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Chromatography, Workflows
& Data evaluation
BASICS
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.
Overview
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
octanol soluteneutral
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
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
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
Hydrophobic mobile
HILIC
phase (organic solvent)
Non-polar compounds
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)
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.
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.
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
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.
+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
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
118.92
120.92
100
50
122.92
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.
MS/MS
Spectrum of unknown
compound
Spectrum of unknown
compound
Library reference
spectrum
Triple quadrupole MS
Quantifier
Qualifier
Quantifier
Target compound:
Signal
Signal
Known ratio of quantifier Qualifier
and qualifier
RT m/z
Signal
and qualifier
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