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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?

2

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

5

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!