MSc Chemistry
Analytical Sciences
Master Thesis
Advanced Characterization of Polyols by Combining
Chromatographic and Spectrometriv Techniques
Introducing Liquid Chromatography-Ion Mobility-Electrospray
Ionization Time-of-Flight Mass Spectrometry (LC-IM-ESI TOF MS)
and Laser Desorption Ionization-Fourier Transform-Ion Cyclotron
Resonance Mass Spectrometry (LDI-FT-ICR MS)
by
Ioanna Voulgari
June 2015
Supervisor:
Peter Schoenmakers
dr. W.T. Kok
Daily Supervisor
dr. W. Genuit
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Advanced
Characterization of
Polyols by Combining
Chromatographic and
Spectrometric
Techniques
Introducing Liquid
Chromatography-Ion Mobility-Electrospray Chromatography-Ionization
Time-of-Flight Mass Spectrometry
(LC-IM-ESI TOF MS) and Laser Desorption
Ionization-Fourier Transform-Ion
Cyclotron Resonance Mass
Spectrometry (LDI-FT-ICR MS)
Polyether Polyols with molecular mass (MM) up to
10000Da are used as a precursor for the synthesis
of polyurethanes. Analytical techniques routinely
applied for polyol analysis are Nuclear Magnetic
Resonance (NMR), Size Exclusion Chromatography
(SEC) and Liquid Chromatography-Mass
Spectrometry (LC-MS). Mass Spectrometry offers
additionally structural analysis compared to the
other techniques. However, polyols and other
polymers produce complex mass spectra in ESI –
MS, due to the appearance of multiple charge
states of ions. As the MM of a molecule increases
so does the probability of multiply charged ion
formation resulting in overlapping peaks and
convoluting spectra which are difficult to interpret.
The aim of this project was to simplify the
accumulated MS spectrum by introducing a third
separation dimension, Ion Mobility Spectrometry
(IMS), to an existing LC-MS method, and optimize
the proposed analytical method. Chromatographic
separation was greatly improved when Ion
Exchange Chromatography (IEC) was introduced
instead of Reverse Phase Chromatography (RP LC).
Derivatization of the free hydroxyl groups of the
Page | 3
polyol components into acid groups enabled the
IEC separation of the polyol samples into fractions
according to the functionality of the molecules. In
negative ESI-MS mode, charge multiplicity was
controlled by the number of acid groups and so by
the functionality of the polyol molecules. IMS
offered additional mobility separation and detailed
information about the structural composition of
the different polyol samples. The polyols were also
analyzed with another analytical technique, Laser
Desorption Ionization-Fourier Transform Ion
Cyclotron Resonance Mass Spectrometry
(LDI-FT-ICR MS).
Ioanna Voulgari
November 2014-June 2015
Wim Genuit
GSNL-PTD/TASE - Analytical Problem Solving
Shell Global Solutions International B.V.
Grasweg 31, 1031 HW Amsterdam, The
Netherlands
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Table of Contents
1.
Introduction ... 6
1.1. Polyols ... 6
1.1.1.
Chemistry of Polyols ... 6
1.1.2.
Basic chemistry of Polyurethanes ... 8
1.1.3.
Polyol Synthesis ... 9
1.1.4.
Polyol Characterization ... 11
1.2. High Performance Liquid Chromatography (HPLC) ... 12
1.2.1.
Reverse Phase Chromatography (RP HPLC) ... 12
1.2.2.
Ion Exchange Chromatography (IEC HPLC) ... 12
1.3. Mass Spectrometry ...14
1.3.1.
Electrospray Ionization Time-of-Flight Mass Spectrometry (ESI TOF-MS)
14
1.3.2.
Laser Desorption Ionization - Fourier Transform - Ion Cyclotron
Resonance Mass Spectrometry (LDI-FT-ICR MS) ...19
1.4. Ion Mobility - Mass Spectrometry (IM-MS) ... 20
1.4.1.
Ion Mobility Spectrometry (IMS) ... 20
1.4.2.
Travelling Wave Ion Mobility Spectrometry (TWIMS) ... 24
1.5. Aim of the project ... 27
2.
Experimental Section ... 27
2.1. Samples and Reagents ... 27
2.2. Sample preparation ... 27
2.3. Derivatization of Polyols ... 27
2.4. Instruments ... 29
2.4.1.
HPLC Chromatography ... 29
2.4.2.
Ion Mobility-Mass Spectrometry ... 30
2.5. Data Analysis ... 31
3.
Results and Discussion ... 32
3.1. Chromatographic Separation- Reverse Phase Chromatography (RP HPLC-ESI MS positive mode) without Ion Mobility ... 38
3.2. Chromatographic Separation- Reverse Phase Chromatography (RP HPLC-ESI MS positive mode) with Ion Mobility (IMS) ... 42
3.3. Ion Mobility-Mass Spectrometry (IM-ESI MS negative mode) of derivatized polyols (No Chromatographic Separation) ... 47
3.4. Chromatographic Separation-Reverse Phase Chromatography (RP HPLC-ESI MS negative mode) with Ion Mobility (IMS) ... 52
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3.5. Chromatographic Separation-Ion Exchange Chromatography (IEC HPLC-ESI MS
negative mode) with Ion Mobility (IMS) ... 57
3.6. LC-IM-MS data processing and interpretation ... 62
3.7. LDI-FT ICR MS data processing and interpretation ... 81
4.
Conclusions ... 88
5.
References ... 90
6.
Appendix A ... 93
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1. I
NTRODUCTION
1.1. Polyols
1.1.1. Chemistry of Polyols
In chemistry, an alcohol is any organic compound that has a hydroxyl
functional group (-O-H) attached to a saturated carbon atom R3COH.1 A
polyol is an alcohol that contains multiple hydroxyl functional groups. In organic and polymer chemistry, a polyol with one hydroxyl functional group is referred to as a monol, with two hydroxyl groups as a diol, with three hydroxyl groups a triol, with four a tetraol and so on. Figure 1-1 demonstrates some examples of these polyols as well as their chemical structures.
Figure 1-1 : Ethylene Glycol, Water and Propylene Glycol contain two hydroxyl functional groups, thus referred as diols. Glycerol contains three hydroxyl groups, thus a triol. Pentaerythitol and Sorbitol contain four and six hydroxyl groups accordingly.
These monomeric polyols are often used as a starting material or an initiator for the synthesis of polymeric polyols. For example, if glycerol is to be used as an initiator, it can react with alkylene oxides such as ethylene oxide (EO) or propylene oxide (PO) (Figure 1-2) in the presence of a catalyst such as potassium hydroxide (KOH) or a double metal cyanide catalyst such as the
zinc hexacyanocobaltate-t-butanol complex (DMC). 2, 3 The result is the
polymerization of PO or EO as we will discuss in 1.1.3, and the outcome is the formation of polyether polymeric polyols, (Figures 1-3, 1-4, 1-5).
Page | 7
Figure 1-2 : Alkylene oxides such as Propylene Oxide (PO) and Ethylene Oxide (EO) are used as the repeating monomer unit for the formation of polyalkylene oxide polyether polyols
Figure 1-3 : An idealized structure of a 750 Da MM ethylene oxide triol based on glycerol.
Figure 1-4 : An idealized structure of a 960 Da MM propylene oxide triol based on glycerol.
Figure 1-5 : An idealized structure of a triol based on glycerol with propylene oxide and end-capped with ethylene oxide.
These polymeric polyether polyols primarily are used in polymer chemistry as a reagent for the formation of even larger polymer molecules called
polyurethanes. Polyurethanes are a special group of heterochain polymers, which contain the following structural unit:
Page | 8 The main applications of polyurethanes (PU) include the manufacture of foams (flexible, semiflexible and rigid foams) and foam matresses; soft solid or low density elastomers used for gel pads and print rollers or for athletic shoes; hard solid or flexible plastics are used as electronic instrument bezels and structural parts or as straps and bands. Moreover, polyurethanes are used in the production of elastomeric wheels and tires as well as surface coatings,
adhesives, sealants and fibres.5
Taking into account the various practical applications of polyurethanes in industry and manufacturing, they can be divided in two main categories: elastic PU and rigid PU. This classification is primarily based on the structure of the polymeric polyether polyol that is used as a starting material.
The physical properties (e.g. flexible foams or hard plastic and coatings) of polyurethanes are determined by the MM and functionality of the polyols. The MM of the polyols used for the formation of polyurethanes varies between 300-10000 Da, which typically includes the region of low MM polymers (oligomers). Additionally, the number of hydroxyl functional groups per molecule of low MM polyol (the polyol functionality) is in general between 3-8 hydroxyl groups per mole.
For example, for the formation of elastic polyurethane, a low functionality polyol (2-3 hydroxyl groups/mole) with a high MM of 2000-10000 Da is more favorable. On the contrary, a high functionality polyol (3-8 hydroxyl
groups/mole) with a low MM of 300-1000 Da leads to the formation of rigid
polyurethanes.3, 4.
1.1.2. Basic chemistry of Polyurethanes
The urethane structure unit that is shown below, which gives the name to this specific group of polymers, is the product of a reaction between a hydroxyl group and an isocyanate.
The general scheme of the reaction that is taking place is the following:
In order to increase the polyurethane formation reaction, organo-metallic catalysts are often used such as stannous octoate and dibutyltin dilaurate. The role of these catalysts is to form an intermediate complex with an isocyanate
Page | 9
1.1.3. Polyol Synthesis
Polyalkylene oxide polyether polyols thus far are the most important group of polyols from the formation of polyurethanes. They are obtained by
polymerization of propylene oxide (PO) and/or ethylene oxide (EO) in the presence of an initiator such as glycerol or water, which were previously demonstrated in Figure 1-1.
Polyols synthesized by PO monomer units contain less reactive secondary hydroxyl groups. In order to obtain more reactive primary hydroxyl functional groups, EO is added near the end of the polymerization reaction (EO ‘tipping’). The percentage of EO in the total polymer chain synthesized is less than 20%. In order to initiate the polymerization reaction for the formation of polyols, the presence of a catalyst is essential. The most important catalyst for industrial scale synthesis of polyols is potassium hydroxide (KOH). Although the use of KOH as a catalyst leads to the production of high MM polyether polyols and block PO-EO copolymers, a high amount of unsaturations are also produced which may affect the quality of the final polyurethane product. Another catalyst group type used is the one of dimetallic catalysts based on a
nonstoichiometric complex of Zn3[Co(CN)6]3*ZnCl2 (DMC). The performance
of this catalyst group compared to KOH is significantly higher, producing higher quality PO polymer products with low presence of unsaturation. On the other hand, DMC catalysts are insufficient for the EO polymerization or the synthesis of PO-EO block copolymers due to the formation of side products
such as cyclic ethers.3, 6, 7
The polymerization reaction of PO, with propylene glycol as the initiator in the presence of KOH as the catalyst, leads to the formation of polyether diol, polypropylene glycol (PPG) as shown in figure 1-6:
Figure 1-6 : Polymerization of PO with propylene glycol as initiator leads to the formation of polyether diol, polypropylene glycol (PPG)
Water is also considered an initiator for the formation of polyether diols. The first step involves the hydrolysis of the oxiranic ring of propylene oxide which leads to the formation of propylene glycol. The second step starts with the polymerization of PO with the use of the produced propylene glycol as initiator.
Consequently, the presence of water in raw materials or in the monomers (PO and EO) always leads to the formation of polyether diols. This has an effect in the functionality of the synthesized polyether polyols that have a functionality higher than f=2 such as polyether triols. The expected functionality of such a polyol is usually 3. However, the actual functionality has slightly decreased. Additionally, if the amount of water present in the monomers (PO or EO) is
Page | 10 more than 0.05-0.1%, then any attempt of synthesizing high MM polyether
diols and triols is no longer possible.3
As previously mentioned, for a successful PO polymerization the presence of a catalyst such as KOH is required. The very presence of the KOH leads to the production of unsaturations, especially as the MM of the polyols increases. KOH is highly reactive with regards to the polymerization reaction, thus responsible for the isomerization of PO to allyl alcohol, as shown in figure 1-7. An allyl alcohol has one hydroxyl group, thus a monol as discussed in 1.1.1. Consequently, according to the same principle the allyl alcohol acts as an initiator for the formation of polyether monol.
Figure 1-7 : PO polymerization of a diol or a triol with KOH as catalyst results in the synthesis of polyether diol or triol accordingly (top reaction). KOH catalyzes also the isomerization of PO to allyl alcohol, which produces a polyether monol (bottom reaction).
If the starting material is a triol such as glycerol, the polymerization reaction that takes place is shown in figure 1-8 and the end product is a polyether triol.
Page | 11
Figure 1-8 : Polymerization of PO with glycerol as initiator leads to the formation of polyether triol.
The expected functionality of the produced polyether triol with glycerol as initiator should be three (f=3). Instead, due to all the reasons mentioned earlier the final product is not pure polyether triol but consists of three polymeric species:
1. Polyether triols produced from the reaction of PO with glycerol (Figure 1-8)
2. Polyether diols produced from the reaction of PO with water present in raw materials or monomers (PO or EO) (Figure 1-6)
3. Polyether monols (allyl ether type) produced by PO isomerization (Figure 1-7)
The actual functionality is lower than 3 but in general is in the range 2 < f < 3. The monols and diols are responsible for the decrease in functionality of the
triols.3
1.1.4. Polyol Characterization
Characterization of polyol mixtures with respect to their properties such as molecular mass distribution (MMD), end group type and functionality is essential as they determine the quality of the final polyurethane product. The analysis of these mixtures can be conducted in several ways using various analytical techniques. Liquid Chromatography (LC), Mass Spectrometry (MS), Nuclear Magnetic Resonance (NMR), Infrared Spectroscopy (IR) and many other techniques have been used and reported in literature for the
determination of all the properties mentioned above. 8, 9, 10, 11
Currently at Shell, the analytical methods of choice are either NMR or
comprehensive two-dimensional liquid chromatography (2D-LC). 12, 13 These
analytical techniques provide information on the relative amounts of PO/EO and the overall functionality of the polyol mixture. Although, NMR and 2D-LC provide an average molecular mass distribution of the mixture no further information can be extracted regarding the molecular mass distribution of monols, diols and triols individually.
On the other hand, mass spectrometry is a useful and powerful analytical technique for analyzing and determining the molecular structure of organic compounds. Consequently, mass spectrometry provides this information, thus is complementary to NMR and 2D-LC.
Page | 12 Previous research of Shell polyol products has already been conducted and
reported. 14, 15 MALDI-TOF and LC-ESI MS were used to determine the
molecular mass distribution, functionality and relative amounts of PO/EO. Due to either low resolution of the instrument, limited mass range or extensive amount of data produced the determination was either not possible or
incomplete. Recently STCA purchased a High Resolution Electrospray
Ionization Time-of-Flight Mass Spectrometer (ESI TOF MS) with the addition of Ion Mobility Spectrometry (IMS).
The implementing of IMS and ESI-TOF MS in addition to liquid
chromatography (LC) may be able to provide structural information of such complex polyol mixtures. Consequently, this combined analytical technique may provide both qualitative and quantitative information that are essential for the quality of the produced polyurethanes.
1.2. High Performance Liquid Chromatography (HPLC)
1.2.1. Reverse Phase Chromatography (RP HPLC)
The principle of liquid chromatography is that it separates molecules based on different affinity properties. The analyte-stationary affinity is determined by a number of factors, one of them being molecular size or mass. Ideally, in a polyol mixture, all polyol material is completely resolved and every time a single polyol species is sent to the mass spectrometer. The result would be a less complex spectrum. Unfortunately, polyols with high MM are not fully resolved when using HPLC.
Rissler et al. found for (poly) ethylene glycol (PEG) that adequate resolution
can be achieved for oligomers with MM up to 3000Da. 39, 40 In his research, he
used reversed-phase high performance liquid chromatography (RP-HPLC) on a octadecasilyl silica gel (C18 ) stationary phase using a binary gradient
composed of acetonitrile (ACN) and water. Based on previous research
conducted at Shell, 15 polyols with MM higher than 3000Da tend to “stick” to
the column and elute as a bulk at the end of the run or even later runs when a nonprotic organic solvent such as ACN is used. On the other hand, when methanol (MeOH) was used as the organic solvent this tendency was no longer observed.
Alternatively, Size Exclusion Chromatography (SEC) is commonly used for separating polyol mixtures but the use of tetrahydrofuran (THF) as an eluent at SEC prevents direct coupling of SEC with any type of mass spectrometer. In many cases, offline use or post-column mixing of the THF solvent coming from HPLC with a buffer solution is needed.
1.2.2. Ion Exchange Chromatography (IEC HPLC)
The principle of ion exchange chromatography (IEC) is that it separates ionized molecules based on differences in charge properties. When being derivatized, monols, diols and triols will obtain one, two or three acid groups, respectively. Thus, in principle, with the use of derivatization and ion
exchange chromatography we can separate the different components based on the number of hydroxyl groups (or their functionality).
Page | 13 Choosing a suitable ion-exchange matrix is probably the most important factor in developing any ion-exchange method. It is based on various parameters with some of them being
a) Desired ion-exchanger charge or strength b) Sample properties
c) Linear flow rate/sample volume
Moreover, ion-exchange functional groups are divided into two categories. For anion exchange chromatography, positively charged diethylaminoethyl
(DEAE) or quartenary ammonium (Q) are commonly used. On the other hand, negatively charged carboxymethyl (CM), sulphomethyl (SM) and sulphopropyl (SP) are routinely used in cation exchange chromatography. Both categories can be classified further as either “strong” or “weak”. Strong ion-exchangers are fully ionized over an extensive working pH whereas weak ion-exchangers are partially ionized over a limited pH range.
Table 1 shows an overview of ion-exchanger properties that are commercially available.
Exchange Type Ion-Exchange Group
Functional Group Buffer Counter Ions
pH Range Strong Anion Quarternary
Ammonium (Q)
CH2N+(CH3)3 Cl-, HCOO-,
CH3COO-,
SO4
2-2-12
Weak Anion Diethylaminoethyl (DEAE)
O(CH2)2N+H(C2H5)2 Cl-, HCOO-,
CH3COO-,
SO4
2-2-9
Strong Cation Sulphopropyl (SP) (CH2)3SO3- Na+, H+, Li+ 4-13
Weak Cation Carboxymethyl (CM)
OCH2COO- Na+, H+, Li+ 6-10
Table 1 : Overview of commercial ion-exchanger properties available.41
Since our molecules are negatively charged, the type of ion exchange functional groups can be either diethylaminoethyl (DEAE) or quartenary ammonium (Q). The latter is a strong ion-exchanger and is fully ionized over a broad working pH range. During anion-exchange chromatography, the
derivatized polyols can be competitively displaced by the addition of negatively charged formate ions (e.g. from ammonium formate). Before the introduction of the polyol mixture to the ion exchange column, the stationary phase is in equilibrium with the formate anions in the mobile phase. Once the sample has been injected into the column, it is adsorbed by displacing the formate anions of the mobile phase. By gradually increasing the concentration of the salt in the mobile phase, a new equilibrium state is constantly
established between trapped and released polyol. At any given time the affinity of interaction between the salt and the stationary phase is in constant
competition with the one that exists between the derivatized polyols and the stationary phase. Consequently, the derivatized species will be displaced and
Page | 14
1.3. Mass Spectrometry
1.3.1. Electrospray Ionization Time-of-Flight Mass Spectrometry (ESI
TOF-MS)
Electrospray Ionization (ESI) is an ionization technique that is mainly used for organic molecules or large molecules like peptides, proteins and polar
synthetic polymers. ESI is a soft ionization technique. Its principle is based on the fact that ions are formed either by the addition of a cation or the removal of a proton. Ions provide information regarding the nature and structure of
their precursor molecule. 16
One notable difference of ESI compared to other ion sources is that it produces multiply charged ions from large molecules with multiple potential ionizable sites. Obtaining ions with multiple charge states leads to the reduction of their mass-to-charge ratio (m/z) compared to singly charged ions. The acquirement of multiply charged ions is a great advantage as it both improves the detector’s sensitivity and allows the analysis of high MM molecules using analyzers with a limited mass range such as quadrupoles.
On the other hand, the very same advantage that offer multiply charged ions to the analysis of high MM molecules may be a disadvantage when it comes to the interpretation of the mass spectrum obtained. The accumulated mass spectrum in a number of cases may be quite complex and confusing due to the large amount of peaks recorded and requires tools and algorithms to be developed in order to successfully assign all the peaks to the right molecules. As a rule, the number of charges produced on a molecule by ESI depends on the molecule’s MM and the number of ionizable sites available. As the MM of a molecule increases so does the probability of multiple charge ion formation. For an example, polymers are large molecules that their MM varies from a couple of hundred Daltons up to a few thousand Daltons. Whereas low MM polymers may exhibit mostly singly charged ions, high MM polymers may form
doubly, triply and multiply charged ions. 19, 47
Some examples of what was previously described can be seen in figures 1-9, 1-10 and 1-11.
Page | 15
Figure 1-9: RP LC-MS mass spectrum of polyol PPG425 standard in positive ESI mode. One charge state is observed for the polyol.
Page | 16
Figure 1-10: RP LC-MS mass spectrum of polyol PPG1200 standard in positive ESI mode. Two different charge states are observed for a single polyol.
Page | 17
Figure 1-11: RP LC-MS mass spectrum of polyol PPG2700 standard in positive ESI mode. Four different charge states are observed for a single polyol.
Moreover, the number of charged states observed also depends on a number of parameters non-related to the analyte such as sample concentration, selection of solvents to be used, instrument settings and ions present in the solution. Consequently, the analysis of a polyol mixture may produce a very complex mass spectrum (Figure 1-12) originated from the following:
The mixture contains different polyol species (monols, diols, triols, etc.)
Each of these polyol species has a molecular weight distribution (MWD)
As well as a charge state distribution
As a result, various techniques have been applied or need to be further developed in order to improve and simplify the interpretation of ESI mass spectra of polyol mixtures.
Page | 18
Figure 1-12: DI-MS mass spectrum of polyol sample A24 in positive ESI mode. A24 polyol sample contains monols, diols and triols, thus producing a very complex mass spectrum.
Page | 19
1.3.2. Laser Desorption Ionization - Fourier Transform - Ion Cyclotron
Resonance Mass Spectrometry (LDI-FT-ICR MS)
(Matrix-assisted)Laser Desorption Ionization (MALDI) has been developed for the analysis of large biomolecules and polar synthetic polymers. LDI is another efficient method for producing gaseous ions. In general, laser pulses yielding
from 106 to 1010 W cm-2 are focused on a small sample surface. In recent years,
the LDI technique is increasingly used for the chemical imaging of surfaces. The principle of LDI (figure 1-13) is based on the fact that these laser pulses ablate material from the surface of the sample; a microplasma is created of ions and neutral molecules which may react among themselves in the dense vapor phase near the sample surface. The laser pulse is responsible for both the vaporization and the ionization of the sample. Since the signals produced from LDI are very short, simultaneous detection analyzers or time-of-flight
analyzers are required. 17, 18
Figure 1-13: Principle of Laser Desorption Ionization
The general principle of Ion Cyclotron Resonance (ICR)(figure 1-14) is based on the fact that ions under the influence of an electromagnetic field are able to be “trapped” on a circular trajectory. When the energy of the wave has the same frequency as an ion in the cyclotron, then that energy is transferred to ion; resulting in an increase in its kinetic energy and thus the radius of the trajectory. For ion detection, ions of a given mass must circulate at tight packets in their orbits. Ions of the same mass excited to the same energy will be in the same orbit and rotate with the same frequency. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) is a technique that
Page | 20 is able to simultaneously determine all the frequencies of those ions by Fourier transformation of the cyclotron-signal voltage, in a way similar to how the free induction decay from a Fourier-transform NMR spectrometer is converted into an NMR spectrum. Moreover, FT-ICR is a technique known for its high mass
resolution, which is the highest of all types of mass spectrometers. 17, 18, 19
Figure 1-14: Schematics of an ICR cell.
FT-ICR MS is routinely applied in samples with high complexity such as those
found in proteomics, metabolomics and petroleomics. 9, 20, 21, 22, 23Its high mass
resolution and simultaneous detection compared to the other mass
spectrometers offers recording of mass spectra that can be used for detailed interpretation and data analysis, high mass accuracy and resolution.
It was mentioned at the end of 1.2.1., that the analysis of a polyol mixture may produce a very complex mass spectrum due to the fact that it contains
different polyol species and its one of them has a molecular weight distribution as well as charge and isotope distribution. Thus far, there have been many
reports in literature 9, 24, 25 where natural or synthetic polymers or copolymers
have been investigated using FT-ICR MS as a method of detection.
Consequently, the use of FT-ICR MS may offer the necessary mass resolution that the TOF MS may lack due to the high complexity of the polyol mixture.
1.4. Ion Mobility - Mass Spectrometry (IM-MS)
1.4.1. Ion Mobility Spectrometry (IMS)
Ion mobility spectrometry (IMS) is an analytical method that separates and characterizes ions based on their transport and interactions in a gas buffer
under the influence of electric fields. 26, 27 When coupled to mass
spectrometers (MS), IMS also separates on the basis of their size-to-charge ratios which in turn can be used for structural analysis of molecules. For the past decades ion mobility has become a powerful technique in separating and characterizing highly complex samples. It has found applications in different
Page | 21
metabolomics, 29, 30 in polymers 31, 32, in environmental analysis 26, in
pharmaceuticals and in medical diagnostics26 or in forensics science33.
The coupling of IMS with MS is commonly referred to as Ion Mobility-Mass Spectrometry (IM-MS) due to the fact that the two analytical methods are both complementary and instrumentally matched to each other. An IM-MS
instrument must be able to perform five processes: sample introduction, ionization of the compound, ion mobility separation, mass separation and ion detection.
Currently, there are four methods of ion mobility separation that are coupled with mass spectrometry. These are:
a. Drift-time ion mobility spectrometry (DTIMS), b. Aspiration ion mobility spectrometry (AIMS),
c. Differential-mobility spectrometry (DMS) which is also called field-asymmetric waveform ion mobility spectrometry (FAIMS) and d. Travelling-wave ion mobility spectrometry (TWIMS).
Table 2 and figure 1-15 show a summary and schematics of the first three methods used in ion mobility spectrometry. TWIMS will be introduced separately in 1.3.2. as it is a relatively new approach in Ion Mobility compared to the other three approaches. Additionally, the polyol mixtures to be investigated at Shell will be analyzed with a commercial instrument that is developed based on the method of TWIMS.
Time-of-Flight IMS AIMS DMS or FAIMS
Sample Introduction
Direct inlets for gaseous compounds
Thermal desorption (in combination with solid phase extraction or trap techniques) Gas chromatography
Liquid chromatography Membranes
Ion Formation
Beta Radiation (RI) Photoionization (PI) Corona discharge ionization (CDI)
Electrospray ionization (ESI)
Ion Transfer to drift tube
Ions are transferred as pulsed packets via a shutter grid
Ions are transferred as a continuous beam via the buffer gas
Ion Separation
Time of Flight Aspirator Differential or Field
Asymmetric
The electric field applied is constant and uniform and runs gradient along cylindrical
electrodes that are inside the drift tube The ion packet
moves through the
The ions enter the drift tube continuously Ions move through the
voltage gradient assisted by the gas stream towards a plane which is opposite to the plane that were initially introduced
The gas flow through the drift tube is in the same direction as the ion motion
The electric field applied is time varying and is applied
Page | 22 voltage gradient
towards the detector The flow of the
buffer gas is in the opposite direction of the ion motion
E= 200-400 V cm-1
E=20-40 V cm-1 orthogonally to the
direction of the ion motion
Emax= 10.000-30.000
V cm-1
Ion Detection
Faraday Plate with aperture grid
Faraday Plate Collecting Plates
Table 2 : Ion Mobility Determination using three different approaches; Time-of-Flight IMS, AIMS and DMS or FAIMS 26, 34
In all methods, samples must first be delivered to the ionization region of the drift tube either by direct sample introduction or after pretreatment of the sample. For example, vapor samples can be directly introduced through a membrane or with gas chromatography. Semi volatile compounds can be thermally desorbed from collection filters or traps. Liquid samples are mostly directly infused into the IM-MS or as eluents from liquid chromatography. Solid samples can be inserted to the drift tube either by depositing them in sample plates or by using pyrolysis.
Gas-phase ions can be created in IM-MS by various methods. Vapor samples can be ionized using either a radioactive ionization (RI) source or
non-radioactive sources such as corona discharge ionization (CDI), photoionization (PI) and electrospray ionization (ESI). For liquid samples, electrospray
ionization (ESI) is the primary ionization method due to the fact that they can be introduced directly to the IMS, whereas for solid samples matrix assisted laser desorption ionization (MALDI) and laser desorption ionization (LDI) are more preferable.
Page | 23
Figure 1-15: Schematics of different types of ion mobility spectrometers. Adapted from ref.34
As mentioned earlier, four types of ion mobility spectrometers are currently available that can be combined with mass spectrometers. Drift time,
differential, aspiration and travelling-wave. Drift time IMS is operated either in ambient or reduced pressure conditions. Differential and aspiration IMS are operated under ambient pressure conditions whereas the travelling wave IMS operates under reduced pressure conditions.
The resolving power of an Ion Mobility Spectrometer (IMS) depends on the number of collisions an ion has with the atoms or molecules of the buffer gas or depends on the potential through which the ion travels. Consequently, for instruments that operate under reduced pressure conditions, increasing the length of the drift tube results in increasing resolving power. In such cases, the diameter of the drift tube is required to be larger; otherwise we exhibit high radial diffusion rates of ions at reduced pressures.
According to a review article by Kanu et al., which compares the various types of ion mobility-mass spectrometers that are commercially available, DTIMS provides the highest IMS resolving power and is the only method out of the four that can measure directly collision cross-sections (is derived by structural parameters such as size, shape and charge location or distribution). AIMS is a
Page | 24 low resolution IMS method but offers the ability of monitoring ions
continuously. DMS or FAIMS offers continuous ion monitoring as well as orthogonal ion mobility separation with high-separation selectivity. All these types of IMS can be combined with various mass spectrometers (quadrupole, time-of-flight, Fourier transform ion cyclotron, ion trap) currently available producing a variety of IMMS instruments given the
compatibility of each approach. 26, 34, 35
1.4.2. Travelling Wave Ion Mobility Spectrometry (TWIMS)
Travelling wave ion mobility spectrometry (TWIMS) is a new mobility method
that was commercially released by Waters Corporation in the SynaptTM
High-Definition Mass Spectrometry (HDMS) system. A schematic of the Synapt system is shown in figure 1-16. The main configuration includes three TW-enabled stacked-ring ion guides, thus the mobility cell, which is embedded to a
quadrupole-orthogonal acceleration-TOF mass analyzer.36
Figure 1-16 : Schematic of a travelling wave ion mobility-mass spectrometer (IM-MS). 38
The figure illustrates that the ions travel from left to right. Starting from the left is located the ionization source (ESI), which introduces aqueous or non-aqueous samples coming from a high-performance liquid chromatography (HPLC) or by direct infusion (Analyte Spray). As the electrosprayed ions enter the MS, they are bent in a “Z” manner in order to eliminate the solvent and other neutral material, which otherwise would contaminate the skimmer, and focus the ions into a travelling wave ion transfer lens. After that the ions enter a quadrupole MS (QMS). The use of QMS allows the selection of either a single mass or a wider range of masses that are directly sent to the triwave assembly. The triwave assembly is a construction of three travelling wave cells. The first one can be operated either as an ion transfer cell or as a collision-induced dissociation (CID) cell. From there, the ions are transferred to the second cell which can serve either as a transfer cell or as an IMS for mobility separation. Following the mobility separation, the ions are transferred to a third travelling wave cell similar to the first one that was described above. Finally, the ions are
transferred to a high-resolution TOF-MS.26
Although in this design the construction of the mobility cell is similar to that of a DTIMS the fundamentals of ion mobility migration is completely different.
Page | 25 In DTIMS the electrical field that is applied is continuous and has low
intensity. On the other hand, in TWIMS a high field is applied to one segment of the mobility cell and is moving sequentially through the cell one segment at a time in the direction of ion migration resulting in the separation of ions based in their mobility.
According to the review article by Kanu et al., despite the fact that the resolving power of the ion mobility cell in the Synapt is not large enough, the TOF-MS that is coupled to it is analytically powerful. Moreover the fact that is commercially available has made the Synapt system widely accepted in various
research communities.35 This success is only contradicted by the fact that the
fundamentals of TWIMS are only qualitatively understood thus far. A first attempt of developing a theory that could explain the foundations of TWIMS
was published by Shvartsburg et al.37. The introduced theory is developed by
using derivations and ion dynamics simulations. The propagating waves may be responsible for pushing all ions along the drift tube but the ion motion depends on c.
Equation 1
𝑐 =𝐾 × 𝐸𝑚𝑎𝑥
𝑠
In Equation 1, c is the ratio of the maximum ion drift velocity at the steepest wave slope to wave speed, K is the ion mobility, Emax is the electric field strength, and s is the wave velocity. In their model, Shvartsburg et al. treated
ion diffusion as independent from drift,37so that the separation parameters
depended only on the drift, while diffusion determined the ion packet width
and consequently the resolution.26
Figure 1-17 shows a picture of a travelling-wave ion mobility cell as
demonstrated by a commercial video of Waters Corporation.38 As it can be
seen, inside the drift 1tube there are multiple neighboring rings which are connected in opposite phase of a radiofrequency (RF) field. The purpose of the
RF field is to produce a radially confined potential well.37A high voltage pulse
is superimposed on one ring (Synapt G1) or two rings (Synapt G2) which results in ion motion. After that the pulse is moved to the neighboring ring(s) where it relaxes. This motion pattern is propagated through the ring structure towards the end of the drift tube at a speed between 300 and 1300 m/s. The procedure is repetitive which leads to ions in the tube to be moved by mobility
in pulses as waves of the RF field pass through them (figure 1-18)38.
The ion motion pattern mentioned above may be described similarly to a cork floating in the sea. Slow waves will move the cork with them (c<1), whereas the cork will only slightly bob in case of encountering a fast wave (c>1). In the occasion of c≈1, the cork will behave like a surfer following the wave but
Page | 26
Figure 1-17 : Picture of the Triwave cell design of Synapt G2 by Waters
A key parameter in understanding the ion motion in TWIMS is through c (Equation 1), which is as previously mentioned the ratio of ion drift velocity at the steepest wave slope to wave speed. At certain parameters, ions with low mobility slip over the wave more than those ions with high mobility. Consequently, separation of ions is achieved by controlling parameters including the travelling wave velocity, the pulse height and the pressure of the gas. When the ratio has a low value, the ion transit velocity (Equation 1) is proportional to the squares of the mobility (K) and electric field intensity (E). At high values of the ration, the transit velocity asymptotically approaches the wave speed. Finally, the resolving power of TWIMS is mobility dependent,
scaling as K1/2 in the lowest values of the ratio and less at the highest values of
the ratio.26, 37
Figure 1-18 : Travelling wave propelling ions in the Synapt G2 system by Waters
RINGS
Ions Wave height
Page | 27
1.5. Aim of the project
The aim of the project is to analyze a series of polyol samples produced either by Shell or by competitors using various combinations of chromatographic and spectrometric techniques; introducing Liquid Chromatography-Ion Mobility-Electrospray Ionization Mass Spectrometry (HPLC-IM-ESI TOF MS) and Laser Desorption Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (LDI-FT-ICR MS). The main goal is to gain information on the content of the polyol sample; various parameters such as polyol functionality, MWD, end groups and possible additives are important in determining the quality of the polyol material and thus, the final polyurethane
product. The polyol samples (Table 10, Appendix A) contain monols, diols and triols which are predominantly PO based polyols; although they might also contain small amounts of EO. Additionally, for optimization of the methods, PPG standards with known MWD were used.
2. E
XPERIMENTAL
S
ECTION
2.1. Samples and Reagents
Poly (propylene glycol) standard materials with number-average molecular mass (MN)
between 425Da and 4000Da, Formic Acid (LC/MS ultra) and Ammonium Formate (LC/MS ultra), both eluent additives for LC/MS, Phthalic Anhydride (ACS reagent, ≥99%), Imidazole and 1,4-diazabicyclo[2.2.2]octane (DABCO) were purchased from Sigma-Aldrich (Steinheim, Germany). Methanol Li chro solv 99.8% (MeOH) and Acetonitrile Li chro solv 99.9% (ACN), both hypergrade for LCMS, were purchased from Merck Chemicals (Darmstadt, Germany). Ultrapure water was obtained from a Millipore Direct Q-3 water purification system (Molsheim, France). Polyol samples were either produced at Shell or acquired from competitor companies (Appendix A, Table 10).
2.2. Sample preparation
For direct infusion-mass spectrometry (DI-MS) and ion mobility-mass spectrometry (IM-MS), Liquid Chromatography-Mass spectrometry (LC-MS) and Liquid
Chromatography-Ion Mobility-Mass Spectrometry (LC-IM-MS) the samples, both underivatized and derivatized, were diluted in a 80:20%v/v MeOH/water or in a 70:30 %v/v ACN/water solution at a concentration of 100ppm. The type of cationization agent and its concentration varied between 10-30mmol for ammonium acetate and ammonium formate used for the detection of positive ions, 0,1-0.2% v/v for formic acid and ammonium hydroxide used for the detection of negative ions. In all measurements the cationization agent was added post-column to the eluents.
2.3. Derivatization of Polyols
When the underivatized polyol compounds are ionized by electrospray, the charge state of the observed ions depends on the number of cations attached to the analyte molecule which in turn depends on the MM of the molecule, as mentioned in section 1.2.1. The polyol samples that were investigated have an average MM of 3000-4000 mass units, thus, the formation of multiple charge states is inevitable which makes the interpretation of the mass spectra more challenging.
After a brief search in literature 42, 43, 44 we found some applications where polymers
Page | 28 polyols we used a simple procedure where phthalic anhydride was used as the
reagent10, 48 . Seven steps were required before the sample was ready to be analyzed.
The steps are the following:
a. Weight 0.1-0.4 g of the polyol sample (not diluted) into a 2 ml vial.
b. Add 1 mL of the reagent mix and homogenize at least for one minute using
a Vortex. The composition of the reaction mix is shown in table 3.
c. 30 minutes at 90 degrees Celsius
d. Cool down to room temperature
e. Dilute 100 times in acetonitrile: water (70%:30% v/v)
f. 30 minutes at 55 degrees Celsius
g. Cool down to room temperature
h. Inject
For the preparation of 20mL of the reaction mix in acetonitrile we used:
mMol g/mol mg weighted
Phthalic Anhydride
1 M 20 148.12 2962
DABCO 0.6 M 12 112.17 1346
Imidazole 0.3 M 6 68.08 408
Table 1: List of reagents that were used and weighted amounts for the preparation of 20mL of the reaction mix in acetonitrile.45
Phthalic anhydride contains two acyl groups bonded to the same oxygen atom. As shown in the schematics below, phthalic anhydride reacts with the hydroxyl groups of the polyol to afford an ester. In most cases, a basic compound as pyridine or imidazole
is added to speed up the esterification.10
If the polyol to be derivatized is a diol the reaction that takes place is the following:
Page | 29 In order to make sure that the derivatization will be completed and all the polyol material will be derivatized the excess of the reaction mix should be at least 3 to 5 times higher compared to the concentration of the polyol.
For the derivatization procedure the mixing was made by means of shaking the sample by hand for several minutes. This proved to be insufficient since a lot of underivatized material was detected when they were injected to the TOF MS. Consequently, for the next derivatizations a Vortex was used for at least a minute, which allowed better mixing.
2.4. Instruments
IM-MS and online LC-IM-MS measurements were performed on an Acquity UPLC system connected to the Synapt G2 HDMS (Waters, Manchester UK).
2.4.1. HPLC Chromatography
Reverse Phase (RP) separation was conducted using either a Synergi 2.5μm MAX-RP 100Å reverse phase C12 column (50x2.00 mm) or a 3μm Luna 100Å reverse phase C18 column (100x2.00 mm) or a 2.6μm Kinetex 100Å reverse phase C8 column (50x2.10 mm), (Phenomenex, UK). The column temperature was set at 40°C. Solvent A was either pure water or water with 0.1% v/v formic acid, solvent B was either methanol (MeOH) or acetonitrile (ACN). The flow rate was 300μL/min. Gradient elution was used with eluent B starting between 50% and 80% up to 100% in 20-30min. The injection volume was set to 1μL. Additionally, a solution of methanol: water (80%: 20% v/v) with 10-30mmole of ammonium acetate or ammonium formate was added post-column by
infusion.
Ion Exchange Chromatography (IEC) was conducted using an Agilent BioHPLC column Agilent PL-SAX 8μm 150x46 mm 1000Å (strong anion
exchange). The column temperature was set at 40°C. Solvent A was acetonitrile (ACN): water (70:30 %v/v) and solvent B was acetonitrile (ACN): water
(70:30 %v/v) with 50mmole ammonium formate. The flow rate was 300μL/min. Gradient (exponential) elution was used with eluent A starting at 98% and eluent B at 2% up to 100% in 20-30min. The injection volume was set to 1μL. Additionally, a solution of acetonitrile: water (70%: 30% v/v) with 0.1% of
Page | 30 Conditioning of the column is critical in IEC, thus, after the end of the
gradient, extra 10 minutes of flushing with solvent B and after that another 10 minutes of flushing with solvent A in order to have the exact same conditions for every sample.
The optimum parameters for programming the gradient are shown in Table 4:
Time Flow (mL/min) %A solvent %B solvent
0 0.3 98 2 0.2 0.3 98 2 4 0.3 94 6 7 0.3 87 13 10.3 0.3 70 30 13.1 0.3 40 60 14.5 0.3 0 100 20 0.3 0 100 21 0.3 98 2 33 0.3 98 2
Table 2 : List of optimum parameters for programming the exponential gradient in Ion exchange chromatography
2.4.2. Ion Mobility-Mass Spectrometry
Experiments were carried out on the Synapt G2 HDMS using an electrospray ionization source. The Synapt G2 HDMS configuration includes a quadrupole-ion mobility-time of flight Q-IMS-TOF combinatquadrupole-ion which allows within a single run the separation of ions according to their collision cross section Ω as well as their m/z values. The ion mobility cell, called TriWAVE as described in 1.3.2. section, is composed of three consecutive travelling-wave (TW) cells, called trap, IMS cell and transfer, respectively. The trap and transfer are filled with helium with a flow of 3mL/min, whereas the IMS cell is filled with nitrogen with a flow of 90mL/min.
Wave parameters, called IMS wave height WH and wave velocity s, can be independently controlled in all three cells. Prior to the IMS cell there is a small-sized cell filled with helium at a flow rate of 180 mL/min.
The system was used in both positive and negative ionization electrospray mode (ESI) with “resolution” set to V-mode (M/ΔM 20.000) in the range of 50-5000Da. The scan time was set at 1 scan per second. For the m/z calibration a solution of sodium iodide (NaI) was used at 2μL/mL. A solution of leucine enkephaline at 2ng/mL was used for the lock mass signal. For the CCS
calibration of ion mobility a solution of polyalanine-acetaminophen at 2ng/mL was used. Mass spectrometric settings and Ion mobility settings were varied. Table 5 shows a summary of the optimized parameters for the ionization source and the mobility cell in order to obtain a higher sensitivity and resolving power.
Page | 31
ESI TriWave Fluidics
Source Trap Sample Flow
Control Capillary (kV) 2 Wave velocity (m/s) 311 Infusion Flow Rate 10μl/mL Sampling Cone
40 Wave height 40 Flow State Combined
Source offset 80 IMS Lockspray Temperature Wave velocity (m/s) 450 Lockspray Capillary Control (kV) 2.00
Source 120 Wave height 40
Desolvation 400 Lockspray
Flow Control
Transfer Infusion Flow
Rate
10μl/mL
Gas Flows Wave
velocity (m/s)
380 Flow State Infusion
Cone Gas (L/h) 0 Wave height 40 Des0lvation Gas (L/h) 450 Nebulizer (Bar) 6.5
Table 3 : Optimized parameters for ESI and TWIMS-MS Analysis of Polyol Samples
2.5. Data Analysis
The mass spectra were recorded and analyzed using MassLynx version 4.1. Then, they were exported in txt format. The txt file was read by Polymerix software (Sierra Analytics, USA) which is used for characterizing homopolymer and copolymer molecular mass distributions as well as for mixture deconvolution. Mobility data were acquired and interpreted using DriftScope 2.1. The automatic detection of peaks tool of DriftScope, was used for peak detection with a minimal intensity of 20 counts and a mass resolution of 20000. The detected peaks were exported in txt format and were processed using Polymerix software. The exported results from Polymerix were exported to an excel file for further analysis.
Figure 2-1 shows a screenshot of the “Peak Detection tool” Window of DriftScope as well as the optimum parameters used for peak detection.
Page | 32
Figure 2-1 : Screenshot of the “Peak Detection tool” Window in DriftScope
3. R
ESULTS AND
D
ISCUSSION
A series of 22 different polyol samples marked as PPG2000 standard, A20-A34, J, K, M, P, G800 and PEG300 (Table 6) was used for developing an analytical method that is suitable for polyol characterization. As a start, all standard samples and samples produced by Shell
Page | 33 or competitors were measured using settings from previous conducted and reported
research at Shell. 15
Name Type/MM Functionality EO present Unsaturates present
Side product diol present
A20 PPG2000(S) Only diol NO NO NO
A21 G3000(S) Only triol NO NO NO
A22 G3000(S) Triol+Diol NO NO NO
A23 G3000(S) Triol+Diol YES NO NO
A24 G3500(S) Triol+Diol YES NO NO
A25 PPG4000(S) Only diol NO NO NO
A26 PPG3500(S)+
EO-tip(K)
Only diol NO NO NO
A27 PPG2000(K) Only diol NO YES YES
A28 PPG1000(K) Only diol NO YES YES
A29 PPG400(K) Only diol NO YES YES
A30 G3000(K) Triol NO YES YES
A31 G3000(K) Triol YES YES YES
A32 G5000(K) Triol YES YES YES
A33 G5000(K) Triol YES YES YES
A34 G600 Triol YES YES YES
J Triol+Diol YES YES YES
K Triol+Diol YES YES YES
M Triol+Diol YES YES YES
P Triol+Diol YES YES YES
G800 G800 Only triol NO NO NO
PEG300 PEG300 ONLY EO NO PO NO NO
PPG2000 standard
PPG2000 Only diol NO NO NO
Table 4 : List of polyol samples
Direct infusion experiments (DI-MS) of the polyol samples and PPG2000 standard in both ESI (+) and ESI (-) were performed. Figure 3-1 and Figure 3-1 (A) show the accumulated mass spectrum of PPG2000 standard (polyol with two hydroxyl groups) in positive mode (bottom, red) and the derivatized PPG2000 standard in negative mode (top, green). The main peaks are separated by 58 mass units, which correspond to a PO unit. The intensity of the observed signal is strong in both modes. However, in positive mode we observe multiple charge states. In the case of the standard polyol PPG2000 the mass
spectrum is relatively easy to interpret despite the presence of multiple charges (1+ -4+).
When observing the mass spectrum of the derivatized PPG2000 standard the mass spectrum is quite simplified. The majority of the observed ions are doubly charged.
Page | 34 This can be explained by the fact that the PPG2000 has two hydroxyl groups that have been
derivatized using phthalic anhydride 10. Measuring in negative ESI mode, ions are formed
by removal of a proton, thus, the loss of two protons forms a doubly negatively charged polyol.
Figure 3-1 : DI-MS mass spectra of PPG2000 standard in positive ESI mode (bottom, red) and the derivatized dPPG2000 standard in negative ESI mode (top, green). The ESI (+) spectrum shows a distribution of 1+-4+ ions.
Page | 35
Figure 3-1 (A): A magnification of the DI-MS mass spectra of PPG2000 standard in positive ESI mode (bottom, red) and the derivatized dPPG2000 standard in negative ESI mode (top, green). The horizontal axis shows all the mass peaks detected in the sample (m/z) and the vertical axis represents the intensity of the detected peak.
Figure 3-2 and Figure 3-2 (B) show the accumulated spectrum of polyol sample A24 in positive mode (bottom red) and the derivatized A24 in negative mode (top, green). Again, in positive mode we observe multiple charge states due to the high MM of the polyol sample. In addition, the mass spectrum is quite complex and there is significant overlapping which makes the interpretation rather challenging. On the other hand, the mass spectrum of the derivatized A24 is somehow complex due to the presence of EO in the sample, though the observed ions are limited to doubly and triply charge states.
Page | 36
Figure 3-2 : DI-MS mass spectra of polyol sample A24 in positive ESI mode (bottom, red) and the derivatized dA24 in negative ESI mode (top, green). The ESI (+) spectrum shows a distribution of 1+-3+ ions and the mass
spectrum is quite complex with no way of distinguishing the components of the sample. The ESI (-) shows only the triple and double charged ions.
Page | 37
Figure 3-2 (B) : A magnification of the DI-MS mass spectra of polyol sample A24 in positive ESI mode (bottom, red) and the derivatized dA24 in negative ESI mode (top, green). The horizontal axis shows all the mass peaks detected in the sample (m/z) and the vertical axis represents the intensity of the detected peak.
Page | 38
3.1. Chromatographic Separation- Reverse Phase Chromatography (RP
HPLC-ESI MS positive mode) without Ion Mobility
In the early stages of research, the organic solvent that was used for the RP LC
separation was acetonitrile combined with a 3μm C18 RP column. For the optimization of the LC gradient and the column selection, polyol sample A24 was used as well as PPG2000 standard. The average MM of PPG2000 standard is easily comparable to that of the polyol samples, and thus the polyol sample A24. Consequently, they are expected to behave in a similar way with regards to multiple charging,
The gradient that was used started at 75% acetonitrile/25% milliQ water (+ 10mmole ammonium formate) up to 100% acetonitrile in 20 minutes with additional extra 7 minutes of flushing with 100% acetonitrile. In that case, the first components of the polyol sample A24 start to elute after 5 minutes. The high MM polyols, though, do not elute even with the additional 7 minutes of flushing with the acetonitrile or by modifying the starting point of the gradient. A possible explanation for this
phenomenon might be related to the solvation of the high-MM polyols. Acetonitrile (ACN) is an aprotic solvent, so its affinity with the ether bonds in PO/EO polymers is possibly lower than that of methanol (MeOH). The efficiency on a C18 column with ACN/water gradient is better than for MeOH/water gradient when looking at the lower-MM polyols. However, for 2000 g/mol of polyol material and upwards it is found
that with ACN not all polyols elute. 12
Since acetonitrile (ACN) proved to be unable to provide successful separation of the polyol sample A24, methanol (MeOH) was used instead. Additionally, the analysis was performed using a 2.6μm C8 RP column which offers less retention against the non-polar polyols and it is expected that every single component would be sufficiently separated and fully eluted from the column by the end of the run.
In Figure 3-3 and Figure 3-3 (A) we can see the performance of the C8 column under the same optimized conditions as the C18 column for polyol A24. The gradient starting point is set at 75% MeOH using the C8 column. The separation could be considered acceptable and every single component elutes from the column before the end of the run.
Page | 39
Figure 3-2 : TIC Chromatogram and Contour plot of a RP LC-MS analysis of polyol A24 using a 2.6μm
50x2.10mm C8 RP column and with the gradient starting at 75% methanol up to 100% in 20 minutes with extra 7 minutes of flushing with methanol. The horizontal axis is the retention time (minutes) of the RP LC separation. The vertical axis is the m/z values as obtained from the ESI (+) mode. The color of the contour plots represents the intensity of the signal obtained.
Page | 40
Figure 3-3 (A) : A close-up image of the contour plot from Figure 3-3. The horizontal axis is the retention time (minutes) of the RP LC separation. The vertical axis is the m/z values as obtained from the ESI (+) mode.
In Figure 3-4 and Figure 3-4 (B) we can see the performance of the C8 column for PPG2000 standard under the same optimized conditions as for polyol A24. The
gradient starting point is set at 75% MeOH using the C8 column. We can observe up to
5+ charge states, with 1+, 2+ and 3+ being more distinct, of the PPG2000 standard as well
as some monols with allyl endgroups. The contour plot is significantly less complex than that of the polyol A24 since it is a standard material that is made up of only one repeat unit (PO).
Page | 41
Figure 3-4 : Contour plot of a RP LC-MS analysis of PPG2000 standard using a 2.6μm 50x2.10mm C8 RP column and with the gradient starting at 75% methanol up to 100% in 20 minutes with extra 7 minutes of flushing with methanol. The horizontal axis is the retention time (minutes) of the RP LC separation. The vertical axis is the m/z values as obtained from the ESI (+) mode. The color of the contour plots represents the intensity of the signal obtained.
Page | 42
Figure 3-4 (B) : A magnified section of the contour plot from Figure 3-4. The horizontal axis represents the retention time (minutes) of the RP LC separation. The vertical axis shows the m/z values as obtained from the ESI (+) mode.
3.2. Chromatographic Separation- Reverse Phase Chromatography (RP
HPLC-ESI MS positive mode) with Ion Mobility (IMS)
As demonstrated in 3.1., the chromatographic separation of polyols was greatly improved, especially for the low MM monols and diols when using the 2.6μm
50x2.10mm C8 RP column with MeOH as the solvent gradient and the gradient starting point set at 75% MeOH. For the high MM diols and triols that elute much later, it proved to be insufficient as demonstrated in figure 3-3. Consequently, adding a second separation dimension after the LC separation was required in order to improve the separation of the higher MM diols and triols. The SYNAPT G2 HDMS by Waters comes
with an Ion Mobility Cell situated in front of the time-of-flight mass analyzer,46 which
in our case serves as a second separation dimension for the polyols.
In figure 3-5 (top), we can see the 2D plot of PPG2000 standard. The horizontal axis shows the retention time in minutes and represents the RP LC separation, using the C8 RP column with the optimized gradient. The vertical axis shows the drift time in milliseconds and represents the ion mobility separation. PPG2000 standard is separated in the first dimension based on its polarity and in the second dimension based on its size-to-charge ratio. After the ions exit the drift tube they are sent to the TOF analyzer where they are separated based on their mass-to-charge ratios. Thus, the accumulated mass spectrum is shown in the bottom of Figure 3-5. Table 12 (Appendix A) shows a list of the accumulated masses and the assignment of them from the recorded spectrum of figure 3-5, (bottom) after data processing with Polymerix software.
Again, the main peaks are separated by 58 mass units, which correspond to a PO unit. With the addition of the ion mobility as a second separation dimension we can observe
up to 6+ charge states of the PPG2000 standard as well as some monols with allyl
endgroups. Moreover, we witness that the singly charged species of the polyol extend much further than the drift time range of the second dimension, thus, we miss
Page | 43 important information. Consequently, adjustment of the Travelling Wave Velocity (the velocity which the ions obtain when they enter the drift tube) is required.
Figure 3-5 : Contour plot of a RP LC-IM-MS analysis of PPG2000 standard. The horizontal axis is the retention time (minutes) of the RP LC separation (based on MM) and the vertical axis is the drift time (milliseconds) of the IM separation (based on size-to-charge ratio). The 2D-plot shows a distribution of 1+-6+ ions. The color of
Page | 44
Figure 3-5 : (Middle): TIC chromatogram of PPG2000 standard in ESI (+).The horizontal axis represents the retention time (minutes) and the vertical axis represents the recorded intensity of the Total Ion Current (TIC) chromatogram. (Bottom): Accumulated MS spectrum of PPG2000 standard in ESI (+).The horizontal axis shows all the mass peaks detected in the sample (m/z) and the vertical axis represents the intensity of the detected peak.
We know thus far that PPG2000 standard has an average MM of 2000, while the actual polyols have an average MM that reaches up to 3500. Additionally, the sample does not contain a single repeat unit as it was demonstrated from the RP LC-MS analysis of polyol A24 in figure 3-3. So, it is only logical that the 2D-plot of the RP LC-IM-MS analysis of sample A24 will be much more complex with multiple charge states that will
Page | 45 actually test the boundaries of the ion mobility with regards to the multiple charge states. Figure 3-6 shows the contour plot, TIC chromatogram and accumulated mass spectrum of polyol A24.
-Figure 3-6 : (Top): Contour plot of a RP LC-IM-MS analysis of polyol A24. The horizontal axis is the retention time (minutes) of the RP LC separation (based on polarity) and the vertical axis is the drift time
(milliseconds) of the IM separation (based on size-to-charge ratio). The 2D-plot shows a distribution of 1+-4+
Page | 46
Figure 3-6 : (Middle): TIC chromatogram of polyol A24 in ESI (+).The horizontal axis represents the retention time (minutes) and the vertical axis represents the recorded intensity of the Total Ion Current (TIC) chromatogram. (Bottom): Accumulated MS spectrum of polyol A24 in ESI (+). The horizontal axis shows all the mass peaks detected in the sample (m/z) and the vertical axis represents the intensity of the detected peak.
As we can see the low MM monols and part of the diols are separated in the first chromatographic dimension (RP LC separation). Additionally, we can observe most of the main peaks which are separated by 58 mass units and correspond to a PO unit (red
Page | 47 arrows). On the other hand, in the same mobilogram we can distinguish the different charge states which ion mobility provides in the second chromatographic dimension (Ion Mobility separation).
Moreover, since polyol sample A24 also contains EO units (44 mass unit difference) ion mobility can separate them from the PO units in the second chromatographic
separation (yellow arrows). But with the higher MM diols and all the triols, ion mobility proves to be insufficient to separate the multiple charge states and the EO units, thus, the interpretation of the accumulated mass spectrum becomes very difficult.
Finally, with regards to instrumental equipment and software the addition of ion mobility to the chromatographic separation and measuring in ESI (+) added extra information which in turn produced large data files (3-4 Gb) for a single run. The DriftScope 2.1 software used for the data interpretation required a significant amount of time to load and process the data and even in some occasions was unable to process them by giving errors or “crashing” the whole system of the TOF MS instrument. Taking all these observations and conclusions into account, we conclude that even with the addition of the ion mobility to the RP LC separation, measuring the polyols in ESI (+) create a lot of multiple charge states, the components cannot be easily
distinguished, especially for the high MM diols and triols. Additionally, due to peak overlapping the assignment of the polyol series becomes very challenging and at best the exported results may not be representative compared to the actual composition of the polyol mixture.
Thus, it is necessary to move on from measuring polyols in ESI (+) to ESI (-), where the produced mass spectra are less complex, once a derivatization procedure is applied, as demonstrated earlier in section 3. Searching in literature there are many applications were polyol samples have been derivatized in order to be measured with a UV detector, as mentioned in 2.3. In this case, applying a relatively simple derivatization procedure would solve the problem of the multiple charge states and would also provide extra possibilities with regards to the LC separation.
3.3. Ion Mobility-Mass Spectrometry (IM-ESI MS negative mode) of
derivatized polyols (No Chromatographic Separation)
Direct infusion experiments of the derivatized polyol samples and PPG2000 standard were performed in ESI (-). Figure 3-7 (bottom) shows the accumulated mass spectrum of the derivatized PPG2000 standard in negative mode. When observing the mass spectrum of the derivatized PPG2000 standard we see that is quite straightforward. The majority of the observed ions are doubly charged, meaning that the diol has been fully derivatized and both phthalic groups have lost a proton. The pH of the solvent that enters the electrospray source is between pH 8 and pH 8.5 due to the presence of the buffer solution of ammonium formate. In order to make sure that the pH remains basic and “force” all the phthalic groups to be completely deprotonated, a solution of
ammonium hydroxide is added post-column by infusion. However, a very small amount of singly charged ions is observed which points to the fact that only one of the two phthalic groups has lost a proton. This is likely a result of the higher “charge-to-surface” ratio of the low MM-polyol. The charge density influences significantly the final result of the mobilogram, meaning that for small molecules this ratio is higher
