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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt


Technische Universität München

Chromatography, Workflows
& Data evaluation

BASICS

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

What to expect from the following slides?


• General definitions: Becoming familiar with workflows and
different kinds of screenings
• Distribution coefficients: Using logP/ logD as additional
information in liquid chromatography
• Chromatographic methods: Understanding the mechanisms of
reversed phase and hydrophilic interaction chromatography
• Chromatographic setups: Improving separation by using
advanced separation techniques
• Peak characterisation: Determining the mass to charge ratio
(m/z) and isotopic patterns
• Tandem mass spectrometry (MS/MS): Analysing the
fragmentation pattern to learn about the compound’s structure

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Workflow Definition
A workflow consists of a progression of steps, which comprise a particular work process.
Proceeding from one step to the next can only be achieved when the requirements of the
previous step(s) are fulfilled.

The workflows discussed and explained here aim for the finding, identification and finally
quantification of target substances by means of chromatographic separation (reversed
phase and/or HILIC) and mass spectrometric detection. The procedure comprehensively
illustrates the standard approaches of target screening, suspected-target screening and
non-target screening and describes how they are applied in many analytical laboratories.

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Overview

What steps do you have to take for each screening method?

Target Suspected Non-target Information


Screening target Screening quality
Screening
Mass of interest → MS full scan, X
trends
Structure proposition → mass X
filtering, peak detection, formula
fitting
Tentative candidate → isotope X X
pattern, RT, fragmentation
information
Plausible structure → database/ X X
literature search
Confirmed structure → reference X X X
standard

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

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)

𝐶𝑖 𝑜𝑐𝑡𝑎𝑛𝑜𝑙
Partition coefficient (log P) = 𝐶𝑖 𝑤𝑎𝑡𝑒𝑟

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

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

logD value
Distribution coefficient
log D refers to ionizable molecules (Lipophilicity and therefore solubility changes as
a function of pH for ionisable compounds). Depending on the pH, a molecule can be
present in a charged or neutral state (species).
𝑛
𝑖=0 𝐶𝑖 𝑜𝑐𝑡𝑎𝑛𝑜𝑙
Distribution coefficient (log D) = 𝑛 𝐶
𝑖=0 𝑖 𝑤𝑎𝑡𝑒𝑟

octanol 𝑠𝑜𝑙𝑢𝑡𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑎𝑙𝑙 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝑑𝑖𝑠𝑠𝑜𝑙𝑣𝑒𝑑 𝑖𝑛 𝑜𝑐𝑡𝑎𝑛𝑜𝑙


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

water soluteionized + un-ionized


log D < 0 log D > 0
Hydrophilic Hydrophobic
-2 -1 0 1 2

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Chromatography
Definition: 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 definite direction.

Mobile phase

Component mixture
Stationary phase

Separated components

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

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 LogP/ LogD which is a measure
of an analytes partition between two immiscible solvents (usually octanol and water)
 compare slides „Polarity and Solubility“
• The higher the LogP/LogD, the more hydrophobic is the molecule.
Non-polar

For gradient elution:


Mobile phase Starting with highly aqueous content.
Increase of organic content over time.

Non-polar to semi-polar analytes

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Hydrophobic mobile
HILIC
phase (organic solvent)
Non-polar compounds

In contrast to reversed phase chromatography,


Polar where non-polar compounds are retained on the
stationary column, in HILIC polar compounds remain in a
phase water-layer, which is adsorbed onto the hydrophilic
stationary phase. Polar compounds prefer the water
layer over the organic solvent/ hydrophobic mobile
phase. Non-polar compounds will not enter the
Flow water layer and 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
PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große
Liquid Chromatography
Comparision of the separation of two analytes either with a 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 will not 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; S. Große
Chromatographic setups
One column (1)
Mixing of solvents A and B

Pump
detector (e.g. MS)

Sample injector
or autosampler

RP-Column
B
A
detector (e.g. UV)
PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Chromatographic setups
One column (2)
By using a reversed-phase column and a gradient elution, one would
Reversed Phase Chromatography: usually start with a mainly aqueous mobile phase.
Extracted ion chromatograms Introducing a complex mixture to this setup will result in the adsorption of
of mass spectrometrically detected non- and semi-polar compounds onto the non-polar stationary phase,
m/z* Polar, highly water soluble compounds will remain in the mobile phase.
This results in the retention of the adsorbed molecules, which are
Increasing hydrophobicity gradually eluted from the column through the increase of the organic
MS intensity (counts)

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.

It is also possible to separate a sample with a HILIC column to capture


0 5 10 15 20 25
polar compounds. However with this setup one would disregard non-
Retention time polar compounds, since these will not adsorb onto the polar stationary
phase. They remain in the mobile phase, which is at the beginning of the
experiment highly organic using a HILIC column. HILIC is therefore
basically the opposite to RP.

* m/z: mass to charge ratio (compare slide 19)


PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große
Chromatographic setups
Two columns (coupling setup) (1)

B
A
Mixing of solvents A and B

Mixing of solvents
detector (e.g.
Pump A and B MS)

Pump

Sample injector
or autosampler

detector
B RP-Column HILIC-Column (e.g. UV)
A
PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Chromatographic setups
Two columns (coupling setup) (2)
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.
Please find 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

The application of this system results in the separation of polar compounds, which elute within the first minutes of
the run, separated by the HILIC column (grey area). After this, non-polar compounds elute, which were retained by
the RP column (orange area).
Compounds retained by the HILIC column mainly possess a logP/ logD < 0, whereas compounds retained by the
RP have logP/ logD values > 0.
PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

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.

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Mass spectrometric detection


For the detection of compounds separated with liquid chromatographic techniques, different
detectors can be utilizied. In case of the later discussed screening strategies, mass
spectrometers (MS) are most commonly used. Depending on the type of MS, different
information about detected compounds can be derived (see also Level 1 slides „Mass
spectrometry_mass analyzer“), ranging from accurate mass over fragmentation patterns to
structural information. The most commonly used types of information are explained in the
following section.

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Mass to charge ratio

Depending on the mass accuracy of the utilized MS instrument, the mass to charge
ratio (m/z) of the detected compound can be utilized to gain information about its
chemical composition. For such experiments, highly accurate MS instruments are
required (TOF, Q-TOF, FTIR-MS, Oribitrap, etc.)
In case of ibuprofen, which is most likely negatively charged due to its carboxyl
group, the m/z would be 205.12339. So if the m/z is present within the sample,
ibuprofen might be contained. However they are a variety of other molecules, which
have the same m/z, but an entirely different structure and so completely different
compounds.

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Isomeric compounds

Although two molecules have the same molecular weight, their physiochemical properties
might be different.
Both molecules, shown below are chargeable, depending on the pH value in the solution.
As a consequence the logD values of both compounds could be different (compare slide 9
and 10 and slides „Polarity and Solubility“). 4-hydroxyphenyl hexyl ketone has a logD of
3.56 at pH 7.4, whereas ibuprofen has a logD of 1.34 at pH 7.4. Thus ibuprofen has
(although still rather hydrophobic) a better water solubility than 4-hydroxyphenyl hexyl
ketone. Furthermore at pH 7.4 a much higher percentage of ibuprofen will be negatively
charged compared to 4-hydroxyphenyl hexyl ketone. Consequently the interaction of the
latter compound with the stationary phase of a RP-column would be much stronger, which
would results in a later elution from the RP column. As a result the elution order of both
compounds in RPLC would be ibuprofen followed by 4-hydroxyphenyl hexyl ketone

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Isotopic pattern (1)

A further indicator of whether or not an observed peak is the target compound is its
isotopic pattern, which can be detected with highly accurate mass spectrometers like
a Time-of-Flight-MS (Please find more information in chapter „Mass spectrometry“,
slides „Mass spectrometry Basics“)

Isotopes
Natural abundance of isotopes and their effect on molecular weight, e.g. Carbon C:
Stable carbon isotopes 12C and 13C naturally occur with an abundance of 98.9:1.1,
i.e. 1.1% of C are 13C, whereas approx. 98.9% are 12C. Their mass differs by 1 Da.

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Isotopic pattern (2)


A molecule, which contains e.g. a Cl atom has a different isotopic pattern than one
which only contains C, H, O atoms. This is due to the probability of the 35Cl being
exchanged with 37Cl.
~75% of a compound containing 1 chlorine contain 35Cl, whereas ~25% contain 37Cl.

Cl Cl2 Cl3 Cl4

+2 +2 +4 +4 +4 +8
This results in a adjacent peak with a m/z +2 Da compared to the m/z of the target
compound, due to 37Cl being 2 Da heavier than 35Cl. In case of chlorine the 37Cl is
approx. 1/3 the height of the 35Cl peak. Compounds containing more than 1 chlorine
therefore possess distinct isotopic patterns, which can be clearly distinguished from
another and from compounds containing no Cl. This also applies for compounds
containing e.g. Br or S or a combination of them, all of which resulting in unique
isotopic patterns. PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Isotopic pattern (3)

Some isotopes and their abundance


M-1 M M+1 M+2 Isotopic mass [u] Absolute
abundance [%]

1H 1.0078 99.99
2H 2.0141 0.01
12C 12.0000 98.90
13C 12.0034 1.10
14N 14.0031 99.63
15N 15.0001 0.37
32S 31.9721 95.02
33S 32.9715 0.75
34S 33.9679 4.21
35Cl 34.9689 75.77
37Cl 36.9659 24.23
79Br 78.9183 50.69
81Br 80.9163 49.31

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Isotopic pattern (4)


For example CHCl3

118.92
120.92
100

50

122.92

119.92 121.92 123.92 124.92

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

MS/MS
To verify the presence of a particular compound in a sample, its fragmentation pattern can be
used. It provides information about the chemical structure of a compound. By means of utilizing
e.g. a triple quadrupole mass spectrometer, the m/z of a target compound can be filtered (Q1)
from a complex sample matrix, whereupon it is fragmented (Q2) (see also Level 1 slides „Mass
spectrometry_mass analyzer“). The fragments are then focussed and sorted in Q3 and detected
afterwards. By means of this method a unique fragmentation spectrum is generated. By
comparing the obtained spectrum with spectra from a library (if available) containing known
reference substances, the identification of a compound can be considered conclusive (compare
next slide). Some MS/MS-databases are publically available in the internet or (e.g. MassBank“)
or provided by vendors of MS/MS instruments. In addition, such libraries can also be created in
the lab, by analysing known compounds.
In case of an unknown compound, there may be no reference spectra available. In this case the
fragment m/z´s may provide information about the chemical structure of the analyte, e.g.
presence of functional groups etc.

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

MS/MS
Spectrum of unknown
compound

Bad library match


Library reference
spectrum

Spectrum of unknown
compound

Good library match

Library reference
spectrum

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Fragmentation pattern – Multiple reaction monitoring


MRM (1)
The analyte’s m/z is selected, whereupon it is separated from other m/z by Q1 after
its introduction to the MS.

By means of utilizing e.g. a triple quadrupole mass spectrometer, two stages of


mass filtration can be applied (see also Level 1 slides „Mass spectrometry_mass
analyzer“). The target compound is selected in quadrupole 1 (Q1), whereupon it is
fragmented in Q2. In Q3 not the entire range of fragments is passing but usually two
which are called quantifier and qualifier and are detected later on.

Triple quadrupole MS

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Fragmentation pattern – multiple reation monitoring


MRM (2)
The abundance of those particular fragments (quantifier and qualifier) and their ratio
is distinct and independent of the analyte concententration. It can therefore be used
for its identification.

Quantifier

Qualifier

PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Quantifier
Target compound:

Signal

Signal
Known ratio of quantifier Qualifier
and qualifier

RT m/z

Different ratio of quantifier Qualifier Quantifier


Signal

Signal
and qualifier

 Not the target


compound
RT m/z

Quantifier
Qualifier is missing
Signal

Signal
Qualifier
missing
 Not the target compound

RT m/z
PD Dr. J Graßmann; PD Dr. T. Letzel; S. Große