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2D Liquid Chromatography
2D Liquid Chromatography
Chromatography Basics
(more information on slides „Normal phase and Reversed phase chromatography“)
Chromatography is a physical method of separation in which the components to be separated
are distributed between two phases, one of which is stationary (stationary phase) while the other
(the mobile phase) moves in a defined direction.
Sample, which dontains The analytes adsorb to Changing the mobile phase composition results in the
different analytes, is the stationary phase elution of the analytes. E.g. using a RP colum, the
introduced to the column differently depending organic solvent proportion is increased over time,
on their hydrophobicity, wherefore the least non-polar compounds elute first,
their structure etc. followed by medium non-polar and finally the most non-
polar ones.
2D Liquid Chromatography
Reversed-Phase Chromatography
• Adsorption Chromatography
• The retention is mainly based on the hydrophobicity of an analyte molecule
• Depending on the organic solvent content of the mobile phase the non-polar analyte either adsords
to the stationary phase or is solved in the increasingly non-polar mobile phase and thus eluted.
• The hydrophobicity of an analyte can be expressed as Log P which is a measure of an analytes
partition between two immiscible solvents (usually octanol and water) compare next slides and slides
„Polarity and Solubility“
• The higher the Log P, the more hydrophobic is the molecule.
Non-polar
2D Liquid Chromatography
logP value
Partition coefficient
log P refers to neutral, non-ionizable molecule species
Lipophilicity/hydrophilicity of a molecules is determined according to its distribution in a
biphasic system, consisting of two immiscible solvents (e.g. octanol and water)
2D Liquid Chromatography
logD value
Distribution coefficient
log D refers to ionizable molecules (Lipophilicity and therefore solubility changes as a
function of pH for ionisable compounds)
2D Liquid Chromatography
HILIC
Hydrophobic mobile
Polar phase (organic solvent)
stationary Non-polar compounds
phase
2D Liquid Chromatography
Chromatographic setups
One column (1)
Mixing of solvents A and B
Pump
detector (e.g. MS)
Manual sample
injector
or autosampler
RP-Column or
HILIC column
B
A
detector (e.g. UV)
PD Dr. J. Graßmann; PD Dr. T. Letzel Flow
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
2D Liquid Chromatography
Chromatographic setups
One column (2)
By using a reversed-phase column and a gradient elution, one usually
Extracted ion chromatograms
starts with a mainly aqueous mobile phase.
of mass spectrometrically detected m/z Introducing a complex mixture to this setup will result in the adsorption of
non- and semi-polar compounds onto the non-polar stationary phase,
whereas polar, highly water soluble compounds will remain in the mobile
Increasing hydrophobicity phase. This results in the retention of the adsorbed non-polar molecules,
MS intensity (counts)
which are gradually eluted from the column through the increase of the
organic solvent proportion of the mobile phase.
However most polar compounds will be flushed through without
interaction with the stationary phase. Thus those compounds often can
not be chromatographically separated using a RP column and are
therefore disregarded.
2D Liquid Chromatography
Comparision of the separation of two analytes either with an Reversed-phase or a HILIC column
By using a RP column, the polar analyte (logP = -1.4) elutes very early,
due to the mainly aqeuous mobile phase at the beginning. The non-polar
analyte (logP = 2.5) is retained due to its low water solubility. It elutes later
with increasing organic solvent content of the mobile phase.
By using a HILIC column, the non-polar compound elutes very early. Due
to its hydrophobicity, it won´t adsorb onto the polar stationary phase of the
column and remains in the non-polar mobile phase. In contrast the polar
compound interacts with the polar stationary phase. It elutes with
increasing water content of the mobile phase.
- logD calculation was performed with „MarvinSketch 14.8.25.0“, ChemAxon (www.chemaxon.com)
- Chromatograms: Greco and Letzel, Sep. Sci. 2013, HILIC Solutions No.1
PD Dr. J. Graßmann; PD Dr. T. Letzel
Greco and Letzel, Sep. Sci. 2013, HILIC Solutions No.1
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
2D Liquid Chromatography
Introduction of multidimensional chromatography
Experimental setup
Pump
1. Dimension
Sample
Column
2. Dimension
Detector
2D Liquid Chromatography
Introduction of multidimensional chromatography
Why more than more dimension?
- Samples with high background signals (e.g. due to complex matrix or impurities)
2D Liquid Chromatography
Definition of Coelution
Intensity
Intensity
2D Liquid Chromatography
Introduction of multidimensional chromatography
Experimental setup
1. Dimension
Sample
Column
2. Dimension
2D Liquid Chromatography
2D Liquid Chromatography
“Comprehensive” LCxLC
Definition „comprehensive“ separation:
Every bit of the sample, which was separated by means of the first column
(„first dimension“) is transferred onto the second column („second
dimension“) to achieve further separation.
1st dimension
Requirements:
2D Liquid Chromatography
“Comprehensive” LCxLC
Process of fractionation and transfer to the second dimension:
2D Liquid Chromatography
“Comprehensive” LCxLC
Process of fractionation and transfer to the second dimension:
2D Liquid Chromatography
“Comprehensive” LCxLC
Process of fractionation and transfer to the second dimension:
2D Liquid Chromatography
“Comprehensive” separation
Process of fractionation and transfer to the second dimension, exemplarily displayed for
fractions 1 and 4:
Peak of compound „red“ or
The fractions are
Each individual
„green“ as they will appear fraction is transferred transferred to the 2nd
after chromatographic to one of the loops column, where a second
separation with the 1st chromatographic
column separation is performed.
Fractions
1 2 3 4 ...
2D Liquid Chromatography
“Comprehensive” LCxLC
Result after 2D chromatographic separation of the sample
2nd dimension
2D Liquid Chromatography
“Heart-cutting LC-LC”
Definition „heart-cutting LC-LC“:
In contrast to a „comprehensive“ 2D-LC, in which every bit of the sample is transferred from the 1st
to the 2nd dimension, with heart-cutting only one or a few peaks are collected and transferred onto
the second column.
By means of this method only specific peaks of interest are introduced to the 2nd column, whereas
the rest is disregarded. Furthermore a collected fraction may contain an entire peak or only a
defined portion of the peak.
Fraction of on an entire peak Fraction of peak front Fraction of peak middle part Fraction of peak end
2D Liquid Chromatography
“Multiple Heart-cutting LC-LC”
Definition „multi heart-cutting LC-LC“:
As the name implies, multiple peaks are selected and fractioned to be introduced to the second
dimension of chromatographic separation. Comparable to „comprehensive“ LCxLC, more than one
sample loop ought to be used (at least two or more), so one peak can be introduced to the second
column from the first sample loop, while another peak is simultaneously collected and stored within
the second sample loop. The content of the second loop will only be transferred onto the second
column as soon as the chromatographic separation of the first fraction is completed and the mobile
phase gradient has returned to its starting conditions.
2D Liquid Chromatography
“Serial LC-LC coupling”
Definition „serial LC coupling of two columns“ (e.g. Reversed phase and
HILIC):
Pump 2
1. Dimension 2. Dimension
Pump 1 Column Column Detector
Sample
2D Liquid Chromatography
“Serial LC-LC coupling”
„Serial LC coupling“ can be used to capture polar and non-polar compounds in one run by means
of coupling a reversed-phase and a HILIC column.
2D Liquid Chromatography
“Serial LC-LC coupling”
2D Liquid Chromatography
“Serial LC-LC coupling”
Sample introduction at starting conditions:
The sample, which contains polar as well as non-
polar compounds, is introduced to the system by
means of a manual sample injector or an
autosampler ( 3 ) and is taken along towards
the RP-column.
Due to the mainly aqueous mobile phase in this
3 section of the setup, polar compounds will remain
solved (hydrophilicity!) and won´t interact with the
non-polar stationary phase of the RP column,
whereas non-polar compounds will be retained
here. Thus polar compounds flow freely through
4 the RP column and get to the T-piece ( 4 ),
which connects the RP-mobile phase with the
HILIC mobile phase. The latter consists of 100%
acetonitrile, wherefore the solubility of the polar
compounds is reduced in the now highly non-
polar mobile phase. Consequently they interact
with the stationary phase of the HILIC column, i.e.
they are being retained.
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München
2D Liquid Chromatography
“Serial LC-LC coupling”
2D Liquid Chromatography
“Serial LC-LC coupling”
Increasing hydrophilicity Increasing hydrophobicity
MS intensity (counts)
0 5 10 15 20 25 30 35
Retention time
Using a system, in which two columns (HILIC and RP) are coupled consecutively, allows the retention of polar and
non-polar compounds in one single run.
The application of this system results in the separation of polar compounds, which elute first, separated by the
HILIC column (grey area). Afterwards non-polar compounds elute, which were retained by the RP column (blue
area). Compounds retained by the HILIC column mainly possess a logP < 0, whereas compounds retained by the
RP have logP values > 0.
2D Liquid Chromatography
“Serial LC-LC coupling”
Comparison of chromatographic separation obtained either by means of a serial coupling
of RP and HILIC column or by using only one RP column:
One RP column:
non-polar compounds interact with the non-polar stationary phase and
are thus being retained. They elute with increasing organic solvent
proportion of the mobile phase.
Polar, but also medium polar compounds are not or only slightly
retained, since they are not interacting with the stationary phase. Most of
them pass through the column and elute right at the beginning of the
experiment (box).
2D Liquid Chromatography
Multidimensional chromatography
A variety of different multidimensional couplings are possible :
1st dimension data recording &
- GC x GC processing
- LC x LC (comprehensive) Injector
- LC – LC (heart-cutting)
- GC x GC x GC
- ……………..
Column 1 Column 2