analysis of multiply charged poly(ethylene oxide- co

Analytical SciencesAdvance Publication by J-STAGEReceived July 19, 2018; Accepted September 19, 2018; Published online on September 28, 2018DOI: 10.2116/analsci.18P332

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Original Papers

Analysis of Multiply Charged Poly(Ethylene Oxide-co-Propylene

Oxide) Using Electrospray Ionization-Ion Mobility Spectrometry-Mass

Spectrometry

Kanako ITO,* Shinya KITAGAWA,*† and Hajime OHTANI*

* Department of Life Science and Applied Chemistry, Graduate School of Engineering, Nagoya

Institute of Technology, Gokiso, Showa, Nagoya 466-8555, Japan

† To whom correspondence should be addressed.

E-mail: [email protected]


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Abstract

Poly(ethylene oxide), poly(propylene oxide), and their copolymer (poly(EO-co-PO)) were

analyzed by electrospray ionization-ion mobility spectrometry-mass spectrometry

(ESI-IMS-MS). ESI produced multiply charged analytes of 2 to 5 Na+ additions, and they were

separately observed in a 2D map of m/z value vs. drift time. The collision cross-section of the

analyte polymers was almost linearly proportional to (molecular weight)0.644, except for the

analytes with 2Na+ addition; a nonlinear relation called “folding” was significantly observed for

the analytes with 2Na+ addition. An increase in electrostatic repulsion, because of the increase in

Na+ addition, suppressed the folding of the polymer. Analyses of poly(EO-co-PO) with different

EO compositions revealed that the copolymer with high EO composition tended to show folding.

The separation of highly multiple charged poly(EO-co-PO)s with different EO contents by

ESI-IMS-MS was successfully demonstrated.

Keywords: multiple charges, copolymer, electrospray ionization, ion mobility, mass

spectrometry, folding, polymer composition.


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Introduction

Ion mobility spectrometry (IMS) is a method for separating ions having the same

mass-to-charge (m/z) ratio but different collision cross-sections (CCS) and/or charge numbers

based on the difference in ion mobility (or drift time, tD) in a thin gaseous atmosphere,

analogous to gel-electrophoresis in the liquid phase.1-6 IMS is often combined with MS for

determining both tD and m/z values in the gas phase, and this technique is known as ion mobility

spectrometry-mass spectrometry (IMS-MS). IMS-MS is frequently used for structural

characterization and analysis of the conformational dynamics of various compounds.

Fundamental studies and applications of IMS-MS are frequency reported for various

compounds form small compounds to proteins.2-5,7-9 In the case of synthetic polymer, in 1995,

von Helden et al. reported a study of IMS behavior of poly(ethylene oxide) (PEO,

polymerization degree, n = 5 to 19) of single Na+ addition generated by matrix-assisted laser

desorption ionization (MALDI).9 Further applications of IMS-MS to synthetic polymers have

also been reported and most previous studies on synthetic polymers using IMS-MS have

focused on homopolymers and their mixtures.10-19

Meanwhile, copolymers are polymerized from two or more monomer species to control its

characteristics, and used generally in various engineering products. In a case of copolymer,

molecules having the same or approximately the same molecular weight but different

compositions and/or arrangements are generally synthesized. Therefore, copolymer analysis in

MS is often difficult to interpret due to complexity of the mass spectrum. To solve this problem,

we focused on a potential of IMS-MS for analyses of copolymers. If the CCS of the monomers

(CSS per unit mass, strictly) used for synthesis of the copolymer is not the same, thus the CCS

of the copolymer depends on its composition even though their m/z values are the same. In

addition, when the copolymer composition and arrangement affect the conformation (or CCS) in


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a gaseous phase, the difference in the composition/arrangement can be identified in IMS.

Furthermore, if the affinity to an attaching ion depends on the monomer unit species, thus the

copolymer composition affects the charge number, which can be identified by IMS. If the

charge density attached on a polymer affects the intramolecular interaction electrostatically, thus

the CCS of the polymer is varied. Therefore, it is concluded that IMS-MS has a potential for the

composition analysis of copolymers. However, the study of IMS-MS for copolymers is

insufficient as described above.

In this research, PEO, poly(propylene oxide) (PPO), and copolymers of poly(ethylene

oxide-co-propylene oxide) (poly(EO-co-PO)) were analyzed in IMS-MS for investigating an

effect of the charge density on CCS. Two types of triblock copolymers (PEO-b-PPO-b-PEO and

PPO-b-PEO-b-PPO) and a random copolymer (poly(EO-r-PO)) were analyzed, and effect of the

composition, or EO unit content, on IMS behavior was investigated. In addition to the

fundamental study, the composition-based separation of poly(EO-co-PO)s in IMS-MS was

demonstrated.

Experimental

Reagents

PEO (average molecular weight (Mw) = 2000, Shodex, Tokyo, Japan), PPO (Mw = 2000,

Wako, Osaka, Japan), poly(EO-r-PO) (Mw = 2500, EO unit 70wt%, Sigma-Aldrich, St. Louis,

MO), PEO-b-PPO-b-PEO (Mw = 2000, EO unit 10wt%, Sigma-Aldrich), and

PPO-b-PEO-b-PPO (Mw = 2000, EO unit 50wt%, Sigma-Aldrich) were used as analytes. The

polymers were dissolved (0.1 mg/mL each) in the mixture of acetonitrile/water (50/50, v/v)

containing 0.01% sodium trifluoroacetate (Wako).


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Apparatus and procedure

Waters Synapt G2 HDMS (Waters, Milford, MA) equipped with an ESI source was used

for analyses in the positive mode (capillary voltage: 3.0 kV, sampling cone voltage: 40 V). This

apparatus has a T-wave type IMS function (TWIMS). The sample solutions were introduced into

the ESI source via direct infusion at a flow rate of 20 µL/min using a microsyringe pump (11

Plus, Harvard Apparatus, Holliston, MA). The basic parameters for TWIMS were as follows:

bias voltage, 50 V; wave height, 40 V; start velocity, 800 m/s; end velocity, 1000 m/s; and

recorded m/z range, 50-2000. For the analysis of the mixture of the poly(EO-co-PO)s, the

parameter set 36 V, 40 V, 600 m/s, 1500 m/s, and 50-1200 was employed.

Results and Discussion

Analysis of PEO and PPO mixture by ESI-IMS-MS

Prior to the analysis of poly(EO-co-PO), a mixture of PEO and PPO was analyzed using

ESI-IMS-MS, and the result is shown in Fig. 1. Figure 1A shows a two-dimensional tD - m/z

distribution, where the signal intensity is presented as a heat map. As seen in this figure, several

groups could be observed in the 2D distribution when a mixture of PEO and PPO was analyzed.

The extracted mass spectrum of area #1 in Fig. 1A is shown in Fig. 1B. The mass spectrum

indicates area #1 including the signals of PEO and PPO with various Na+ additions,

[PEO+3Na]3+, [PEO+4Na]4+, [PEO+5Na]5+, [PPO+2Na]2+, and [PPO+3Na]3+. Here, the

charge number (z value) was identified from the difference of m/z value between the isotopic

peaks, and the polymerization degree and adduct ions species were identified by the exact mass

value. The other areas (#2 - #6) in Fig. 1A are also identified from the extracted mass spectra. It

was clear that various kinds of ion adducts were formed by ESI and partially separated by IMS.

As shown in Fig. 1A, both Na+ and H+ addition to the polymer were observed in zones #4, #5,


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and #6. However, for simplified discussion, we focused only on the analytes with Na+ addition

in this paper.

Figure 2A shows the relationship between molecular weight of polymer (m/z × z - mNa × z,

where mNa is a mass of Na cation) and drift time, without information about the signal intensity.

As clearly shown in Fig. 2A, the drift time increased with an increase in the molecular weight,

or polymer chain length. In addition, the increase in z reduced the tD, and a curved relationship

between the molecular weight and tD was observed in each series.

The CCS value, , in TWIMS is given by the following equation:20

= +.

(1)

where e, mi, mN, kb, T, P, N, A, and B are the elementary charge, mass of the analyte, mass of the

collision gas, Boltzmann constant, temperature of the gas, buffer gas pressure, number density

of the gas, correction factor for the electric field parameters, and compensating factor for the

non-linear effect of the TWIMS device, respectively. Under certain experimental conditions, eq.

1 is transformed into eq. 2.

= +(2)

where A’ is a constant depending on the experimental conditions.

=.

(3)

In our system, the B value was empirically estimated to be as 0.644 based on the analysis of

polyaniline. Therefore, the value proportional to the CCS (relative CCS defined as ’) is given

by the following equation.

+ .

(4)


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The relationships between ’ and the molecular weight value of PEO/PPO are shown in Fig. 2B.

A nearly linear relationship between ’ and the molecular weight of the polymers is seen, i.e.,

the CCS is proportional to molecular weight of PEO/PPO, except [PEO+2Na]2+ and

[PPO+2Na]2+. For [PEO+2Na]2+ and [PPO+2Na]2+, nonlinear relationships are clearly observed

(a slightly curved relationship is also seen for [PEO+3Na]3+ in the higher molecular weight

region). This nonlinear relationship between molecular weight and CCS is referred to as

“folding”, which is often observed in the analysis of polymers by IMS.11,21-23 The folding

behavior is discussed in further detail in the next section.

As shown in Fig. 2B, the ’ values for 3, 4, and 5 Na+ addition were approximately the

same for the same molecular weight for each polymer. That is, the charge number, or number of

Na+ additions, did not affect significantly the polymer morphology when the molecular weight

of the polymer was the same. On the other hand, the ’ value of the analytes with 2Na+ addition

was significantly small in the region of molecular weight > 1500 due to folding, whereas the ’

value for molecular weight < 1500 would be the same as that for 3 to 5 Na+ additions. Figure 2C

depicts the relationship between the polymerization degree and ’. The ’ value for PPO is

larger than that for PEO at the same n. The side methyl groups in PPO enhanced the molecular

volume and/or suppressed the intramolecular interaction of the PPO main chain.

Effect of charge number on CCS

Winter et al. reported that folding caused by the extension of the chain length during the

analysis of polylactide by IMS.22 As shown in Fig. 2, folding of both PEO and PPO was

observed for their 2Na+ adducts. However, there was no notable folding in the cases of 3 to 5

Na+ additions. Since the morphology of multiply charged polymers in the gaseous state will be

affected by electrostatic interactions,22 the charge density of PEO and PPO was evaluated. The

average chain length per unit Na+ addition, n/z, was calculated as a measure of charge density.


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For example, 2Na+ addition was observed for PEO with the polymerization degree ranging from

24 to 50, and the n/z value was calculated as 12 to 25.

Table 1 shows the n/z values of PEO and PPO for 2, 3, and 4 Na+ additions. Moreover,

folding is cited in terms of three magnitudes (folding, slight folding, and no folding). The n/z

value for the folding region is also determined, as summarized in Table 1. In the case of

[PEO+2Na]2+, folding was observed for n = 32 - 50, as shown in Fig. 2C, and the n/z ranged

from 16 to 25. As clearly shown in Table 1, the charge density on the polymer increased with an

increase in the z value, i.e., a higher z value produced a lower n/z value. Interestingly, folding

was observed in the region with a relatively large n/z, or in the low charge density region (n/z

range: 15 to 25). This phenomenon suggested that a high charge density (n/z < 15) would result

in the electrostatic repulsions, thus preventing folding. As shown in Fig. 2C and Table 1, the

folding effect was more clearly observed in PEO rather than in PPO, suggesting that the methyl

side group in PPO inhibits the intramolecular interaction to induce folding.

Analysis of poly(EO-co-PO) by ESI-IMS-MS

In this section, we describe the analysis of different types of poly(EO-co-PO) using

ESI-IMS-MS. Figure 3A shows the typical results for PEO-b-PPO-b-PEO (EO unit 10wt%) in a

2D heat map. The extracted mass spectrum for zone #1 in Fig. 3A is shown in Fig. 3B. Similar

to Fig. 1A, the charge number was identified from the m/z difference between the isotope peaks

and both the additive type and the polymer composition were identified from the exact mass

value. In zone #1, the copolymers corresponding to 2, 3, and 4 Na+ additions were observed and

marked by diamonds, circles, and triangles, respectively. The number of EO and PO units were

described for each peak as [NEO, NPO], e.g. [5, 23] and [3, 25] for m/z = 547.3564 and 837.5098,

respectively.

Figure 4A illustrates the relationship between ’ and the molecular weight of


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PEO-b-PPO-b-PEO with 2, 3, and 4 Na+ additions. Slight folding was observed only for 2Na+

addition, similar to Fig. 2A. The copolymers of PPO-b-PEO-b-PPO (EO unit 50wt%) and

poly(EO-r-PO) (EO unit 70wt%) also analyzed by ESI-IMS-MS, and their results for 2 to 4 Na+

additions are shown in Figs. 4B and 4C, respectively. Similar to the case of the PEO and PPO

homopolymers shown in Fig. 2, the increase in z, or Na+ addition, inhibited the folding of the

polymer. The average chain length per unit Na+ addition, n/z, was calculated as a measure of the

charge density, akin to both PEO and PPO. Since the analyte is a copolymer, the sum of the

number of EO and PO units is used to calculate the n/z value.

As shown in Table 1, the increase in z led to an increase in the charge density on the

polymer, similar to the case of PEO and PPO homopolymers. The folding effect was not

observed for the analytes with 4Na+ addition due to electrostatic repulsion. In the case of 2Na+

addition, the folding effect was observed for all copolymers. Interestingly, the folding effect was

notable in the order of poly(EO-r-PO), PPO-b-PEO-b-PPO, and PEO-b-PPO-b-PEO. This order

was consistent with that for the EO content in the copolymers. Therefore, the folding behavior

of poly(EO-co-PO) copolymer depends on its EO unit content. At the same molecular weight,

the polymer chain length, or total monomer unit number, increased with the increase in EO

content, because mass of EO is smaller than that of PO. The decrease in the charge density due

to the increased chain length will be effective for folding.

Composition-based separation of poly(EO-co-PO) by ESI-IMS-MS

At the same molecular weight, the drift time for PEO with 3Na+ addition was almost the

same as that for PPO, as shown in Fig. 2A. In the case of 2Na+ addition, the difference in drift

time between PEO and PPO was found only in the region with the folding phenomenon. On the

other hand, in the case of 4Na+ addition, the drift time for PEO was slightly larger than that for

PPO in entire region. That is, the drift time of PEO was different from that of PPO under high


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Na+ addition conditions. Therefore, composition-based separation of poly(EO-co-PO)

copolymers may be achieved by ESI-IMS-MS analysis with highly charged analytes.

Figure 5 shows the analytical results for the mixture of PEO-b-PPO-b-PEO (EO unit

10wt%) and poly(EO-r-PO) (EO unit 70%) in a 2D heat map. The analytical conditions were

optimized for observing the highly charged analytes. Under these conditions (Fig. 5A), only two

zones were observed, in contrast to Fig. 1, and this will be suitable for achieving simple

separation based on the difference in polymer composition by IMS-MS. The extracted mass

spectra from regions #1 and #2 in Fig. 5A are shown in Figs. 5B and 5C, respectively. Zone #1

included the poly(EO-co-PO) of approximately 75% EO content with 4 and 5 Na+ additions and

zone #2 included that of 15-20% EO content with 3 and 4 Na+ additions. Therefore, it concluded

that zone #1 included poly(EO-r-PO) (EO unit 70wt%) while zone #2 included

PEO-b-PPO-b-PEO (EO unit 10wt%). That is, poly(EO-co-PO) copolymers with different EO

content were successfully separated by ESI-IMS-MS.

Conclusions

ESI-IMS-MS was applied to the analysis of multiply charged poly(EO-co-PO)s. The effect of

charge number on the drift time and folding behavior was similar to that reported for

homopolymers.10-19 The folding behavior was dominated by the charge density of the multiply

charged polymer. The EO content in the copolymer affected the folding behavior, that is, higher

EO content resulted in a more notable folding effect. When the molecular weight was the same,

in the case of poly(EO-co-PO), the EO content dominated the polymer main chain length or

polymerization degree, that is, a higher EO content resulted in a longer main chain and the

decrease in the charge density. The difference in the main chain length or charge density would


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be the key factor influencing the folding behavior of multiply charged polymers.

In addition, the side chain also affected the CCS of the polymer. It is reasonable that the

CCS of PPO is larger than that of PEO when the polymerization degree is the same, as shown in

Fig. 2C. On the other hand, when the molecular weight is the same, the CCSs of PEO and PPO

depended on the charge number, as shown in Fig. 2B. In the case of z = 4, the CCS for PEO is

slightly larger than that for PPO. This behavior was exploited for the successful

composition-based separation of poly(EO-co-PO)s, as demonstrated in Fig. 5.

In the case of poly(EO-co-PO), the differences in structure, molecular weight, and

chemical property between EO and PO are not very large. When the differences between the

properties of the monomer units are significant, composition-based separation by ESI-IMS-MS

will become easy. Further investigation in this regard is ongoing in our laboratory.

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Table 1 Average chain length per unit Na+, n/z, for PEO, PPO, and poly(EO-co-PO)a.

Polymer 2Na+ 3Na+ 4Na+ PEO 12 ~ 25

FDb (16 ~ 25) 8 ~ 19 SFDb (15 ~ 19)

8 ~ 15 NFDb

PPO 11 ~ 18 SFD (15 ~ 18)

6 ~ 17 NFD

8 ~ 13 NFD

poly(EO-r-PO) (EO unit 70wt%) 12 ~ 23 FD (18 ~ 23)

13 ~ 22 FD (17 ~ 22)

11 ~ 19 NFD

PPO-b-PEO-b-PPO (EO unit 50wt%) 12 ~ 24 FD (16 ~ 24)

9 ~ 19 SFD (16 ~ 19)

8 ~ 13 NFD

PEO-b-PPO-b-PEO (EO unit 10wt%) 12 ~ 22 SFD (16 ~ 24)

9 ~ 16 NFD

8 ~ 12 NFD

a. In the case of copolymers, the sum of the number of EO and PO units was used for

calculating the n/z value.

b. FD: folding, SFD: slight folding, NFD: no folding


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Figure Captions

Fig. 1 Analysis of PEO and PPO mixture by ESI-IMS-MS. 2D heat map of the m/z value and

drift time is shown in (A) and the extracted mass spectrum of zone #1 is shown in (B). The term

n in (B) is polymerization degree of polymers.

Fig. 2 Effect of molecular weight on (A) drift time and (B) relative CCS, ’, for PEO (red) and

PPO (blue) with multiple Na+ additions. The relationship between polymerization degree and

relative CCS is also shown in (C).

Fig. 3 Analysis of PEO-b-PPO-b-PEO (EO unit 10wt%) by ESI-IMS-MS. The 2D heat map of

the m/z value and drift time is shown in (A), and the extracted mass spectrum of zone #1 is

shown in (B).

Fig. 4 Effect of molecular weight on relative CCS, ’, for (A) PEO-b-PPO-b-PEO (EO unit

10wt%), (B) PPO-b-PEO-b-PPO (EO unit 50wt%), and (C) poly(PEO-r-PPO) (EO unit 70wt%)

with different Na+ additions.

Fig. 5 Separation of PEO-b-PPO-b-PEO (EO unit 10wt%) and poly(EO-r-PO) (EO unit 70wt%)

by ESI-IMS-IMS. The 2D heat map of the m/z value and drift time is shown in (A), and the

extracted mass spectra of zones #1 and #2 are shown in (B) and (C), respectively.


15

Fig. 1 Analysis of PEO and PPO mixture by ESI-IMS-MS. 2D heat map of the m/z value and

drift time is shown in (A) and the extracted mass spectrum of zone #1 is shown in (B). The term

n in (B) is polymerization degree of polymers.


16

Fig. 2 Effect of molecular weight on (A) drift time and (B) relative CCS, ’, for PEO (red) and

PPO (blue) with multiple Na+ additions. The relationship between polymerization degree and

relative CCS is also shown in (C).


17

Fig. 3 Analysis of PEO-b-PPO-b-PEO (EO unit 10wt%) by ESI-IMS-MS. The 2D heat map of

the m/z value and drift time is shown in (A), and the extracted mass spectrum of zone #1 is

shown in (B).


18

Fig. 4 Effect of molecular weight on relative CCS, ’, for (A) PEO-b-PPO-b-PEO (EO unit

10wt%), (B) PPO-b-PEO-b-PPO (EO unit 50wt%), and (C) poly(PEO-r-PPO) (EO unit 70wt%)

with different Na+ additions.


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Fig. 5 Separation of PEO-b-PPO-b-PEO (EO unit 10wt%) and poly(EO-r-PO) (EO unit 70wt%)

by ESI-IMS-IMS. The 2D heat map of the m/z value and drift time is shown in (A), and the

extracted mass spectra of zones #1 and #2 are shown in (B) and (C), respectively.


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Graphical Index

analysis of multiply charged poly(ethylene oxide- co

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