOF-MS in Full Scan mode
• Working plan:
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1D and 2D Real Life Sample Analysis - Results
0
500
1000
1500
2000
2500
3000
3500
dryness diluted conc. direct 1D separation
Summed de-convoluted peak count
ESI+ ESI-
• 2D-LC yields 15-20 times more de-convoluted peaks than 1D-LC
• Corresponds well with surface coverage corrected nc
• Fraction preparation only of minor influence
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
20 30 40 50 60 70
direct diluted concentrated dryness
Deconvoluted peaks in 2D per fraction (ESI-)
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1D-LC vs. 2D-LC in Environmental Analysis – Part 1
Juri Leonhardt, Thorsten Teutenberg, Duisburg
Measurement of a reference standard
Cytostatics
Antibiotics
Beta-blockers
Pesticides, Mycotoxins Radio contrast
agents Corrosion inhibitors
Steroids The reference standard contains 162 compounds in a concentration of 1 mg L-1.
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1D-LC vs. 2D-LC in Environmental Analysis – Part 2
Primary treatment
Aeration basin
Final clarification Outlet
River Ruhr
Sample collection
Measurement of a waste water sample with a high matrix load
Sample from feed of waste water treatment plant
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Nano-LC dimension (1st ) Column: Thermo Scientific™ Hypercarb™ 50 x 0.1 mm, 5 µm Temperature: 60 °C Mobile Phase A: H2O + 0.1% FA Mobile Phase B: MeOH + 0.1% FA Flow rate: 200 nL min-1 Injection volume: 1.5 µL Gradient: 0 - 8 min: 1% B 8 - 53 min: 1% B - 99% B 53 - 88 min: 99% B 88 - 93 min: 99% B – 1% B 93 -101 min: 1% B Capillary-LC dimension (2nd) Column: ChromaNik® SunShell® C18 50 x 0.3 mm, 2.6 µm (CHROMANIK ® Technologies) Temperature: 6 0°C Mobile Phase A: H2O + 0.1% FA Mobile Phase B. ACN + 0.1% FA Flow rate: 40 µL min-1 Injection volume: 200 nL Gradient: 0.0 - 0.5 min: 3% B - 97% B 0.5 - 0.6 min: 97% B 0.6 - 0.7 min: 3% B 0.7 - 1.0 min: 3% B
Analytical-LC dimension Column: Luna® C18 (Phenomenex, Inc.) 150 x 2.0 mm, 3.0 µm Temperature: 30 °C Mobile Phase A: H2O + 0.1% FA Mobile Phase B: ACN + 0.1% FA Flow rate: 200 µL min-1 Injection volume: 20 µL Gradient: 00.00 - 03.00 min: 02% B 03.01 - 18.00 min: 02% B - 98% B 18.01 - 24.00 min: 98% B 24.01 - 24.50 min: 98% B - 2% B 24.51 - 30.00 min: 02% B
20 µL 1.5 µL
Method Details – On-line Nano-Micro 2D-LC
2D-LC/MS
QTOF-detection in 2D
1D-LC/MS
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Comparison of standard and waste water sample for 1D- and 2D-LC
1D-LC standard
1D-LC sample
2D-LC standard
2D-LC sample
1D-LC vs. 2D-LC in Environmental Analysis – Chromatograms
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1D-LC vs. 2D-LC in Environmental Analysis – Results
• Several analytes from reference standard get lost with 2D method
• 2D method is superior with complex real life sample (but not scaling to nc)
Reference standard
Exact mass +/- 5 ppm
Exact mass +/- 5 ppm
Retention time +/- 2.5%
MS/MS data base hit
Reference standard Waste water sample
1D-LC 2D-LC
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Considerations and Conclusions
• Information content of complex sample fingerprinting and 1D-LC proteomics workflows scales with peak capacity.
• Peak capacity in 2D-LC has a different quality than in 1D-LC and strongly depends on coverage of 2D retention space, additional selectivity provides value.
• In targeted analysis: 2D-LC and high peak capacities pay off with very complex samples and tough matrices.
• There is significant value in methods with high peak
capacity, in particular for non-targeted analysis of highly complex samples.
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Acknowledgement
• Co-Authors from Thermo Fisher Scientific • Remco Swart, Evert-Jan Sneekes (Proteomics) • Michael Heidorn (TCM)
1D-LC proteomics workflows scales with peak capacity
• IMP Vienna (HeLa Cell Line Protein ID) • Karl Mechtler • Thomas Köcher
• TU Munich (Food Metabolimics Collaboration)
• Maria Grübner, Andreas Dunkel • Thomas Hofmann
• IUTA Duisburg (Environmental Analysis) • Juri Leonhardt • Thorsten Teutenberg
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Thank You!