CASA

Advances in 2D-LCAN INTRODUCTION to 2D-LC AND THE RECENT DEVELOPMENTS

Bob Pirok

HTC-16

Thursday, 30 January 2020, Ghent, Belgium

HEART-CUT ONLYD1 PUMPS

InjectorINJECTOR COLUMN 1 DETECTOR

WASTE

DETECTORCOLUMN 2

D2 PUMPS

2D-LC: Hardware

REQUIRED HARDWARE RELATIVE TO 1D-LC Second (binary) pump (comprehensive: UHPLC pref.) Modulation interface (e.g. valve) Heart-cut: Second detector

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2D-LC: Modes

HEART-CUT ONLYD1 PUMPS

InjectorINJECTOR COLUMN 1 DETECTOR

D2 PUMPS WASTE

DETECTORCOLUMN 2

COMPREHENSIVE 2D-LC (LC×LC) All 1D effluent subjected to a 2D separation. 2D limiting analysis time

2D usually under UHPLC conditions. May complicate MS hyphenation.

Full characterization of complex samples Group-type analysis, etc.

(MULTIPLE) HEART-CUT 2D-LC (LC-LC or mLC-LC) Fraction(s) of 1D effluent subjected to a 2D separation. Flexible and intuitive relative to comprehensive

Readily combined with MS(/MS) For moderately complex samples

Popular: 1D peak-purity assessments

×-

HEART-CUT 2D-LCLC-LC

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Heart-cut 2D-LC (LC-LC)

Taking (multiple) Heart-Cut(s) Subjecting one or a few fractions to a

second separation mechanism. More peak capacity

LC-LC𝑛𝑛𝑡𝑡𝑡𝑡𝑡𝑡 = 𝑛𝑛𝐷𝐷1 + 𝑛𝑛𝐷𝐷2

(m)

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1D-Column

2D-Column

Waste

2D-Pump

Loop

Loop

Multiple Heart-cut 2D-LC (mLC-LC)

6

1D-ColumnWaste

LOOP DECK A

LOOP DECK B

2D-Column

2D-Pumps

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USER-SPECIFIED1D BASED AUTO SELECTION

Multiple Heart-cut 2D-LC (mLC-LC)

7

Van Herwerden et al., 2020, University of Amsterdam

Convenient use of 2D-LC Contemporary software packages allow

user to select 1D peaks of interest. Automated peak selection also possible

Trigger by 1D detector Real-time curve-resolution

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Heart-cut 2D-LC (LC-LC)

8

Very high resolving power Added selectivity from second (“orthogonal”) dimension Choice from many different retention mechanisms Enhanced purification of target analytes Preparative separations possible Greatly reduced uncertainty of peak assignments Readily combined with MS and MS/MS techniques

Rigorous assessment of peak purity is tantamount in (bio-) pharmaceutical industries

(Spatial) comprehensive two-dimensional (and three-dimensional) LC

For qualitative analysis high-resolution hyphenated techniques (LC-MS or LC-MS/MS) are usually preferred

Somewhat increased conceptual and instr. complexity Analysis time is increased (especially when multiple

fractions are selected for analysis in the second dimension)

Possibly reduced detection sensitivity Phase-system incompatibility issues

STRENGTHS WEAKNESSES

OPPURTUNITIES THREATS

COMPREHENSIVE 2D-LCLC×LC

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Peak capacity

10

Statistical overlap theoryPeaks are spaced randomly on complex chromatograms.

Davis & Giddings, Anal. Chem. 1983, 55, 418-424

ln𝑝𝑝 = ln𝑚𝑚 −𝑚𝑚𝑛𝑛𝑐𝑐

𝑚𝑚 = number of analytes𝑝𝑝 = number of singular peaks

EXAMPLEFor a separation of 1000 peaks (𝑚𝑚) with a peak capacity of 1000 (𝑛𝑛𝑐𝑐) the number of separated peaks will be: 368!

To separate 95% of the peaks, a peak capacity will be required of 20000!

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Peak capacity and particle size

11

5 μm3 μm2 μm1 μm

Estimated maximum attainable peak capacity as a function of the gradient time(s) for HPLC separation of peptides.

𝑇𝑇 = 40◦C, Δ𝑃𝑃 = 40 MPa, starting composition 5% acetonitrile in water.

Smaller particles will improve your peak capacity for fast separations. Up to a limit!

Pirok et al. J. Sep. Sci., 41(1), 2018, 68-98 (Open Access)

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Peak capacity & Orthogonality

12

In LC×LC, the peak capacity is the product of the peak capacities of the individual dimensions. It may be corrected for Undersampling factor, 𝛽𝛽 Incomplete surface coverage, 𝑓𝑓coverage

𝑛𝑛𝑐𝑐,2𝐷𝐷 = 1𝑛𝑛𝑐𝑐 ⋅ 2𝑛𝑛𝑐𝑐

Practice TheoryCWOT

× =

𝑛𝑛𝑐𝑐,2𝐷𝐷∗ =

1𝑛𝑛𝑐𝑐⋅ 2𝑛𝑛𝑐𝑐⋅𝑓𝑓coverage𝛽𝛽

To maximize the use of the available peak capacity, the two separation mechanisms must be orthogonal (i.e. be statistically independent from each other.

J. Calvin Giddings Analytical Chemistry,, 1984, 56, 1258A

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Data formatting

13

RAW DATA

Pirok, Edam, Halmans, van Heuzen, 2015, Shell Global Solutions

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How to read a LC×LC chromatogram

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How to read a LC×LC chromatogram

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0 10 20 30 40 50 60

retention time (min)

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FIRST-DIMENSION CHROMATOGRA

B.W.J. Pirok, Making Analytically Incompatible Approaches Compatible, 2019, Amsterdam

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Sampling rate

16

Pirok et al. J. Sep. Sci., 41(1), 2018, 68-98 (Open Access)

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Undersampling

17

First-dimension First-dimension

Reconstructed 1D Reconstructed 1D

Pirok et al. J. Sep. Sci., 41(1), 2018, 68-98 (Open Access)

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High peak capacities (1,000 – 10,000) routinely possible High peak-production rates (typically 1 peak per second) Choice from many different retention mechanisms Added selectivity from second (“orthogonal”) dimension Structured, readily interpretable chromatograms “Group-type” separations of classes of analytes Readily combined with MS and MS/MS techniques Greatly reduced uncertainty of peak assignments (in

comparison with 1D-LC)

Increased need for detailed characterization of complex samples from many fields

“Spatial” comprehensive two-dimensional (and three-dimensional) LC

High-resolution hyphenated techniques (LC-MS, LC-IMS-MS, IMS-MS) may compete for certain applications

Added conceptual and instrumental complexity Rather long analysis times (typically 30 min – 2 h) Possibly reduced detection sensitivity Phase-system incompatibility issues Data-analysis software needed Difficult and time-consuming method development

STRENGTHS WEAKNESSES

OPPURTUNITIES THREATS

Comprehensive 2D-LC (LC×LC)

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Technical 2D-LC database

19

www.multidlc.org

Comprehensive platform for multi-dimensional separations: all information in a single place

Now released: Searchable database of all technical 2D-LC literature.

Collaboration between Gustavus Adolphus College (Dwight Stoll) and University of Amsterdam (Pirok).

METHOD DEVELOPMENT

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Retention mechanisms

21

MECHANISM ACRONYM SELECTIVITY1 Reversed phase RP Hydrophobicity, Chain length, carbon skeleton

1 Ion pairing IP Hydrophobicity, suppression of analyte ionization (acid/ bases)

1 Hydrophobic interaction

HIC Hydrophobicity

2 Normal phase NP Polarity, Functional groups2 Argentation AgLC Degree of saturation, cis-trans isomers

2 Hydrophilic interaction HILIC Hydrophilicity, Polar character

3 Ion exchange IEX Charge, Ionic interactions

4 Size exclusion SEC Molecular size, Molecular weight

5 Mixed mode MM Combination of retention mechanisms

6 Chiral Chiral Selector-specific chirality

7 Affinity Affinity Selector-specific affinityPirok et al. J. Sep. Sci., 41(1), 2018, 68-98 (Open Access)

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Challenges and advantages in 2D-LC

22

MEANING DESCRIPTIONAdsorption Lengthening of elution time due to injection solvent. Applies exclusively to SEC.

Breakthrough/Peak distortion Anomalous early elution of analytes injected from 1D to 2D.Easy to modulate Ease of developing active-modulation methods (e.g. trap columns or solvent

admixing).Fast separation Method with short analysis times (e.g. <1 min)High-resolution separation Method capable of high peak capacity.Isocratic Possibility of (easily) running isocratic methods, reducing the complexity of the

setup.MS compatible Possibility of using volatile mobile-phase additives and achieving good MS

sensitivity.Orthogonal Degree of independence of two separation mechanisms, assuming that the analyte

mixture exhibits sample dimensions targeted by the two dimensions.Applicability Usefulness of the resulting separation.Column re-equilibration Speed of column re-equilibration.Selectivity/Specificity Capability of the separation method to separate based on chemical characteristics

of sample components (e.g. shape, orientation, composition/ sequence)Solvent compatibility Extent of (in)compatibility of 1D effluent and 2D eluent.Pirok et al. J. Sep. Sci., 41(1), 2018, 68-98 (Open Access)

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Selection of retention mechanisms

23

Pirok et al. J. Sep. Sci., 41(1), 2018, 68-98 (Open Access)

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Sample dimensionality

24

The sample dimensionality is the number of independent variables that must be specified to identify the components of the sample. The absolute starting point for any method in 2D-LC.

J. Calvin Giddings Journal of Chromatography A, 1995, 703, 3-15

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Ion pairs & orthogonality

25

NN

HO

S

SHN

O

O

O

O O

N

N

NN

HO

S

SHN

O

O

O

O O

N

N

NN

HO

S

SHN

O

O

O

O ON

N

NN

HO

S

SHN

O

O

O

O O

NO ION-PAIR TMA TEA TBA

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Gradients and modulations

0 25 50 75 100 125 150 175 200

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First Dimension retention (min) - Anion-Exchange / Mixed Mode

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First Dimension retention (min) – Anion-Exchange/Mixed-Mode

Seco

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) –Re

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C18

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Gradients and modulations

0 25 50 75 100 125 150 175 200

175

150

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75

50

25

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First Dimension retention (min) - Anion-Exchange / Mixed Mode

Seco

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First Dimension retention (min) – Anion-Exchange/Mixed-Mode

Seco

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) –Re

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C18

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Gradient assemblies

time (min)frac

tion

of s

tron

g so

lven

t, φ

time (min)frac

tion

of s

tron

g so

lven

t, φ

time (min)frac

tion

of s

tron

g so

lven

t, φ

MULTI-STEP SHIFTED GRADIENT

CONSTANT GRADIENT

SHIFTING GRADIENT

time (min)frac

tion

of s

tron

g so

lven

t, φ

PARALLEL GRADIENT

THE IDEAL GRADIENT ASSEMBLY?

Pirok et al., J. Sep. Sci., 41(1), 2018, 68-98.

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Breakthrough phenomenon

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Exclusion effect withinsolvent plug

Fraction of the analytes

elutes too soon

Focusing effect as plug

dilutes

Peak splitting or nullification of retention Result of injection solvent / volume Gradient may refocus the disturbed retention bands (if arriving in time).

Strong injection solvent inhibits

adsorption

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First-dimension breakthrough

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10 μL

5 μLx1 x2

y2

y1

first dimension retention time (min) – mixed-mode

seco

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) –re

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2 D re

tent

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time

(s)

2 D re

tent

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time

(s)

1D retention time (min)

1D retention time (min)

20 μL

Pirok, Gargano & Schoenmakers, J. Sep. Sci., 41(1), 2018, 68-98.

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Second-dimension breakthrough

31

B.W.J. Pirok, Making Analytically Incompatible Approaches Compatible, 2019, Amsterdam

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Examples of Applications

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DyesPirok et al. 2019

Therapeutic AntibodiesStoll et al. 2018

NanoparticlesPirok et al. 2017

Anthocyanins in WineWillemse et al. 2015

PolymersUliyanchenko et al. 2012

SurfactantsGargano et al.,

2016

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Example of state of the art

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Stoll et al. J. Chromatogr. B, 1134-1135, 2019, 121832

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2D-LC Application Database

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www.2dlcdatabase.com

Searchable database of all 2D-LC publications available in literature.Over 500 applications.

Collaboration between groups at University of Amsterdam (Pirok) and Gustavus Adolphus College (Stoll).

B.W.J. Pirok, D.R. Stoll and P.J. Schoenmakers, Anal. Chem. 2019, 91(1), 240-263 (Open Access)

www.multidlc.org

MODULATION

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Passive modulation

1D-Column

2D-Column

Waste

2D-Pump

Loop

Loop

1D-Column

2D-Column

Waste

2D-Pump

Loop

Loop

Schematic: Pirok, Stoll and Schoenmakers, Anal. Chem. 2019 , 91(1), 240-263 (Open Access)

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Waste

μL∙min-1 mL∙min-1

FIRST DIMENSION SECOND DIMENSION

The Modulator – Heart of the 2D-LC

37

Injector Column 1 Column 2

PumpsWaste

Injector

Pumps

Detector

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Stationary-phase assisted modulation (SPAM)

38

Gargano et al. Anal. Chem. 88, 2016, 1785−1793

Reduction of analysis time Removal of incompatible fraction Relies on retention on traps

PASSIVE SPAM

Baglai et al. Anal. Chim. Acta 2018, 1013, 87–97

PASSIVE SPAM

SURFACTANTS STEROIDS IN URINE

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Active-solvent modulation (ASM)

Stoll et al. Anal. Chem. 2017, 89 (17), 9260–9267.

In ASM, 1D column effluent is admixed with weak eluent of the 2D separation method. Developed for RPLC as second dimension. Significantly reduces occurrence of breakthrough

effects. On-column focusing of injected analytes.

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Active-solvent modulation (ASM)

Stoll et al. Anal. Chem. 2018, 90(9), 5923-5929

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Vacuum evaporation modulation (VEM)

1D-Column

2D-Column

2D-Pump

VACUUM

Loop

Loop

1D-Column

2D-Column

2D-Pump

VACUUM

Loop

Loop

Incompatible solvent is evaporated May be vacuum-asssiunder a vacuum. Only demonstrated for combinations of NPLC and RPLC. Analyte loss a concern.

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Use of retention mechanisms in 2D-LC

B.W.J. Pirok, D.R. Stoll and P.J. Schoenmakers, Anal. Chem. 2019, 91(1), 240-263 (Open Access)

RPLCIon-PairHILICIEXHICNPLCSECSFCChiralVarious

FIRST DIMENSION SECOND DIMENSIONNON-COMPREHENSIVE NON-COMPREHENSIVE

COMPREHENSIVECOMPREHENSIVE

LC×LC: 1. RPLC×RPLC (~35%), 2. HILIC×RPLC(~15%), 3. IEX×RPLC (~5%), 4. SEC×RPLC (~4%)LC-LC: 1. RPLC-RPLC (~36%), 2. SEC-RPLC(~11%), 3. IEX-RPLC (~7%), 4. HILIC-RPLC (~7%)

MOST COMBINED

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Use of modulation strategies in 2D-LC

Pirok, Stoll and Schoenmakers, Anal. Chem. 2019, 91(1), 240-263

0

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NON-COMPREHENSIVE

COMPREHENSIVE Use of detectors in 2D-LC

010203040

1990 1995 2000 2005 2010 2015 2020

PASSIVEASM/FSMSPAM

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Overview of modulation techniquesTECHNIQUE STRENGTHS WEAKNESSESStationary-phase-assisted modulation (SPAM)

Eliminates incompatible solvent Improves detection sensitivity

(reduces dilution factors) Modulation volume no longer

limiting factor.

Trap robustness Discrimination Operation and optimization is sample

dependent Method development may be challenging

Active-solvent modulation (ASM)

Dilutes incompatible solvent On-column focusing in the second

dimension: potential sensitivity improvement

Robust

Modulation volume still a limiting factor. Mainly useful with RPLC in second

dimension

(Vacuum-) Evaporation modulation (VEM)

Evaporates incompatible solvent Fast operation appears possible

(under vacuum conditions) Membrane can be used to prevent

loss of analytes

Discrimination: Loss of volatile analytes during evaporation not investigated (without membrane)

Some analytes (e.g. polymers) may redissolve slowly

Highly experimental

Based on: Groeneveld et al. Faraday Discussions, 1450, 2019, 29-37

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Summarizing recent advancements

45

MODULATIONMS HYPHENATION

HILICThe use of HILIC as 2D

receives significant attention. Studies suggest that reproducible 2D HILIC

modulations seems feasible.

OPTIMIZATIONResearch continues into

developing methods compatible with MS.

There is a move towards smaller, more efficient

column technology.

Method-development is particularly costly for

comprehensive 2D-LC. Several groups work on

optimization tools to automate this.

Active-modulation interfaces are emerging.

These greatly reduce sensitivity effects and solvent incompatibility.

ASM particularly robust.

Active-modulation interfaces are emerging.

These greatly reduce sensitivity effects and solvent incompatibility.

ASM particularly robust.

DATA ANALYSISINSTRUMENTATIONRobust hardware is

increasingly commercially available.

CONCLUSIONS

46

2D-LC has matured well enough for routine application in analytical labs. There are a large number of applications known in literature. Robust instrumentation now commercially available.

2D-LC: A POWERFUL, MATURE TECHNIQUE

The recent developments in modulation technology unlocked new possibilities in hyphenation of 2D-LC with MS. HILIC appears useful as 2D. Solvent incompatibility often no longer an issue.

RECENT DEVELOPMENTS

THANK YOU FOR LISTENING!

Similar to its bigger brother GC×GC, data processing for LC×LC receives lots of attention, ultimately also facilitating automated method optimization.

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