Two-Dimensional Chromatography

Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt

Technische Universität München
2D Liquid Chromatography
PD Dr. J. Graßmann; PD Dr. T. Letzel

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.

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
For gradient elution:

Starting with highly aqueous content.
Increase of organic content over time.
Non-polar to semi-polar analytes

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)
Partition coefficient (log P) =
𝑠𝑜𝑙𝑢𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑑𝑖𝑠𝑠𝑜𝑙𝑣𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙

𝑠𝑜𝑙𝑢𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑑𝑖𝑠𝑠𝑜𝑙𝑣𝑒𝑑 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟
octanol soluteneutral
log P < 0 log P > 0

water soluteneutral Hydrophilic Hydrophobic
-2 -1 0 1 2
logD value
Distribution coefficient
log D refers to ionizable molecules (Lipophilicity and therefore solubility changes as a
function of pH for ionisable compounds)
Distribution coefficient (log D) =
𝑠𝑜𝑙𝑢𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎𝑙𝑙 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝑑𝑖𝑠𝑠𝑜𝑙𝑣𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙

𝑠𝑜𝑙𝑢𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎𝑙𝑙 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝑑𝑖𝑠𝑠𝑜𝑙𝑣𝑒𝑑 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟
octanol soluteionized + un-ionized
water soluteionized + un-ionized

log D < 0 log D > 0
Hydrophilic Hydrophobic
-2 -1 0 1 2
HILIC
Hydrophobic mobile
Polar phase (organic solvent)
stationary Non-polar compounds
phase
In comparison to reversed phase chromatography, where

non-polar compounds are retained on the column, with
HILIC polar compounds remain in a water-layer, which is
Flow adsorbed onto the hydrophilic stationary phase. Polar
compounds prefer the water layer over the organic
solvent/ hydrophobic mobile phase. Non-polar compounds
won´t enter the water layer and will remain in the organic
solvent. Thus they are not retained, resulting in them being
flushed through the column. By gradually increasing the
water content of the mobile phase, polar compounds will
solubilize in the mobile phase and elute from the column.
Water layer
Polar compounds
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
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.
0 5 10 15 20 25 It is also possible to separate a sample with a HILIC column to capture

Retention time polar compounds. However with this setup one would disregard non-
polar compounds, since they won´t adsorb onto the polar stationary
phase. They remain in the mobile phase, which is at the beginning of the
experiment highly organic. HILIC is therefore basically the opposite to
RP.

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
Greco and Letzel, Sep. Sci. 2013, HILIC Solutions No.1
Introduction of multidimensional chromatography
Experimental setup
Pump
1. Dimension
Pump Column Detector Waste
Sample
Column
2. Dimension
Arrows depict the flow directions
Detector

Why more than more dimension?
- For samples, which are very complex or

possess a very complex sample matrix
Very complex sample,
which contains a multitude
of different compounds
- Sample, which contains substances with Peaks are not sufficiently

similar or equal retention times (i.e. Coelution) separated (i.e. not
baseline separated)
- Samples with high background signals (e.g. due to complex matrix or impurities)

Definition of Coelution
Intensity
Although only one peak is visible in the

chromatogram, more than one compound
may elute at this particular retention time
(=coelution). Retention time
Intensity
 Compounds are not separable with the

applied chromatographic method
PD Dr. J. Graßmann; PD Dr. T. Letzel Retention time

Experimental setup
Chromatogram of first dimension

separation
Pump
Pump Column Detector Waste
1. Dimension
Sample
Column
2. Dimension
PD Dr. J. Graßmann; PD Dr. T. Letzel Detector

Chromatogram of first dimension

separation
 Using 2D chromatography it may be possible to resolve the coeluting peaks by transferring

the chromatographically separated analytes from the first dimension onto a second dimension

“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:
 Fractionation of the chromatographically

separated sample (1st dimension) results in each
peak being separated into 3 to 4 fractions.
Each fraction is transferred to the second dimension
Each peak is separated into fractions
Consecutive transfer of fractions to 2nd dimension

Process of fractionation and transfer to the second dimension:

First fraction, containing a portion of the

red peak is collected in „Loop 1“, which
is not connected to the 2nd column.
 The fraction is collected but not yet
transferred to the 2nd column

By means of switching the valve into a new position, loop 1 is

connected to the 2nd column. While the second fraction of the red
peak is collected in „Loop 2“, the first fraction of peak „red“ can be
transferred from loop 1 onto the 2nd column (i.e. 2nd dimension)
..... and so on ....
 The 2nd dimension chromatographic separation is thus required

to be faster than the time span it takes to transfer one fraction
into a sample loop.
By means of this techniques the fractions can be consecutively
sampled and introduced to the second column without time delay.
The entirety of the sample can thus be transferred from the first to
the second dimension.

“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 ...
Coeluting peaks of the 1st

dimension can here be
separated.

Result after 2D chromatographic separation of the sample
2nd dimension
1st dimension Final result of 2D-LC

“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
Collected fraction is being transferred to the 2nd dimension

“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.

“Serial LC-LC coupling”
Definition „serial LC coupling of two columns“ (e.g. Reversed phase and
HILIC):
As opposed to „comprehensive“ and „heart-cutting“ 2D LC techniques, in which fractions are

collected from the chromatographically separated sample to be consecutively introduced to the
second dimension, the serial coupling of two chromatographic separations does not involve sample
fractionation and sample storage in loops. On the contrary, the compounds, which were separated
on the first column, are directly and continuously delivered to the second column.
Pump 2
1. Dimension 2. Dimension
Pump 1 Column Column Detector
Sample

„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.

Starting conditions of the setup:

1 Pump 1 delivers a mainly aqueous mobile phase
with a minor flow rate compared to the flow rate of
pump 2, which delivers a 100% organic mobile
2
phase (here: acetonitrile  compatible with
HILIC).
Consequently, at the beginning of the experiment
a highly aqueous mobile phase flows through the
RP column, whereas the mobile phase reaching
the HILIC column is mainly organic with low
Exemplary starting conditions Flow from pump 2:
0.4 mL/min aqueous proportion, due to the dilution of the
= 100% ACN aqueous mobile phase from the RP column by
means of the distinctly higher flow rate of pump 2.
Flow from pump 1: Total flow:

0.05 mL/min 0.45 mL/min
= 10% ACN, 90% aqueous

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.
Mobile phase gradient:

By gradually decreasing the organic solvent
proportion of the HILIC mobile phase ( 6 ) to a
5 more aqueous composition, the polar compounds
will be eluted from the column.
6 Simultaneously the RP mobile phase composition
( 5 ) will become more and more organic, which
causes the non-polar compounds, which interacted
with the RP stationary phase, to elute.
Eluting compounds then reach the detector.

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.
More information in the following publications:

- „Study of the retention behavior in zwitterionic hydrophilic interaction chromatography of isomeric hydroxy- and
aminobenzoic acids” by Greco et al., Journal of Chromatography A, 1235 (2012) 60–67
- Robustness of a method based on the serial coupling of reversed-phase and zwitterionic hydrophilic interaction LC–
MS for the analysis of phenols, Greco et al., J. Sep. Sci. 2014, 37, 630–634

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).
Serial coupling of RP and HILIC column:

 Polar and medium polar compounds interact with the polar stationary
phase of the HILIC column. Thus they are retained and elute with
decreasing organic solvent proportion of the mobile phase.
 Compared to the upper setup, polar and medium polar compounds can
be chromatographically separated.
 The setup provides the possibility to separate non-polar compounds as
well, thus covering a wide range of polarities in one single run.

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)
- LC x SEC (size exclusion chr.)

- LC x GC
- SFC (supercritial fluid chr.) x GC 2nd dimension data
Cut recording &
- SFC x LC processing
- GC x GC x GC
- ……………..
Column 1 Column 2
See also slides „Gas chromatography“

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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

For gradient elution:

Non-polar to semi-polar analytes

PD Dr. J. Graßmann; PD Dr. T. Letzel

Partition coefficient (log P) =

𝑠𝑜𝑙𝑢𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑑𝑖𝑠𝑠𝑜𝑙𝑣𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙

log P < 0 log P > 0

Distribution coefficient (log D) =

𝑠𝑜𝑙𝑢𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎𝑙𝑙 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝑑𝑖𝑠𝑠𝑜𝑙𝑣𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙

water soluteionized + un-ionized

In comparison to reversed phase chromatography, where

0 5 10 15 20 25 It is also possible to separate a sample with a HILIC column to capture

PD Dr. J. Graßmann; PD Dr. T. Letzel

Pump Column Detector Waste

Arrows depict the flow directions

PD Dr. J. Graßmann; PD Dr. T. Letzel

- For samples, which are very complex or

- Sample, which contains substances with Peaks are not sufficiently

PD Dr. J. Graßmann; PD Dr. T. Letzel

Although only one peak is visible in the

 Compounds are not separable with the

PD Dr. J. Graßmann; PD Dr. T. Letzel Retention time

Chromatogram of first dimension

Pump Column Detector Waste

PD Dr. J. Graßmann; PD Dr. T. Letzel Detector

Chromatogram of first dimension

 Using 2D chromatography it may be possible to resolve the coeluting peaks by transferring

PD Dr. J. Graßmann; PD Dr. T. Letzel

 Fractionation of the chromatographically

Each peak is separated into fractions

Consecutive transfer of fractions to 2nd dimension

PD Dr. J. Graßmann; PD Dr. T. Letzel

First fraction, containing a portion of the

PD Dr. J. Graßmann; PD Dr. T. Letzel

By means of switching the valve into a new position, loop 1 is

 The 2nd dimension chromatographic separation is thus required

PD Dr. J. Graßmann; PD Dr. T. Letzel

Coeluting peaks of the 1st

PD Dr. J. Graßmann; PD Dr. T. Letzel

1st dimension Final result of 2D-LC

PD Dr. J. Graßmann; PD Dr. T. Letzel

Collected fraction is being transferred to the 2nd dimension

PD Dr. J. Graßmann; PD Dr. T. Letzel

As opposed to „comprehensive“ and „heart-cutting“ 2D LC techniques, in which fractions are

PD Dr. J. Graßmann; PD Dr. T. Letzel

PD Dr. J. Graßmann; PD Dr. T. Letzel

Starting conditions of the setup:

Flow from pump 1: Total flow:

PD Dr. J. Graßmann; PD Dr. T. Letzel

Mobile phase gradient:

PD Dr. J. Graßmann; PD Dr. T. Letzel

More information in the following publications:

PD Dr. J. Graßmann; PD Dr. T. Letzel

Serial coupling of RP and HILIC column:

PD Dr. J. Graßmann; PD Dr. T. Letzel

- LC x SEC (size exclusion chr.)

See also slides „Gas chromatography“

PD Dr. J. Graßmann; PD Dr. T. Letzel