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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
14
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How to read a LC×LC chromatogram
15
0 10 20 30 40 50 60
retention time (min)
-10000
-8000
-6000
-4000
-2000
inte
nsity
00.
10.
20.
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70.
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rete
ntio
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in)
-600
-400
-200
0
200
intensity
00.
10.
20.
30.
40.
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70.
8
rete
ntio
n tim
e (m
in)
-100
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intensity (mAU)
3
00.
10.
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in)
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102030
intensity
39 41
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rete
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in)
-100
1020
intensity
41
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
175
150
125
100
75
50
25
0
First Dimension retention (min) - Anion-Exchange / Mixed Mode
Seco
nd D
imen
sion r
etent
ion (s
) - Re
verse
d Pha
se C1
8
First Dimension retention (min) – Anion-Exchange/Mixed-Mode
Seco
nd D
imen
sion
rete
ntio
n (s
) –Re
vers
ed P
hase
C18
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Gradients and modulations
0 25 50 75 100 125 150 175 200
175
150
125
100
75
50
25
0
First Dimension retention (min) - Anion-Exchange / Mixed Mode
Seco
nd D
imen
sion r
etent
ion (s
) - Re
verse
d Pha
se C1
8
First Dimension retention (min) – Anion-Exchange/Mixed-Mode
Seco
nd D
imen
sion
rete
ntio
n (s
) –Re
vers
ed P
hase
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
29
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
30
0 50 100 150 200 250 300
175
150
125
100
75
50
25
0 50 100 150 200 250 300
175
150
125
100
75
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25
0 50 100 150 200 250 300
175
150
125
100
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10 μL
5 μLx1 x2
y2
y1
first dimension retention time (min) – mixed-mode
seco
nd d
imen
sion
rete
ntio
n tim
e (s
) –re
vers
ed-p
hase
2 D re
tent
ion
time
(s)
2 D re
tent
ion
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
32
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
33
Stoll et al. J. Chromatogr. B, 1134-1135, 2019, 121832
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2D-LC Application Database
34
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
50
100
150
# of
ap
plic
atio
ns
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
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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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