Polyhedral oligomeric silsesquioxane-based hybrid monolithic columns: Recent advances in their preparation and their applications in capillary liquid chromatography

Accepted Manuscript Polyhedral oligomeric silsesquioxane-based hybrid monolithic columns: Recent advances in preparation and its applications for capillary liquid chromatography Xiaoqiang Qiao, Rui Chen, Hongyuan Yan, Shigang Shen PII:

S0165-9936(17)30228-5

DOI:

10.1016/j.trac.2017.08.006

Reference:

TRAC 14981

To appear in:

Trends in Analytical Chemistry

Received Date: 29 June 2017 Revised Date:

10 August 2017

Accepted Date: 10 August 2017

Please cite this article as: X. Qiao, R. Chen, H. Yan, S. Shen, Polyhedral oligomeric silsesquioxanebased hybrid monolithic columns: Recent advances in preparation and its applications for capillary liquid chromatography, Trends in Analytical Chemistry (2017), doi: 10.1016/j.trac.2017.08.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Polyhedral oligomeric silsesquioxane-based hybrid monolithic columns:

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Recent advances in preparation and its applications for capillary liquid

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chromatography

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Xiaoqiang Qiao a,*, Rui Chen a, Hongyuan Yan b, Shigang Shen c,**

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a

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Diagnosis, Ministry of Education, Hebei University, Baoding 071002, China

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b

College of Public Health, Hebei University, Baoding, 071002, China

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c

College of Chemistry & Environmental Science, Key Laboratory of Analytical Science and

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Technology of Hebei Province, Hebei University, Baoding 071002, China

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College of Pharmaceutical Sciences, Key Laboratory of Medicinal Chemistry and Molecular

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Corresponding author:

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* Associate Professor Dr. Xiaoqiang Qiao, College of Pharmaceutical Sciences, Key

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Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Hebei

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University, Baoding 071002, China

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Tel.: +86-312-5971107

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Fax: +86-312-5971107

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E-mail: [email protected]; [email protected]

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Additional corresponding author:

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Professor Dr. Shigang Shen, E-mail: [email protected]

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ABSTRACT As a naturally molecular level organic-silica hybrid particle, polyhedral oligomeric

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silsesquioxane (POSS) has been proved to be a good choice for preparation of new hybrid

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monolithic columns with the merits of ease of fabrication, ultrahigh column efficiency and

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excellent separation selectivity. In this review, we summarize the recently reported

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POSS-based hybrid monolithic columns, especially those reported in the recent 5 years.

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These POSS-based hybrid monolithic columns are classified based on the reactive groups of

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POSSs as well as the introduced organically functionalized reagents, emphasizing the

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preparation methods and further applications in capillary liquid chromatography.

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Keywords: Polyhedral oligomeric silsesquioxane; Hybrid monolithic column; Preparation

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method; Capillary liquid chromatography; Separation; Chromatographic application

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ACCEPTED MANUSCRIPT Contents

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1. Introduction

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2. Preparation of POSS-based hybrid monolithic columns

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3. POSS-MA or acryl-POSS-based hybrid monolithic columns

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3.1. Reaction with methyl acrylate modified organically functionalized reagents

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3.2. Reaction with acrylic ester modified organically functionalized reagents

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3.3. Reaction with vinyl modified organically functionalized reagents

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3.4. Reaction with thiol modified organically functionalized reagents

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4. VinylPOSS-based hybrid monolithic columns

5. POSS-epoxy-based hybrid monolithic columns

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6. Conclusions

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Acknowledgments

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References

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1. Introduction Monolithic columns have been reputed as the fourth generation of high performance liquid

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chromatography (HPLC) stationary phases. The porous structures of monolithic columns

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dispersed with macropores, mesopores or micropores render them possessing the merits of

5

high permeability, good mass transfer characteristic as well as high specific surface area.

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Since firstly introduced by Hjertén and Svec as a new column technology for fast separation

7

of small molecules and macromolecules in the late 1980s [1-3], many new monolithic

8

columns based on organic polymer, inorganic silica or organic-silica hybrid matrix have been

9

significantly reported [4-13]. Thereinto, organic-silica hybrid monolithic columns, which

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combine both the advantages of inorganic silica and organic polymer-based monoliths,

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including ease of preparation, excellent mechanical stability, and extensive pH range

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tolerance, were paid much attention [14-18].

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Polyhedral oligomeric silsesquioxane (POSS) is a cubic rigid silsesquioxane with the

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general formula of (RSiO1.5)n. The inherent inorganic silicon and oxygen skeleton

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surrounding by reactive organically functionalized groups render it a naturally molecular

16

level of organic-silica hybrid particle (diameter: 1-3 nm) [19,20]. In the recent 5 year, many

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new hybrid monolithic columns based on POSSs with different reactive groups were reported.

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The POSS-based hybrid monolithic columns always displayed good oxidation resistance and

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high thermal & pH stability [21]. Most interestingly, ultrahigh column efficiency (up to

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500,000 plates/m) and good suitability for separation of both macromolecules and small

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molecules were achieved, indicating the good potential for high-efficiency HPLC separation

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[22,23].

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ACCEPTED MANUSCRIPT In order to further understand the POSS-based hybrid monolithic columns, we attempt to

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provide a summary of the recently reported POSS-based hybrid monolithic columns. These

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columns are classified based on the reactive groups of POSSs as well as the introduced

4

organically functionalized reagents, emphasizing the preparation methods and further

5

applications in capillary liquid chromatography.

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2. Preparation of POSS-based hybrid monolithic columns

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For preparation of the POSS-based hybrid monolithic columns, POSSs with different

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reactive groups have been introduced. Up till now, monolithic columns based on POSS

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methacryl

substituted

(POSS-MA)

[16],

acrylopropyl

(POSS-epoxy)

[25]

POSS

or

(acryl-POSS)

polyhedral

[24],

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octaglycidyldimethylsilyl

oligomeric

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vinylsilsesquioxanes (vinylPOSS) [26,27] have been largely reported. In general, three main

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methods have been exploited to prepare POSS-based hybrid monolithic columns, as

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summarized in Table 1.

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The most commonly used method is traditional free radical copolymerization [16] based on

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POSS-MA or acryl-POSS. By introducing multiple monomers or crosslinkers, such as acrylic

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ester- [24], methyl acrylate- [28] and vinyl-containing compounds [29], POSS-based hybrid

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monolithic columns could be facilely prepared via the “one-pot” process with the presence of

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initiator and porogenic solvents. Thus, the method is very simple. Both thermally-initiated

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[16] and photo-initiated [24] polymerization methods have been exploited for preparation of

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these new monolithic columns. For the thermally-initiated polymerization, the fabrication

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temperature will largely affect the permeability and morphology of the monoliths. Thus, the

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method is temperature-sensitive. Furthermore, the preparation time is rather long and could

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ACCEPTED MANUSCRIPT reach 12 h [30]. Compared with thermally-initiated polymerization, photo-initiated

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polymerization is very fast and the preparation time can be reduced to several minutes [24].

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Moreover, the column efficiency of the POSS-based hybrid monolithic column fabricated via

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photo-initiated polymerization is higher than that via the thermally-initiated polymerization

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[31]. The main drawback of the photo-initiated polymerization is that expensive

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UV-transparent capillary is necessary for preparation of the monolithic columns.

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The second most commonly used method is thiol-based click reactions, including thiol-ene

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[27,32], thiol-methacrylate [33] and thiol-acrylate [34]. POSS-MA, acryl-POSS or

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vinylPOSS can be copolymerized with various thiol-containing compounds, such as

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1,6-hexanedithiol

(HDT)

[33],

1-octadecanethiol

(ODT)

[34],

and

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2,2’-(ethylenedioxy)diethanethiol (EDDT) [35] via this method. The thiol-based click

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reactions possess the merits of high yield, mild reaction conditions and insensitive towards

13

water and oxygen. Thus, the reactions are more easily controlled under mild conditions [36].

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Recently, thiol-epoxy click polymerization reaction was also reported for preparation of

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POSS-based hybrid monolithic columns by copolymerization of POSS-epoxy with

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multi-thiol

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trimethylolpropanetris(3-mercaptopropionate) (TPTM) [37].

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pentaerythritoltetrakis(3-mercaptopropionate)

(PTM)

or

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The third method is ring-opening polymerization reaction reported by Zou and coauthors

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by copolymerization of POSS-epoxy with diamines or polyethylenimines [25,38]. Since the

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ring-opening polymerization reaction exhibited progressively phase-separating process, the

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monolithic columns prepared via this method often displayed well-defined 3D skeletal

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microstructure. Thus, ultra-high column efficiency up to 100,000 plates/m could be achieved 6

ACCEPTED MANUSCRIPT [25], outperforming those prepared via traditional free radical polymerization [39].

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Furthermore, ring-opening polymerization reaction is rather easy to achieve even without any

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catalyst due to the strong tension of epoxide [40,41], which is obviously different with that

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via free radical copolymerization or thiol-based click reactions.

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3. POSS-MA or acryl-POSS-based hybrid monolithic columns

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POSS-MA and acryl-POSS were largely reported for preparation of POSS-based hybrid

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monolithic columns. Various organically functionalized reagents with reactive methyl

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acrylate group, acrylic ester group, vinyl group or thiol group [16,24,33,42] were introduced

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as monomers or crosslinkers. These new monolithic columns are summarized in Table 2.

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3.1. Reaction with methyl acrylate modified organically functionalized reagents Zou and coauthors developed a series of POSS-based hybrid monolithic columns by

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introduction of different methyl acrylate modified organically functionalized reagents.

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POSS-MA

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N-(2-(methacryloyloxy)ethyl)-dimethyloctadecylammonium bromide (MDOAB) to fabricate

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the first POSS-based hybrid monolithic column via thermally-initiated free radical

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copolymerization [16]. The new MDOA-POSS hybrid monolithic column could be easily

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fabricated just as the organic polymer-based monolithic columns via the “one-pot” process, as

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shown in Fig. 1. Because of the introduction of the rigid POSS silica core, the new

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MDOA-POSS column exhibited good pH (from 1 to 11) and mechanical stability.

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Furthermore, it possessed mixed-mode reversed-phase liquid chromatography (RPLC)/ anion

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exchange chromatography (AEX) retention mechanisms and the column efficiency reached

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50,000 plates/m for thiourea. The new column was firstly applied for separation of five

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firstly

combined

with

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ACCEPTED MANUSCRIPT alkylbenzenes and baseline-separation with good peak shapes was achieved. Furthermore,

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successful separation of seven protein standards (including ribonuclease B, insulin,

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cytochrome c, ovalbumin, lysozyme, bovine serum albumin (BSA) and enolase) and tryptic

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digests of BSA were also achieved, indicating the good potential of the MDOA-POSS

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column for separation of complex samples.

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Subsequently, with ethylene dimethacrylate (EDMA) or bisphenol A dimethacrylate

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(BPADMA) as the crosslinkers, poly(POSS-co-EDMA) and poly(POSS-co-BPADMA)

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hybrid monolithic columns were further prepared via the same “one-pot” method [43]. With

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five alkylbenzenes as the sample, the authors further evaluated the HPLC performance of the

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two columns. Compared with poly(POSS-co-EDMA) column, the poly(POSS-co-BPADMA)

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column displayed higher column efficiencies (23,600-27,200 plates/m) for alkylbenzenes.

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When they were further used for separation of polycyclic aromatic hydrocarbons (PAHs),

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compounds 4,4-dimethylbiphenyl and pyrene could be only baseline-separated via the

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poly(POSS-co-BPADMA) column. Thus, better separation selectivity could be achieved via

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the poly (POSS-co-BPADMA) column when it was used for separation of aromatic

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compounds.

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Zou and coauthors [31] further compared the photo-initiated and thermally-initiated

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polymerization

methods

for

preparation

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cyclosiloxane-co-polyhedral

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monolithic column. The photo-initiated polymerization exhibited several advantages, such as

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rapid reaction time (within 10 min), fast separation in high flow rate without loss of the

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efficiency. Most importantly, epoxy-MA-POSS column prepared via photo-initiated

oligomeric

the

poly(methacrylate

epoxy

silsesquioxanes)

(epoxy-MA-POSS)

hybrid

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of

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alkylbenzenes), outperforming that fabricated via thermally-initiated polymerization

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(41,100-48,000 plates/m for alkylbenzenes). Thus, photo-initiated polymerization is a good

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choice to the commonly used thermally-initiated polymerization for fast preparation of

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POSS-based hybrid monolithic columns with high column efficiency.

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The separation of intact proteins is vitally important in both “top-down” and “bottom-up”

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based proteome research due to the high complexity of the biological samples. In their recent

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work, Zou et al. focused on preparation of POSS-based hybrid monolithic columns suitable

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for high-efficiency separation of intact proteins. With stearyl methacrylate (SMA), lauryl

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methacrylate (LMA) or benzyl methacrylate (BeMA) as the monomers, four new hybrid

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monolithic

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BeMA-SMA-POSS, were further fabricated via the “one-pot” process [28]. The dual

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organically functionalized monomers (BeMA and SMA) modified BeMA-SMA-POSS hybrid

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monolithic column exhibited good separation selectivity with low back pressure when it was

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used for separation of seven protein standards (lysozyme, cytochrome c, insulin, BSA,

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ovalbumin, myoglobin and ribonuclease B). The effects of separation time, flow rate, column

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length and additives of the mobile phase on the separation efficiency of the

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BeMA-SMA-POSS column were further systematically studied. Under the optimal

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conditions, fast separation of the above seven proteins was achieved within 2.5 min via a

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10-cm long column. The important application of the BeMA-SMA-POSS column is the

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high-efficiency separation of complex Escherichia coli protein extracts. Many protein peaks

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could be observed from 1 µg of protein sample (Fig. 2), indicating the good potential of these

including

SMA-POSS,

LMA-POSS,

BeMA-POSS

and

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columns for proteome research. With pentadecafluorooctyl methacrylate (PDFOMA) as the monomer and POSS-MA as the

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crosslinker, Chen et al. [39] designed a perfluorinated hybrid monolithic column for specific

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affinity and separation of fluorous compounds. The chromatographic performance of the

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POSS-PDFOMA column was initially evaluated with four neutral alkylbenzenes and four

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basic phenyl amines. Both of the two kinds of compounds were well resolved with good peak

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shapes. Furthermore, since strong fluorous-fluorous affinity interaction could be produced by

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the new POSS-PDFOMA column, it displayed enhanced separation selectivity for fluorous

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containing perfluoroalkyl methacrylates, outperforming the traditional C18 hybrid monolithic

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column. Most importantly, the new column was successfully used for separation of

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perfluoroalkyl sulfonates in the mobile phase free of ammonium salt, which could reduce the

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ion suppression effects in the subsequent mass spectrometry (MS) detection. The main

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drawback of the POSS-PDFOMA column is the relatively low column efficiency. It is only

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30,000 plates/m for 1H,1H-heptafluorobutyl methacrylate (HFBMA).

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3.2. Reaction with acrylic ester modified organically functionalized reagents

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With

2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl

acrylate

as

perfluorous

monomer,

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acryl-POSS as the crosslinker, Zou et al. [24] further prepared a new hybrid fluorous

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monolithic column via the “one-pot” process. Fluorobenzene, 1,2,4,5-tetrafluorobenzene,

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1,2-difluorobenzene, 1,2,4-trifluorobenzene, hexafluorobenzene and pentafluorobenzene

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could be successfully separated within 18 min via the developed hybrid fluorous column.

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However, these fluorobenzenes could not be efficiently separated via the traditional

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C12-functionalized hybrid monolithic column under the optimal chromatographic conditions. 10

ACCEPTED MANUSCRIPT Even though it was compared with the previously reported POSS-PDFOMA hybrid

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monolithic column [39], the new column also exhibited higher column efficiencies and

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reached 51,900-76,000 plates/m for fluorobenzenes. One of the important applications of the

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new column is the determination of perfluoroalkyl acids via LC-MS/MS. The limit of

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detection (LOD) and limit of quantification (LOQ) reached 0.1-0.5 ng/mL and successful

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determination of spiked perfluoroalkyl acids in the river water was achieved with the

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recoveries of 64.2-98.0%.

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In our group [22], dipentaerythritol penta-/hexa-acrylate (DPEPA) was combined with

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POSS-MA to fabricate a highly cross-linked DPEPA-POSS hybrid monolithic column via the

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“one-pot” process. The new DPEPA-POSS column was firstly evaluated by the separation of

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thioureas, amides and phenols. The highest column efficiencies of 220,000 and 511,000

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plates/m were achieved respectively for 2-imidazolidinethione and dimethylacetamide,

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outperforming the previously reported poly(LMA-co-DPEPA) column [44]. Furthermore,

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five protein standards (cytochrome c, ribonuclease, myoglobin, lysozyme and BSA) and egg

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white proteins were also efficiently separated with reduced peak tailing. The most important

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application of the DPEPA-POSS column is the successful separation of expressed BARD1

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BRCT domains protein from other protein impurities with high efficiency.

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3.3. Reaction with vinyl modified organically functionalized reagents

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Vinylbenzyl trimethyl-ammonium chloride (VBTA) [42] was firstly exploited to prepare a

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versatile POSS-based hybrid monolithic column by combination of POSS-MA via the

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“one-pot” thermally-initiated free radical polymerization. Since the introduced VBTA could

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provide cationic sites, hydrophilic and π-π interactions while the polar ester groups of 11

ACCEPTED MANUSCRIPT POSS-MA could provide hydrogen-bonding interaction, the new poly (POSS-MA-co-VBTA)

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column exhibited typical mixed-mode retention mechanisms. Thus, a broad analytical range

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from polar phenols, amides, nucleobases and nucleosides to hydrophobic estrogens and

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anthraquinones was achieved. For example, eight polar nucleosides and nucleobases were

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efficiently separated within 12 min. The column efficiency reached 78,000 plates/m for

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thiourea.

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Recently, many imidazolium-based ionic liquids with reactive vinyl groups were combined

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with POSS-MA to prepare a series of new hybrid monolithic columns. With

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1-vinyl-3-(perfluorobenzyl)-imidazolium bromide (VIMPFP) as the monomer, POSS-MA as

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the crosslinker, Xu et al. [45] firstly synthesized a pentafluorobenzyl imidazolium modified

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POSS-VIMPFP hybrid monolithic column via the “one-pot” free radical polymerization.

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Since VIMPFP is a soft monomer while POSS-MA possesses rigid Si-O-Si skeleton structure,

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the new POSS-VIMPFP column exhibited moderate rigidity which could efficiently reduce

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the swelling propensity or fragile characteristic existed in the polymer-based or silica-based

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monolithic columns. The retention mechanisms of the POSS-VIMPFP column were

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systematically investigated by the separation of PAHs, alkylbenzenes, organic acids, bases

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and amphoteric compounds. Mixed-mode π-π, hydrophobic, electrostatic and ion-exchange

18

interactions could be observed. The new POSS-VIMPFP column was further used for

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separation of nine nucleosides and four halogenated compounds. Due to the strong

20

dipole-dipole and ion-dipole interactions produced by the introduced pentafluorobenzyl

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imidazolium groups, baseline separation of the four halogenated compounds could be only

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achieved via the developed POSS-VIMPFP column, which could not be achieved via the

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commercially available C18 or PFP column. The column efficiency of the POSS-VIMPFP

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column is 72,000 plates/m. In the same group [46], cuprous oxide nanoparticles modified

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hybrid monolithic column was further prepared for affinity of electron rich analytes. Firstly,

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the monolithic matrix

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1-vinylimidazole (VIM). Due to the strong interaction existed between cuprous oxide and the

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imidazole groups in the monolith, cuprous oxide could be easily immobilized on the surface

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of the monolith, followed by further transferring into stable cuprous sulfide by flushing with

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20 mg/mL of Na2S for 3 min. The binding capacity of the new column is 0.73 mg/mL, which

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is obviously higher than that of the commercially available Cu2+ (binding capacity: 0.5

10

mg/mL) and Ni2+ (binding capacity: 0.36 mg/mL) IMAC sorbents. The new monolithic

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column was successfully used for enrichment of kanamycin A from complex milk sample.

fabricated

by copolymerization

of POSS-MA with

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In our group, 1-vinyl-3-octylimidazolium bromide (VOI) was firstly combined with

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POSS-MA to prepare a new POSS-VOI hybrid monolithic column via the “one-pot”

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thermally-initiated free radical polymerization [47]. The new column was evaluated by the

15

separation of alkylbenzenes, PAHs, phenol isomers and basic aromatic amines. The highest

16

column efficiencies of 124,000 and 134,000 plates/m were achieved respectively for benzene

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and 1,10-phenanthroline monohydrate, which is much higher than that of the previously

18

reported POSS-VIMPFP hybrid monolithic column [45]. Moreover, the new POSS-VOI

19

column also displayed excellent planar selectivity. Subsequently, 1-allyl-3-vinylimidazolium

20

bromide (AVI) modified periodic imidazolium bridged POSS-AVI hybrid monolithic column

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was facilely prepared via the same method [29]. The column efficiencies of the POSS-AVI

22

column reached 126,000-151,000 plates/m for separation of alkylbenzenes. The POSS-AVI

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ACCEPTED MANUSCRIPT column also exhibited enhanced separation selectivity and broader separation region for

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phenols and PAHs, outperforming the common imidazolium embedded POSS-AEI hybrid

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monolithic column. One of the important applications of the POSS-AVI column is the

4

separation of hydrophilic nucleoside and nucleic acid bases with pure water as the mobile

5

phase, which is often infeasible in the conventional RPLC column.

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Zou et al. [48] developed a β-cyclodextrin (CD) and L-2-allylglycine hydrochloride (AGH)

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dual chiral selectors modified POSS hybrid monolithic column. Firstly, POSS-MA was

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self-polymerized with AIBN as the initiator to form the POSS matrix. Then, vinyl modified

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CD and AGH were directly grafted on the surface of the POSS matrix to form the

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POSS-CD/AGH hybrid monolithic column. The new column can be used for

11

enantioseparation in both RPLC and NPLC modes and it provided enhanced

12

enantioseparation for hydroxyl acids, compared with the singe CD modified monolithic

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column.

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3.4. Reaction with thiol modified organically functionalized reagents

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Thiol-containing compounds were firstly exploited by Zou and coauthors to construct

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POSS-based hybrid monolithic columns via thiol-methacrylate click reaction. With

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POSS-MA and multi-thiol compounds TPTM, HDT or PTM as the crosslinkers,

18

dimethylphenylphosphine (DMPP) as the catalyst, three new hybrid monolithic columns,

19

POSS-TPTM, POSS-HDT and POSS-PTM [33], were facilely prepared via the “one-pot”

20

approach, as shown in Fig. 3. The new POSS-TPTM column exhibited the highest column

21

efficiency for separation of alkylbenzenes (190,000 plates/m for butylbenzene). The

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separation of PAHs, phenols and anilines also exhibited high column efficiencies, which are

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ACCEPTED MANUSCRIPT 94,200-114,000, 55,500-81,400 and 51,400-117,000 plates/m, respectively. The new column

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was further successfully used for separation of EPA 610 or BSA tryptic digests. Based on

3

database search, totally 69 peptides were successfully recognized with the sequence coverage

4

of 67.8%. These results were comparable to those obtained via the C18 particle based packed

5

column. Most importantly, based on these columns, versatile hybrid monolithic columns

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could be further prepared by the introduction of new precursors via both pre- and

7

post-modification.

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Ye et al. [49] further prepared a new hybrid monolithic column based on POSS-MA and

9

1,4-bis(mercaptoacetoxy) butane (BMAB) via the same method. The POSS-BMAB hybrid

10

monolithic column exhibited typical RPLC retention characteristic and the column efficiency

11

reached 73,000 plates/m for benzene. PAHs and phenols were selected to evaluate the

12

chromatographic performance of the POSS-BMAB column and the column efficiencies of

13

53,800-69,900 and 56,600-69,200 plates/m were observed, respectively. Furthermore, even

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though it was used for separation of weakly basic anilines, symmetric peak shapes and

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accepted column efficiencies (53,899-64,500 plates/m) were also achieved.

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Tuncel et al. [50] fabricated a carboxyl functionalized POSS-based hybrid monolithic

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column for hydrophilic interaction liquid chromatography (HILIC). With POSS-MA and

18

mercaptosuccinic

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attached-poly(POSS-MA) hybrid monolithic column was facilely prepared via the “one-pot”

20

photo-initiated click polymerization reaction. The authors systematically studied the HILIC

21

retention behaviors of the new column and it can be used for separation of hydrophilic

22

nucleotides, nucleosides and organic acids with almost retention independent column

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acid

(MSA)

as

the

15

monomer

and

crosslinker,

MSA

ACCEPTED MANUSCRIPT 1

efficiency. Photo-initiated thiol-acrylate click reaction was also exploited for fabrication of new

3

POSS-based hybrid monolithic column by Zou and coauthors [34]. With acryl-POSS as the

4

crosslinker, ODT or sodium 3-mercapto-1-propanesulfonate as the monomers, two new

5

hybrid monolithic columns were ultra-rapid prepared within 5 min. Both the “one-pot”

6

approach and “two-step” approach of which the POSS matrix was firstly fabricated via

7

self-polymerization, followed by further introduction of monothiol monomers via

8

thiol-acrylate click reaction, were exploited for fabrication of the new column, as shown in

9

Fig. 4. Compared with the “two-step” approach, the “one-pot” approach possesses the merits

10

of enhanced preparation reproducibility and fast polymerization rate. The new column was

11

successfully used for separation of a series of compounds, such as alkylbenzenes, basic

12

compounds, phenolic compounds, EPA 610, tryptic digests of proteins ovalbumin, BSA,

13

myoglobin and α-casein or model proteins. The column efficiencies could reach

14

60,000-73,500 plates/m for alkylbenzenes.

15

4. VinylPOSS-based hybrid monolithic columns

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VinylPOSS is a POSS with eight or fewer vinyl groups in the POSS skeleton. Because of

17

the low reactive activities, vinylPOSS is difficult to undergo homopolymerization via free

18

radical polymerization [36]. However, vinylPOSS is more inclined to polymerize with

19

thiol-containing compounds via thiol-ene click reaction. Nischang et al. [26] firstly exploited

20

to prepare a series of hybrid monoliths based on vinylPOSS and a variety of thiol-containing

21

compounds with step-growth mechanism. The new monoliths exhibited a wide range of

22

physical behavior (from rigid materials to gels) and chemical nature (from hydrophilicity to

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ACCEPTED MANUSCRIPT 1

hydrophobicity). Obviously, vinylPOSS exhibits a good potential for preparation of new

2

POSS-based hybrid monolithic columns. The recently reported vinylPOSS-based hybrid

3

monolithic columns are summarized in Table 3. Nischang et al. [35] fabricated two new hybrid monolithic columns by copolymerization of vinylPOSS

pentaerythritol

tetra

(3-mercaptopropionate)

(PETMP)

or

6

2,2’-(ethylenedioxy)diethanethiol (EDDT) via the “one-pot” thiol-ene click reaction, as

7

shown in Fig. 5. By proper choice of preparation conditions, the POSS-PETMP, POSS-EDDT

8

columns could be obtained with accessible near-ideal nanoscale network structures and they

9

were successfully used for separation of uracil, benzyl alcohol, benzene and five

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5

10

with

RI PT

4

alkylbenzenes with symmetric peak shapes.

Ye et al. [32] fabricated a new HILIC based POSS hybrid monolithic column via two steps

12

of photo-initiated thiol-ene click reaction. Firstly, vinylPOSS was modified by 1-thioglycerol

13

so as to improve the solubility of vinylPOSS. Then, the modified vinylPOSS was further

14

polymerized with dithiothreitol (DTT) to form the final poly(vinylPOSS-co-DTT) hybrid

15

monolithic column. The HILIC nature of the new column was evaluated for separation of

16

toluene, dimethylformamide, formamide, thiourea and the column efficiencies reached

17

60,000-65,000 plates/m. One of the important applications of the monolith is the enrichment

18

of glycopeptides via HILIC or hydrazide chemistry after it was further modified by hydrazine

19

groups. Fig. 6 are the representative matrix-assisted laser desorption/ionization time-of-flight

20

(MALDI-TOF) MS spectra of IgG tryptic digests (Fig. 6a) and that enriched via hydrazine

21

chemistry method followed by deglycosylation with PNGase F (Fig. 6b). Two glycopeptides,

22

EEQFN#STFR and EQTN#STYR, were successfully identified at m/z 1158.5 and 1190.5,

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17

ACCEPTED MANUSCRIPT 1

indicating the good potential of the monolith for glycoprotein analysis.

2

5. POSS-epoxy-based hybrid monolithic columns POSS-epoxy are often used to prepare hybrid monolithic columns by copolymerization

4

with diamines, polyethylenimine (PEI) or multi-thiol containing compounds via ring-opening

5

polymerization [25] or thiol-epoxy click polymerization [37], as summarized in Table 4.

RI PT

3

Zou and coauthors did a lot of excellent work for preparation of POSS-epoxy based hybrid

7

monolithic columns. For the first time, with POSS-epoxy as the monomer, four new hybrid

8

monolithic columns were facilely fabricated via “one-pot” ring-opening polymerization with

9

diamines 1,6-diaminohexane (HDA), 1,8-diaminooctane (DAO), 1,10-diaminodecane (DAD)

10

or 1,12-diaminododecane (DADD) [25], as shown in Fig. 7. Since the ring-opening

11

polymerization reaction exhibited progressively phase-separating process, the new monolithic

12

columns displayed well-defined 3D skeletal structure. Thus, ultra-high column efficiency (up

13

to 140,000 plates/m) could be achieved for alkylbenzenes. The separation performance of the

14

POSS-DADD hybrid monolithic column was also evaluated for separation of anilines,

15

phenols, PAHs, BSA tryptic digests and satisfactory results were achieved. Subsequently,

16

another

17

1,4-butanediamine (BDA) [51]. The separation of five alkylbenzenes showed with good peak

18

shapes and the column efficiency reached about 100,000 plates/m. Excellent separation

19

performance was also achieved for PAHs, phenols and benzoic acids, purines and

20

pyrimidines, anilines and the column efficiencies reached 68,000-79,000, 215,000-226,000,

21

73,200-82,000 and 35,800-52,700 plates/m, respectively. One of the important characteristics

22

of these columns is that numerous primary and secondary amine groups are still present in the

monolithic

column

was

further

fabricated

by

introduction

of

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hybrid

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ACCEPTED MANUSCRIPT 1

monoliths. Thus, they could be further modified by different reactive groups so as to prepare

2

new specific monolithic columns. PEI is a polymer with repeating amine units. Because of the high density of the amine

4

groups, PEI could provide extensive interaction sites and post-modification sites when it was

5

used for preparation of POSS-based hybrid monolithic columns. Zou and coauthors [38]

6

further prepared a new POSS-PEI hybrid monolith via the “one-pot” ring-opening

7

polymerization of POSS-epoxy with PEI. Furthermore, based on the residue amino groups on

8

the surface of the monolith, γ-gluconolactone or 1,2-epoxyoctadecane modified POSS-PEI

9

hybrid monolithic columns and cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC)

10

physically coated hybrid monolithic column were also fabricated, as shown in Fig. 8. The

11

POSS-PEI hybrid monolithic column can be used for RPLC or HILIC separation and the

12

column efficiency reached 110,000 plates/m for alkylbenzenes. The γ-gluconolactone

13

modified hybrid monolithic column possesses remarkably increased hydrophilicity and was

14

successfully applied for HILIC separation of six phenols, four basic drugs, six small peptides

15

and a mixture of seven pyrimidines, purines, nucleosides with good peak shapes. The column

16

efficiencies could reach 60,000-80,000 plates/m for phenols. The CDMPC modified column

17

was used for separation of nine basic racemates and almost all these racemates could be

18

baseline-separated with good peak shapes. Wu et al. [52] further modified the POSS-PEI

19

hybrid monolithic column with iodomethane (IM) or bromoacetic acid (BAA) via

20

nucleophilic substitution reaction for separation of neutral polar dimethylformamide, toluene,

21

formamide, thiourea and a series of halogen benzoic acids.

22

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In their recent work, cystamine dihydrochloride was further combined with POSS-epoxy to 19

ACCEPTED MANUSCRIPT prepare a new 3D skeleton POSS-based hybrid monolithic column via “one-pot”

2

ring-opening polymerization [53]. The disulfide bonds present in the monolith could be

3

further reduced with 0.2 M DTT for 2 h to form free thiol groups. Thus, a series of

4

alkene-containing monomers could be further introduced into the monolith via thiol-ene click

5

reaction so as to prepare various new POSS-based hybrid monolithic columns. SMA, benzyl

6

methacrylate (BMA) and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium

7

hydroxide (MSAH) modified hybrid monolithic columns were further prepared, as shown in

8

Fig. 9. These new columns were successfully used for separation of alkylbenzenes, phenols,

9

EPA 610 via SMA or BMA modified columns in RPLC or dimethylformamide, benzene,

10

thiourea via MSAH modified column in HILIC. The column efficiencies of the SMA

11

modified column reached 64,000-97,000 plates/m for phenols while the column efficiencies

12

of the MSAH modified column is about 76,700 plates/m for thiourea. Recently, open tubular

13

column [54] was further fabricated by the introduction of aromatic diamine 4-aminophenyl

14

disulfide (APDS) and POSS-epoxy. The new column was successfully used for separation of

15

mouse liver tryptic digests and totally 1234 proteins were identified via LC-MS/MS analysis.

16

However, only 1008 proteins could be recognized when the packed C18 column was used.

AC C

EP

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1

17

Zou et al. [37] further exploited to use thiol-epoxy click polymerization reaction to prepare

18

POSS-based hybrid monolithic columns. With POSS-epoxy as the monomer, PTM or TPTM

19

as the precursors, KOH as the alkaline catalyst, two new hybrid monolithic columns,

20

POSS-epoxy-PTM and POSS-epoxy-TPTM, were readily prepared via the “one-pot”

21

approach. The POSS-epoxy-PTM column exhibited higher column efficiency for

22

alkylbenzenes (182,700 plates/m for butylbenzene) than that of the POSS-epoxy-TPTM 20

ACCEPTED MANUSCRIPT column (103,100 plates/m for butylbenzene), which is also higher than that prepared via

2

ring-opening polymerization and free radical polymerization methods [16,25,38]. The

3

POSS-epoxy-PTM column was also evaluated for separation of PAHs, phenols, basic anilines,

4

benzoic acids, pesticides and the column efficiencies reached 102,400-178,000,

5

45,000-109,000, 37,900-167,700, 35,500-96,600 and 40,000-173,400 plates/m, respectively.

6

Furthermore, the POSS-epoxy-PTM hybrid monolithic column was further used for

7

separation of BSA tryptic digests and totally 53 unique peptides which corresponding to

8

58.65% sequence coverage was recognized, indicating the good suitability of the column for

9

separation of macromolecules.

SC

6. Conclusions

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Herein, the recently developed POSS-based hybrid monolithic columns are systematically

12

summarized. Based on a variety of substituted POSS reagents, including POSS-MA,

13

acryl-POSS, vinylPOSS and POSS-epoxy, many organically functionalized reagents with

14

different characteristics and reactive groups were introduced for fabrication of new hybrid

15

monolithic columns via free radical copolymerization, thiol-based click reactions or

16

ring-opening polymerization. The column efficiency of these new columns could reach more

17

than 500,000 plates/m. Especially, many new columns suitable for separation of

18

macromolecules were specifically designed and reported. However, the applications of these

19

columns for separation of real samples are still preliminary. Thus, the exploitation of further

20

applications of these reported columns for real sample separation are still promising.

21

Furthermore, the introduction of versatile new POSS-based reagents and organically

22

functionalized reagents for preparation of new POSS-based hybrid monolithic columns with

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ACCEPTED MANUSCRIPT 1

multiple retention mechanisms and specific applications will be another hot spot in future

2

research.

3

Acknowledgements We are grateful for the financial support from National Natural Science Foundation of

5

China (21675039), Young Talent of Hebei Province, Project funded by China Postdoctoral

6

Science Foundation (2016M591401), the Young Talent Program in College and University of

7

Hebei Province (BJ2014005) and Hebei University Science Fund for Distinguished Young

8

Scholars (2015JQ06).

9

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1

28

ACCEPTED MANUSCRIPT nano-hydrophilic interaction chromatography, J. Chromatogr. A 1502 (2017) 14-23.

2

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3

silsesquioxane based hybrid monoliths by ring-opening polymerization for capillary LC and

4

CEC, J. Sep. Sci. 36 (2013) 2819-2825.

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6

monolith with anion exchange/hydrophilic interaction mixed-mode via epoxy-amine

7

ring-opening

8

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as

functional

11

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13

capillary columns by in situ ring-opening polymerization and their applications in

14

cLC-MS/MS analysis of tryptic digest, Anal. Chim. Acta 979 (2017) 58-65.

18 19

EP AC C

17

by

TE D

silsesquioxane-based

16

monolith

20 21 22 29

ring-opening

monomer,

10

15

hybrid

polyethylenimine

SC

using

M AN U

polymerization

RI PT

1

polymerization

and

ACCEPTED MANUSCRIPT Fig. 1. Schematic diagram for fabrication of MDOA-POSS hybrid monolithic column via free

2

radical copolymerization. (Reprinted from [16]).

3

Fig. 2. Chromatogram of Escherichia coli proteins separated by the BeMA-SMA-POSS

4

hybrid monolithic column. (Reprinted from [28]).

5

Fig. 3. Schematic diagram for fabrication of POSS-TPTM, POSS-HDT and POSS-PTM

6

hybrid monolithic columns via thiol-methacrylate click reaction. (Reprinted from [33]).

7

Fig. 4. “One-pot” and “two-step” methods for preparation of C18-functionalized POSS-based

8

hybrid monolithic columns via thiol-acrylate click reaction. (Reprinted from [34]).

9

Fig. 5. Schematic diagram for fabrication of POSS-PETMP and POSS-EDDT hybrid

M AN U

SC

RI PT

1

monolithic columns via thiol-ene click reaction. (Reprinted from [35]).

11

Fig. 6. MALDI-TOF-MS spectra of (a) IgG tryptic digests and (b) that enriched via the

12

hydrazine-modified poly(vinylPOSS-co-DTT) hybrid monolithic column. (Reprinted from

13

[32]).

14

Fig. 7. Schematic diagram for fabrication of POSS-HDA, POSS-DAO, POSS-DAD and

15

POSS-DADD hybrid monolithic columns via ring-opening polymerization. (Reprinted from

16

[25]).

17

Fig. 8. Schematic diagram for fabrication and post-modification of POSS-PEI hybrid

18

monolithic column. (Reprinted from [38]).

19

Fig. 9. Schematic diagram for fabrication of POSS-epoxy-based hybrid monolithic column

20

via ring-opening polymerization and further post-functionalizated based on thiol-ene click

21

reaction. (Reprinted from [53]).

AC C

EP

TE D

10

22 30

ACCEPTED MANUSCRIPT 1 2 3

RI PT

4 5 6

SC

7

11 12 13 14

EP

10

Figure 1

AC C

9

TE D

M AN U

8

15 16 17 31

ACCEPTED MANUSCRIPT 1 2 3

RI PT

4 5 6

10 11 12 13

EP

9

Figure 2

AC C

8

TE D

M AN U

SC

7

14 15 16 32

ACCEPTED MANUSCRIPT 1 2 3

RI PT

4 5 6

SC

7

11 12 13 14

EP

10

Figure 3

AC C

9

TE D

M AN U

8

15 16 17 33

ACCEPTED MANUSCRIPT 1 2 3

RI PT

4

6 7

AC C

EP

TE D

M AN U

SC

5

Figure 4

8 9 10 11 34

ACCEPTED MANUSCRIPT 1 2 3

5 6 7

AC C

EP

TE D

M AN U

SC

RI PT

4

Figure 5

8 9 10 11 35

ACCEPTED MANUSCRIPT 1 2 3

RI PT

4 5 6

SC

7

11 12 13 14

Figure 6

EP

10

AC C

9

TE D

M AN U

8

15 16 17 18 36

ACCEPTED MANUSCRIPT 1 2 3

5 6

AC C

EP

TE D

M AN U

SC

RI PT

4

Figure 7

7 8 9 37

ACCEPTED MANUSCRIPT 1 2 3

RI PT

4 5 6

9 10 11

AC C

8

EP

TE D

M AN U

SC

7

Figure 8

12 13 14 15 38

ACCEPTED MANUSCRIPT 1 2 3

RI PT

4 5

8

Figure 9

AC C

7

EP

TE D

M AN U

SC

6

39

ACCEPTED MANUSCRIPT

Table 1 The methods for preparation of POSS-based hybrid monolithic columns Reaction mechanism

Methyl acrylate-containing compounds Acrylic ester-containing compounds Vinyl-containing compounds

Thiol-containing compounds

Reference

Free radical copolymerization

Photo-initiation Thermally-initiation

[16,22,24,28 ,29]

Thiol-methacrylate click reaction Thiol-acrylate click reaction

Photo-initiation Thermally-initiation

[33,34]

Thiol-ene click reaction

Thermally-initiation Photo-initiation

[27,32,35]

Ring-opening polymerization

-

[25,38]

Thiol–epoxy click polymerization

-

[37]

SC

M AN U

TE D

Thiol-containing compounds

Initiation mode

RI PT

Monomer/crosslinker

EP

Diamines, polyethylenimine

Multi-thiol compounds

AC C

POSS reagent

40

ACCEPTED MANUSCRIPT

Table 2 POSS-MA or acryl-POSS-based hybrid monolithic columns Structure

Analytes

RPLC/AEX

Alkylbenzenes, model proteins, tryptic digests of BSA

Column efficiency

50,000 plates/m thiourea

Reference

for

[16]

Alkylbenzenes, PAHs

[43]

Alkylbenzenes

97,000-98,400 plates/m (photo-initiated), 41,100-48,000 plates/m (thermally-initiated) for alkylbenzenes

[31]

TE D

RPLC

23,600-27,200 plates/m for poly(POSS-co-BPADM A) column for alkylbenzenes, 18,900-22,100 plates/m for poly (POSS-co-EDMA) column for alkylbenzenes

Epoxy-MA-POSS hybrid monolithic column

AC C

EP

Poly(POSS-co-BPADMA ), poly(POSS-co-EDMA) hybrid monolithic columns

M AN U

SC

MDOA-POSS hybrid monolithic column

Retention mechanisms

RI PT

Stationary phase

RPLC

41

ACCEPTED MANUSCRIPT

POSS-PDFOMA hybrid monolith column

-

[28]

Alkylbenzenes, phenyl amines, perfluoroalkyl methacrylates, perfluoroalkyl sulfonates

30,000 plates/m HFBMA

Alkylbenzenes, fluorobenzenes, perfluoroalkyl acids

51,900-76,000 plates/m for fluorobenzenes

TE D

M AN U

RPLC/Affinity

EP

RPLC

AC C

-

Model proteins, Escherichia coli protein extracts

RPLC

RI PT

-

SC

SMA-POSS, LMA-POSS, BeMA-POSS, BeMA-SMA-POSS hybrid monolithic columns

42

for

[39]

[24]

ACCEPTED MANUSCRIPT

511,000 plates/m for dimethylacetamide

[22]

Phenols, amides, estrogens, anthraquinones, nucleobases, nucleosides

78,000 plates/m thiourea

for

[42]

Alkylbenzenes, PAHs, nucleosides, halogenated compounds

72,000 plates/m alkylbenzenes

for

RI PT

-

RPLC

M AN U

SC

DPEPA-POSS hybrid monolithic column

Amides, thioureas, phenols, model proteins, egg white proteins, BARD1 BRCT domains proteins

HILIC, AEX, π–π conjunction, hydrogen bonding

TE D

Poly (POSS-MA-co-VBTA) hybrid monolithic column

EP AC C

POSS-VIMPFP hybrid monolithic column

Hydrophobic, π–π, ion-exchange, electrostatic, dipole-dipole, ion-dipole interactions

43

[45]

Affinity chromatography

POSS-VOI hybrid monolithic column

RPLC

Purine, kanamycin A

-

[46]

Alkylbenzenes, phenols, PAHs, aromatic amines

124,000 plates/m for benzene

[47]

Alkylbenzenes, phenols, PAHs

112,000-156,000 plates/m alkylbenzenes

[29]

RPLC

AC C

POSS-AEI hybrid monolithic column

EP

TE D

M AN U

SC

Cu2S modified hybrid monolithic column

RI PT

ACCEPTED MANUSCRIPT

44

for

ACCEPTED MANUSCRIPT

RI PT

RPLC

-

RPLC/NPLC

POSS-TPTM, POSS-HDT, POSS-PTM hybrid monolithic columns

126,000-151,000 plates/m alkylbenzenes

for

[29]

-

[48]

Alkylbenzenes, phenols, anilines, PAHs, EPA 610, tryptic digests of BSA

190,000 plates/m for butylbenzene

[33]

Alkylbenzenes, phenols, anilines, PAHs

73,000 plates/m benzene

[49]

POSS-BMAB hybrid monolithic column

AC C

EP

TE D

RPLC

Hydroxy acids

M AN U

POSS-CD/AGH hybrid monolithic column

Alkylbenzenes, phenols, PAHs, aromatic amines, nucleosides, nucleic acid bases, tryptic digests of BSA

SC

POSS-AVI hybrid monolithic column

RPLC

45

for

ACCEPTED MANUSCRIPT

HILIC

Nucleosides, nucleotides, benzonic acids

-

[50]

-

RPLC RPLC/Cation exchange chromatography (CEX)

Alkylbenzenes, basic compounds, phenolic compounds, EPA 610, model proteins, tryptic digests of proteins myoglobin, BSA, ovalbumin and α-casein

60,000-73,500 plates/m for alkylbenzenes

[34]

AC C

EP

TE D

M AN U

SC

RI PT

MSA-attached-poly(POS S-MA) hybrid monolithic column

46

ACCEPTED MANUSCRIPT

Table 3 VinylPOSS-based hybrid monolithic columns Retention mechanisms

Structure

POSS-PETMP, POSS-EDDT hybrid monolithic columns

Benzyl alkyl uracil

alcohol, benzenes,

Column efficiency

Reference

-

[35]

65,000 plates/m for formamide

[32]

M AN U

SC

RPLC

Analytes

RI PT

Stationary phase

Poly(vinylPOSS-co-DTT) hybrid monolithic column

AC C

EP

TE D

HILIC

47

Toluene, dimethylformamide, formamide, thiourea

ACCEPTED MANUSCRIPT

Table 4 POSS-epoxy-based hybrid monolithic columns Retention mechanisms

POSS-HDA, POSS-DAO, POSS-DAD, POSS-DADD hybrid monolithic columns

RPLC

EP

hybrid

AC C

Poly(POSS-co-BDA) monolithic column

TE D

M AN U

RPLC

48

Column efficiency

Reference

Alkylbenzenes, phenols, anilines, PAHs, tryptic digests of BSA

>100,000 plates/m for alkylbenzenes

[25]

Alkylbenzenes, phenols and benzoic acids, anilines, PAHs, purines and pyrimidines

100,000 plates/m for alkylbenzenes

[51]

Analytes

RI PT

Structure

SC

Stationary phase

POSS-PEI hybrid monolithic column

RI PT

ACCEPTED MANUSCRIPT

Alkylbenzenes

110,000 plates/m for alkylbenzenes

[38]

HILIC

Alkylbenzenes, phenols, basic drugs, purines, pyrimidines, nucleosides, small peptides

60,000-80,000 plates/m for phenols

[38]

RPLC

Alkylbenzenes

-

[38]

M AN U

SC

RPLC/HILIC

EP AC C

1,2-Epoxyoctadecane modified POSS-PEI hybrid monolithic column

TE D

γ-Gluconolactone-modified POSS-PEI hybrid monolithic column

49

CDMPC-coated POSS-PEI hybrid monolithic column

RI PT

ACCEPTED MANUSCRIPT

Basic racemates

90,000 plates/m for benzoin

[38]

RPLC/HILIC/AEX

Halogen benzoic acids, neutral polar analytes

-

[52]

HILIC/AEX

Halogen benzoic acids, neutral polar analytes

56,000 plates/m for neutral polar analytes

[52]

HILIC/AEX

Neutral analytes

-

[52]

M AN U

SC

RPLC/NPLC

hybrid

BAA-PEI-POSS monolithic column

hybrid

AC C

EP

IM-PEI-POSS monolithic column

TE D

PEI-POSS hybrid monolithic column

50

polar

ACCEPTED MANUSCRIPT

hybrid

MSAH-modified monolithic column

hybrid

Poly(POSS-APDS) column

OT

hybrid

AC C

POSS-epoxy-PTM, POSS-epoxy-TPTM monolithic columns

Alkylbenzenes, phenols, EPA 610

64,000-97,000 plates/m for phenols

[53]

RPLC

EPA 610

-

[53]

HILIC

Dimethylformamid e, benzene, thiourea

76,700 plates/m for thiourea

[53]

RPLC

Alkylbenzenes, tryptic digests of mouse liver

-

[54]

RPLC

Alkylbenzenes, phenols, anilines, PAHs, benzoic acids, pesticides, dipeptides, model proteins, EPA 610, tryptic digests of BSA

182,700 plates/m (POSS-epoxy-P TM), 103,100 plates/m (POSS-epoxy-T PTM) for butylbenzene

[37]

RPLC

RI PT

BMA-modified monolithic column

[53]

SC

hybrid

71,000 plates/m for benzene

M AN U

SMA-modified monolithic column

Alkylbenzenes

RPLC/HILIC

TE D

hybrid

EP

POSS-epoxy-based monolithic column

51

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

52

ACCEPTED MANUSCRIPT • The recently reported POSS-based hybrid monolithic columns are summarized. • These columns are classified based on the reactive groups of POSSs.

AC C

EP

TE D

M AN U

SC

RI PT

• This work emphasizes on the preparation methods and further HPLC applications.