TitleDevelopment of stationary phase for separation of inorganicanions and organic compounds in capillary liquidchromatography( 本文(Fulltext) )

Author(s) ROZA, LINDA

Report No.(DoctoralDegree) 博士(工学) 甲第444号

Issue Date 2013-09-30

Type 博士論文

Version ETD

URL http://hdl.handle.net/20.500.12099/47356

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

Development of stationary phase for separation

of inorganic anions and organic compounds in capillary

liquid chromatography

ROZA LINDA

September 2013

Development of stationary phase for separation

of inorganic anions and organic compounds in capillary

liquid chromatography

（キャピラリー液体クロマトグラフィーにおける無機陰イオンおよ

び有機化合物分離のための固定相の開発）

ROZA LINDA

Material Engineering Division

Graduate School of Engineering

Gifu University

JAPAN

September 2013

Development of stationary phase for separation

of inorganic anions and organic compounds in capillary

liquid chromatography

A dissertation submitted to the Gifu University

in partial fulfillment of the requirements for

the degree of Doctor of Philosophy in

Material Engineering

By

ROZA LINDA

September 2013

i

Contents

page

Contents i

Summary v

Chapter 1 Introduction 1

1.1. Principle and Classification of Chromatography 1

1.2. Capillary Liquid Chromatography 4

1.3. Polimer Monolithic Stationary Phase in Liquid Chromatography 5

1.4. Silica Bonded Stationary Phase in Liquid Chromatography 8

1.5. Ion Chromatography 8

1.6. Hydrophilic Interaction Liquid Chromatography (HILIC) 10

1.7. Objectives of Present Research 11

1.8. References 12

Chapter 2 Methacrylate-based Diol Monolithic Stationary Phase for Separation

of Polar and Non Polar Compounds in Capillary Liquid

Chromatography 15

2.1. Introduction 15

2.2. Experimental 18

2.2.1. Apparatus 18

2.2.2. Reagents and chemicals 18

2.2.3. Preparation of poly(GMA-PEGDMA) monolithic capillary column 19

ii

2.3. Results and Discussion 20

2.3.1. Column characterization 20

2.3.2. Chromatographic properties 25

2.4. Conclusions 32

2.5. References 33

Chapter 3 Poly(ethylene oxide)-bonded Stationary Phase for Separation of

Inorganic Anions in Capillary Ion Chromatography 36

3.1. Introduction 36

3.2. Experimental 39

3.2.1. Apparatus 39

3.2.2. Reagents and materials 40

3.2.3. Preparation of PEO-bonded stationary phase 40

3.2.4. Preparation of water and saliva samples 41

3.3. Results and Discussion 42

3.3.1. Column characterization 42

3.3.2. Retention behaviors of anions on PEO-bonded stationary phase 42

3.3.3. Effect of eluent cation and eluent anion 46

3.3.4. Effect of salt concentration 50

3.3.5. Application to real sample 51

3.4. Conclusions 53

3.5. References 53

iii

Chapter 4 Tetraethylene oxide-bonded Stationary Phase for Separation of

Inorganic Anions and Polar Compounds in Capillary Liquid

Chromatography 55

4.1. Introduction 55

4.2. Experimental 58

4.2.1 Apparatus 58

4.2.2 Reagents and materials 59

4.2.3 Preparation of TEGMM-bonded and TEG-bonded stationary phases 60

4.3. Results and Discussion 61

4.3.1. Elemental analysis of the stationary phases 61

4.3.2. Retention behaviors of anions on TEGMM-bonded stationary phase and

TEG-bonded stationary phase 62

4.3.3. Effect of eluent cation 65

4.3.4. Effect of salt concentration 67

4.3.5. The HILIC mode properties of TEG-bonded stationary phase 68

4.4. Conclusions 70

4.5. References 71

Conclusions 73

Figure list 75

Table list 77

List of publications 78

List of presentations 79

iv

Curriculum vitae 80

Acknowledgements 81

v

Summary

Capillary liquid chromatography (cLC) has been increasingly recognized

over the last few decades as a powerful separation technology, as it enables high

performance, rapid and sensitive analysis, and low-cost operation and environmental

compatibility. cLC has established itself as a complementary technique to conventional

LC columns routinely used in high-performance liquid chromatography (HPLC). This is

mainly due to the advantages found in an increasing number of cLC applications.

Stationary phase is one of important factor in LC beside mobile phase. The stationary

phases are mainly polymer-based and silica-based. The increased mass sensitivity of the

miniaturized columns is attractive for the detection of compounds present at low

concentrations and or in limited sample volumes. In this dissertation, we have

developed the stationary phases, packed and monolithic columns, for separation of

inorganic anions and organic compounds.

Polymer-based monolithic columns have gained increasing attention in cLC.

In this research, a monolithic capillary column prepared with glycidyl methacrylate

(GMA) and poly(ethylene glycol)dimethacrylate (PEGDMA) have been investigated

and used in cLC. The polymer monolith was synthesized in the presence of methanol

and decanol as the biporogenic solvents by in-situ polymerization of GMA and

PEGDMA, and the optimum composition of monomer and porogen was investigated.

After polymerization, glycidyl groups were hydrolyzed with sulfuric acid to produce

diol groups at the surface of the porous monolith via epoxy-ring-opening. The GMA

content in the polymerization mixture affected the hydrophilicity of monolith. The

vi

separation capability was evaluated by separation of phenol compounds, phthalic acids,

and polycyclic aromatic hydrocarbons. The poly(GMA-PEGDMA) monolithic capillary

column exhibited satisfactory stability.

Although silica-based packings cannot be used in strongly acidic or alkaline

mobile phases, and have residual silanols which affect separation of biological samples,

they have specific advantages such as resistance to organic solvents, high rigidity, and

fast mass transfer. In addition, the rich silanol groups on the surface of silica gel allow

easy introduction of many functional groups onto the gel. We have prepared two kinds

of bonded silica stationary phases, they are poly(ethylene oxide)-bonded stationary

phase and tetraethylene oxide-bonded stationary phase.

A tosylated-poly(ethylene oxide) (PEO) reagent was reacted with primary

amino groups of an aminopropylsilica packing material (TSKgel NH2-60) in acetonitrile

to form PEO-bonded stationary phase. The reaction was a single and simple step

reaction. The prepared stationary phase was able to separate inorganic anions. The

retention behavior of six common inorganic anions on the prepared stationary phase was

examined under various eluent conditions in order to clarify its separation/retention

mechanism. The elution order of the tested anions was iodate, bromate, bromide, nitrate,

iodide, and thiocyanate, which was similar as observed in common ion chromatography.

The retention of inorganic anions could be manipulated by ion exchange interaction

which is expected that the eluent cation is coordinated among the PEO chains and it

works as the anion-exchange site. Cations and anions of the eluent therefore affected the

retention of sample anions. We demonstrated that the retention of the analyte anions

decreased with increasing eluent concentration. The repeatability of retention time for

the six anions was satisfactory on this column with relative standard deviation values

vii

from 1.1 to 4.3% when 10 mM sodium chloride was used as the eluent. Compared with

the unmodified TSKgel NH2-60, the prepared stationary phase retained inorganic anions

more strongly and the selectivity was also improved. The present stationary phase was

applied to the determination of inorganic anions contained in tap water and saliva.

The shorter chain of ethylene oxide; tetraethylene glycol monomethyl ether

or tetraethylene glycol modified silica was synthesized and investigated as stationary

phase for IC. The tetraethylene oxide bonded stationary phase was prepared by

chemically bonding of the tetraethylene glycol monomethyl ether (TEGMM) and

tetraethylene glycol (TEG) on silica via reaction with 3-

glycidyloxypropyltrimethoxysilane. The present stationary phases were successfully

used for separation of inorganic anions and phenol compounds. The TEG bonded

stationary phase has better selectivity and strong retention for the 5 anions, compared

with the TEGMM bonded stationary phase. Both of the present ethylene oxide bonded

stationary phase could not separate bromide and nitrate, unfortunately. The elution order

of the tested anions was similar as observed in common ion chromatography; iodate,

bromate, bromide and nitrate, iodide, and thiocyanate. It was also found that the TEG

bonded stationary phase has hydrophilic property, due to the hydroxyl group on the end

of TEG chains.

1

Chapter 1

Introduction

1.1. Principle and Classification of Chromatography

Chromatography is a method of separation in which the components to be

separated are distributed between two phases, one of these is called a stationary phase

and the other a mobile phase which moves on the stationary phase in a definite direction.

The history of chromatography can be traced to the turn of the century when the

Russian botanist Mikhail Tswett (1872–1919) used a column packed with a stationary

phase of calcium carbonate to separate colored pigments from plant extracts. Tswett

named the technique chromatography, combining the Greek words for “color” and “to

write.” There was little interest in Tswett’s technique until 1931 when chromatography

was reintroduced as an analytical technique for biochemical separations [1-4].

In the modern method, chromatography is general term for various

separation techniques involving two phases, stationary phase and mobile phase. The

stationary phase can be a solid or a liquid and the mobile phase a liquid, gas or a

supercritical fluid. The sample mixture is introduced into mobile phase and undergoes a

2

series of partition or adsorption interaction at the mobile phase-stationary phase

boundary as it moves through the chromatograph system. The speed of a migrating

sample component depends on whether the component has an affinity for the stationary

or mobile phase. The differences of physical and chemical properties of the individual

components determine their relative affinity to two phases. The components with a

stronger interaction to the stationary phase will elute from the column slowly compared

to the components which prefer to the mobile phase [2,4].

Generally, the classification of chromatography is based upon the mobile

and the stationary phase utilized for the separation. Basis of the nature of mobile phase,

there will be three types of chromatographic techniques; they are liquid, gas and

supercritical fluid chromatography. For the classification taken by the criteria of the

mechanism that is responsible for separation, there will be four distinct types of

chromatography, viz. adsorption, partition, ion-exchange and size exclusion. Based on

the shape of separation bed there are tubular and planar chromatography. The

classification of chromatography is described in Table 1.1 [1].

3

Table 1.1. The classification of chromatography

Type of stationary phase

(CHROMATOGRAPHIC

PRINCIPLE)

Type of

mobile

phase

Type of

separation

media

Type of

chromatography

Adsorption chromatography

Competition between a solid

adsorbent and the mobile phase

Gas Column

Gas-solid

chromatography

(GC/GSC)

Liquid

Column

Column liquid

chromatography

(LC)

High performance

liquid

chromatography

(HPLC)

Planar layer

Thin layer

chromatography

(TLC)

Paper

chromatography

(PC)

Partition chromatography

Competition between a liquid

stationary phase and the mobile

phase

Gas Column

Gas-liquid

chromatography

(GC/GLC)

Supercritical fluid

chromatography

(SFC)

Liquid Column

Liquid-liquid

chromatography

(LC)

High performance

liquid

chromatography

(HPLC)

Ion exchange chromatography

Competition between an ion

exchange resin stationary phase

and liquid mobile phase

Liquid Column

Ion exchange

chromatography

(IEC)

High performance

ion chromatography

(IC/HPIC)

Permeation chromatography

Competition between a polymer

matrix and liquid mobile phase

Liquid Column

Gel permeation

chromatography

(GPC)

4

1.2. Capillary Liquid Chromatography

Miniaturization of separation columns has been investigated in liquid

chromatography (LC) for more than a quarter of a century. The application of capillary

columns had a rapid growth since the invention of fused silica capillary in 1979. The

development history of capillary based separation methods and their related technique

are shown in Table 1.2 [5].

LC can be classified based on the diameters of column. Columns with the

diameter less than 0.075 mm are used in nano-LC and the flow rate is less than 1

µL/min. The column diameter in micro-LC is 0.2-0.8 mm and its flow rate 1-20 µL/min.

Advantages of capillary columns in LC are attributed to narrow column diameter and

low eluent flow rate, which leads to the fact that capillary LC saves solvents, reagents

and packing materials, compared with conventional LC (4–6 mm i.d. columns). It

enables us to use the expensive chemicals and the samples which are available in

restricted amounts such as in the case of biological substances. This situation is quite

friendly to the environment. Increased mass sensitivity due to small-volume detection

can be expected in capillary LC. Low heat capacity of capillary columns permits much

easier control of column temperature, indicating that capillary columns for LC can also

5

be utilized for supercritical fluid chromatography and gas chromatography (GC). It will

also improve column efficiency of LC and facilitate hyphenation with MS, leading to

acquisition of more accurate analytical information [5-7].

Table 1-2. The development history of capillary based separation methods and their

related technique

Year Developed capillary separation

methods and their related technique

1974 μLC

1978 Open-tubular capillary LC

1978 Packed microcapillary LC

1979 Fused-silica capillary

1981 Capillary zone electrophoresis

1985 Electrokinetic chromatography

1987 Capillary electrochromatography

1998 Monolithic silica capillary column

1.3. Polymer Monolithic Stationary Phase in Liquid Chromatography

Monolithic columns for LC were introduced approximately two decades ago

and have been applied in most of the LC separation modes. In a monolithic column, the

microglobular skeleton is interconnected to form a continuous porous stationary phase

that is absent of structural void volumes that are sometimes present in packed columns.

The ease of fabrication of monolithic materials from liquid precursors in miniaturized

6

column formats has spurred the development of monolith stationary phases as an

alternative for packed-bed columns. Whereas the first monolithic columns were

prepared in large i.d. column formats, the preparation and application of capillary

column formats with diameters ranging between 20 and 530 μm are routinely reported

in literature. Furthermore, mass transfer resistance in and out of the stationary phase

support is less in monolithic stationary phases compared to packed bed because the

diameters of the microglobules are typically less than for spherical particles, and there

are more pores through which the mobile phase can flow [6,8].

According to their components, monolithic materials can be divided into

inorganic (i.e., silica-based monoliths) introduced by Tanaka and Nakanishi and organic

polymers materials introd ced b ec and r chet. The inorganic monoliths can be

prepared by the sol–gel method [9], and the organic polymer monoliths are generally

made by in situ polymerization of monomers, cross-linkers, and porogens [10-11]. Due

to their good permeability, fast mass transfer, high stability, and ease of modification,

monolithic materials have been regarded as the new generation matrix for LC.

Organic polymer monoliths have several distinct advantages in life-science

research, including wide pH stability, less irreversible adsorption, facile preparation,

7

and modification. Organic monoliths are generally prepared by thermal or photo-

initiated polymerization. In addition, microwave irradiation and γ-radiation initiated

polymerization have been also employed. Methods used to introduce active groups into

the monolith include copolymerization of cross-linker with a functional monomer,

grafting of pore surface, and a post modification of reactive monoliths. Among the three

methods mentioned previously, copolymerization of cross-linker and functional

monomer is the most straight forward strategy and allows the dynamic binding capacity

of the column to be controlled by adjusting the amount of the functional monomer in the

polymerization mixture [12].

The organic polymer-based monolithic columns can be classified into four

main groups, according to the monomer system used, they are (i) styrene-based

monoliths which are strongly hydrophobic, (ii) polynorbornene monoliths, non-polar

monoliths, (iii) methacrylate-based monoliths, which are moderately polar, and (iv)

acrylamide-based monoliths, which are highly polar [13].

8

1.4. Silica Bonded Stationary Phase in Liquid Chromatography

Silica-based materials are still the most commonly useful LC packing

materials owing to their high chromatographic efficiency and excellent mechanical

stability. In recent years, numerous improvements have been made on silica-based LC

packing materials to deal with problems caused by either the silica supports or the

bonded phases. However, contemporary silica-based reversed-phase (RP) LC packings

still have two major limits. First, basic compounds always suffer from peak tailing and

poor efficiency due to detrimental ionic interactions with acidic silanol groups; second,

the mobile phases are only allowed within the pH range of 2–8 due to the poor chemical

stability of silica-based packings. While improving the chemical stability of silica

particles is significant for extended pH chromatographic application, it is also a

challenging task. During the last decades, many researcher have put lots of efforts into

improving the chemical stability of silica-based stationary phases [6,14-15].

1.5. Ion Chromatography

Ion chromatography (IC) is an analytical technique for the separation and

determination of ionic solutes, where a liquid permeates through a porous solid

9

stationary phase and elutes the solutes into a flow-through detector. Ion exchange

columns are used to separate ions and molecules that can be easily ionized. Separation

of the ions depends on the ion's affinity for the stationary phase, which creates an ion

exchange system. The electrostatic interactions between the analytes, mobile phase, and

the stationary phase, contribute to the separation of ions in the sample. Positively or

negatively charged complexes can interact with their respective cation or anion

exchangers [1,16].

Common packing materials for ion exchange columns are diatomaceous

earth, styrene-divinylbenzene, and cross-linked polystyrene resins with amines or

sulfonic acid groups. Some of the first ion exchangers used were inorganic and made

from aluminosilicates (zeolites), although aluminosilicates are not widely used as ion

exchange resins. Ion-exchange resins are divided into four categories: strongly acidic

cation exchangers; weakly acidic cation exchangers; strongly basic anion exchangers;

and weakly basic anion exchangers. Table 1.3 provides a list of several common ion-

exchange resins [1].

10

Table 1.3. Common ion-exchange resins

Type Functional Group Examples

strongly acidic cation sulfonic acid –SO3–

–CH2CH2SO3–

weakly acidic cation

exchanger

carboxylic acid –COO–

–CH2COO–

Strongly basic anion

exchanger

quaternary amine –CH2N(CH3)3+

–CH2CH2N(CH2CH3)3+

Weakly basic anion

exchanger

amine –NH2

–CH2CH2N(CH2CH3)2

1.6. Hydrophilic Interaction Liquid Chromatography (HILIC)

The name hydrophilic interaction liquid chromatography HILIC was coined

by Alpert in 1990. HILIC is a type of the normal phase LC that uses mobile phases

typically containing more than 50% organic solvent in water. Acetonitrile is strongly

preferred organic solvent in most HILIC applications, as mobile phases containing other

solvents often provide insufficient sample retention and broad or non-symmetrical peak

shapes. HILIC is useful for an analysis of polar solutes whereas RPLC is for nonpolar

solutes. HILIC has a much wider variety of functional groups such as silanol, amine,

amide, diol, cyano, poly-succinimide, sulfoalkylbetaine, and zirconia. All polar

functionalities including anionic and cationic moieties on the HILIC packing material

11

surface can absorb some water (0.5%–1.0%) to form a stagnant water-enriched layer

between the mobile and stationary phases, especially when the water ratio is low in the

mobile phase. This layer is immobilized and can be considered as a portion of the

stationary phase. The transition between adsorption and partition mechanisms is

probably continuous as the water content in the mobile phase gradually increases [17-

19].

The interest in the HILIC technique in the last years has been promoted by

growing demands for the analysis of polar drugs, metabolites and biologically important

compounds in proteomics, glycomics and clinical analysis. HILIC has gradually found

useful applications in the analysis of various small polar compounds, such as

pharmaceuticals, toxins, plant extracts and other compounds important to food and

pharmaceutical industries [19-20].

1.7. Objectives of Present Research

Various types of stationary phases and separation modes have been

developed in capillary LC. The stationary phases are mainly polymer monolith-based

column and silica-based packing column. The ease of fabrication of monolithic

12

materials from liquid precursors in miniaturized column formats has spurred the

development of monolith stationary phases. On the other hand, although silica-based

packings have disadvantages such as inapplicability in strongly acidic or alkaline

mobile phases, and residual silanols which affect separation of biological samples, they

have specific advantages such as resistance to organic solvents, high rigidity, and fast

mass transfer. The rich silanol groups on the surface of silica gel allow easy

introduction of many functional groups onto the gel. Thereby, many researchers have

been interested in developing polymer monolith-based and silica-based stationary

phases. The main objective of present research is to develop a stationary phase for cLC,

which is prepared by a simple step, with low cost and high efficiency for separation of

inorganic anions and also organic compounds.

1.8. References

[1] A. Braithwaite and . J. mith, “Chromatographic Methods”, 5th ed., 1996, Kluwer

Academic Publisher, Dordrecht.

[2] D. Harvey, “Modern Analytical Chemistry”, 2000, McGraw Hill, New York.

13

[3] V. R. Me er, “Practical High performance Liquid Chromatography”, 2004, Willey,

New Jersey.

[4] L. R. Snyder, J. J. Kirkland, and J. W. Dolan, “Introduction to Modern Liquid

Chromatography”, 3rd ed., 2010, Willey, New Jersey.

[5] T. Takeuchi, Chromatography, 26, (2005), 7-10.

[6] D. Coradini, “Hand Book of HPLC”, 2nd ed., 2011, CRC Press, Boca Raton, USA.

[7] T. Takeuchi, Anal. Bioanal. Chem., 375, (2003), 26–27.

[8] G. Zhu, L. Zhang, H. Yuan, Z. Liang, W. Zhang, and Y. Zhang, J. Sep. Sci., 30,

(2007), 792–803.

[9] H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, and N. Tanaka, Anal. Chem., 68,

(1996), 3498–3501.

[10] E. F. Hilder, F. Svec, J. M. J. r chet, J. Chromatogr. A, 1044, (2004), 3–22.

[11] M. Bedair, Z. El Rassi, Electrophoresis, 25, (2004), 4110–4119.

[12] R. Bakry, C. W. Huck, and G. K. Bonn, J. Chromatogr. Sci., 47, (2009), 418-431.

[13] J. Urban, J. Sep. Sci., 31, (2008), 2521 – 2540.

[14] S. Wongyai, Chromatographia, 38, (1994), 485.

14

[15] M. Sun, J. Feng, S. Liu, C. Xiong, X. Liu, and S. Jiang, J. Chromatogr. A, 1218,

(2011), 3743.

[16] C. Eith, M. Kolb, A. Seubert, and K. H. Viehweger, “Practical Ion

Chromatography, An Introduction”, 2001, Methrohm Ltd., Herisau.

[17] P. G. Wang and W. He, “Hydrophilic Interaction Liquid Chromatography (HILIC)

and Advanced Aplication”, 2011, Taylor and Francis Group, LLC.

[18] A. J. Alpert, J. Chromatogr., 499, (1990) 177.

[19] P. Jandera, Analytica Chimica Acta, 692, (2011) 1–25.

[20] Z. Jiang, N. W. Smith, and Z. Liu, J. Chromatogr. A, 1218 (2011) 2350–2361.

15

Chapter 2

Methacrylate-based Diol Monolithic Stationary Phase for

Separation of Polar and Non-Polar Compounds in Capillary Liquid

Chromatography

2.1. Introduction

Polymer-based monolithic columns have gained increasing attention in

capillary liquid chromatography (cLC) and capillary electrochromatography since they

were first prepared in early 1990s [1]. Monolithic columns possess various advantages,

such as simple preparation and wide selection of monomers available with different

functional groups, easy control of permeability, no need to prepare frits, and moderate

phase ratios. Monolithic columns are often prepared by polymerization of functional

monomers for generation of the chromatographic interaction sites, via in-situ

polymerization with a cross-linking agent, monomers and some porogens in a capillary

tube. The polymerization conditions can hardly be changed because each variation of

the solvent composition has significant effect on the structure of the resulting materials

[2, 3].

16

Hydrophilic interaction liquid chromatography (HILIC) uses polar

stationary phases and allows the use of aqueous mobile phases, which is a useful

alternative and rival technique to reversed-phase LC (RPLC) for separating polar

compounds [4,5]. Recently, HILIC monolithic column has attracted increasing attention.

The application of monolithic columns in the HILIC mode has been studied for cLC, it

was successfully used for carbohydrates [6,7], protein [8,9], nucleosides [9,10], phenols

and vitamin C [11], and some small polar analytes [12].

The methacrylate-based polymers are one of the most widely researched

monoliths. Methacrylates are the most popular among polymer chemistries used as

separation media [13,14]. There are several advantages associated with methacrylate-

based monoliths, including high stability even under extreme pH conditions, fast and

simple preparation, a wide selection of monomers available with wide ranging polarities,

and easy functionalization when using glycidyl methacrylate (GMA) as the monomer.

The epoxy group in GMA can be further modified and they were processed successfully

as the stationary phase for the separation of various compounds [13-16].

In this research, we synthesized monolithic capillary columns by using

GMA and poly(ethylene glycol) dimethacrylate (PEGDMA). PEGDMA is a reactive

17

crosslinker which provides good flexibility, physiological inactivity, low toxicity, and

stability under many chemical conditions. The linked alkyl end-groups providing

hydrophobic interaction sites and the poly(ethylene glycol) (PEG) groups providing a

mildly hydrophilic matrix [17]. After hydrolysis of the epoxide functionalities with acid

[18], diol groups formed at the surface of the polymeric skeleton and thus monolithic

HPLC separation media was obtained. The composition of polymerization mixture was

optimized and the separation ability was investigated. The physical properties of the

monolithic column, such as permeability, mechanical stability were characterized. The

poly(GMA-PEGDMA) monolith capillary columns were applied to the determination

and separation of polar compounds such as phenol compounds and phthalic acids. The

poly(GMA-PEGDMA) monolith capillary columns in different composition of

monomer were also used as stationary phase for separation of non-polar compounds

polycyclic aromatic hydrocarbons (PAHs). PAHs are carcinogenic compounds and exist

in a wide range of environmental matrices, and they are normally analyzed with RPLC

methods using silica-based C18 packed columns.

18

2.2. Experimental

2.2.1. Apparatus

For LC separation the eluent was supplied by an L.TEX-8301 Micro Feeder

(L. TEX Corporation, Tokyo, Japan) equipped with an MS-GAN 050 gas-tight syringe

(0.5 mL; Ito, Fuji, Japan). A model M435 micro injection valve (Upchurch Scientific,

Oak Harbor, WA, USA) with an injection volume of 0.2 µL, was used as the injector. A

UV-970 UV-Vis detector (Jasco, Tokyo, Japan) was operated at 254 or 220 nm and all of

the data were collected by a CDS data processor (LASOFT, Chiba, Japan). The inlet

pressure was monitored by an L.TEX-8150 pressure sensor (L.TEX).

2.2.2. Reagents and chemicals

3-(trimethoxysilyl)propyl methacrylate, PEGDMA and 1-decanol were

purchased from Tokyo Chemical Industry (Tokyo, Japan). GMA (97% pure), phthalic

acid, pyrocathecol, uracil, toluene, naphthalene, fluorene, anthracene, fluoranthene,

pyrene, and benzo[a]pyrene were obtained from Nacalai Tesque (Kyoto, Japan). 2,2’-

Azobis(isobutyronitrile) (AIBN), methanol, ethanol, acetonitrile, sulfuric acid, salicylic

acid, isophthalic acid, phenol, and pyrogallol were obtained from Wako Pure Chemical

19

Industries (Osaka, Japan). All other reagents and solvents were of commercially

available highest grade and were used without further purification. The water used for

sample and mobile phase preparation was purified with a Milli-Q deionization system

(Nihon Millipore Kogyo, Tokyo, Japan).

2.2.3. Preparation of poly(GMA-PEGDMA) monolithic capillary column

The poly(GMA-PEGDMA) monolith was prepared by one-step

polymerization method, in which the polymerization was initiated by thermal treatment

of the mixtures of GMA as the functional monomer, PEGDMA as the crosslinker,

methanol and decanol as the binary porogenic solvents and AIBN as the initiator. In the

present study, the polymer monolith was in-situ synthesized in a fused-silica capillary

(150×0.32mm i.d.). Firstly, the inner surface of capillary was derivatized with 3-

(trimethoxysilyl)propyl methacrylate which makes it possible to anchor the polymer to

the wall, and then dried with N2 [8]. Subsequently, the polymerization mixture was

completely mixed by vortexing and ultrasonication to form a homogeneous solution.

The various compositions of the polymerization mixture investigated in this study are

shown in Table 2-1. Besides these compositions shown in Table 2-1, we also prepared

20

the poly(GMA-PEGDMA) containing less GMA, i.e. 2% GMA, while the porogen was

70%.

Finally, the capillary was immediately sealed with teflon tube and the

polymerization took place in water bath at 60 ˚C for 20 h. After polymerization, the

capillary was washed with acetonitrile to eliminate the unreacted porogenic solvents and

other soluble compounds. The resulting monolithic capillary was then flushed with 0.2

mol/l sulfuric acid solution at room temperature or at elevated temperatures (40 ˚C and

60 ˚C) for 2 h to open the epoxide groups in order to form diols at the surface of the

polymeric skeleton [18]. Then, the prepared poly(GMA-PEGDMA) monolithic

capillary was used for evaluation.

2.3. Results and Discussion

2.3.1. Column characterization

It is known that even minor change to the composition of the polymerization

mixture influences the performance of a methacrylate-based monolithic column [12,19].

After the ring-opening reaction, the poly(GMA-PEGDMA) monolith possesses diol

groups at the surface of the polymer skeleton, which contribute to the hydrophilicity of

21

the column, and the composition of GMA contained in the polymer will affect the

hydrophilic properties of the stationary phase. Poly(GMA-PEGDMA) monoliths with

different GMA contents were prepared and evaluated. Firstly, the ratio of the monomer

to the crosslinker (GMA:PEGDMA) was varied by keeping the same porogen content,

where the GMA composition was varied from 6 to 16% (v/v). In order to investigate the

morphology and the hydrophilic properties of the prepared monoliths, scanning electron

microscope (SEM) and LC were used. Figure 2-1 displays SEM photos of the

poly(GMA-PEGDMA) monoliths prepared by 6 and 12% (v/v) GMA, respectively.

Both of the photos show fullpacked monolith and the through pores in the polymer

skeletons are highly interconnected, forming a porous network of channels. And it can

also be observed that the monolith is attached tightly to the inner-wall of the capillary.

The skeleton of monolith approximate range in 0.5-1 µm and 2-3 µm for monolith

content 6% GMA and 12 % GMA, respectively.

22

A. 6 % GMA B. 12 % GMA

Fig. 2-1. Scanning electron microscopy images of the poly(GMA-PEGDMA)

monolithic capillary columns. 6 % GMA (A) and 12% GMA (B).

It was found that increasing the GMA content, i.e. decreased PEGDMA

content, improved the permeability of the columns. When the GMA content increased

from 6 to 12% (v/v), the permeability increased from 4.04 × 10-10

to 4.35 × 10-10

cm2

(Table 2-1). The content of GMA also affected the column efficiency. Increasing GMA

up to 12% (v/v) increases the column efficiency to around 18,000 theoretical plates per

meter at a flow rate of 3μl/min. When the content of GMA was increased to 16% (v/v),

the permeability and efficiency of the column became deteriorated. Therefore, the GMA

fraction was kept constant at 12% (v/v) in further experiments.

23

Table 2-1 Effect of different compositions of the polymerization mixture on the

efficiency and the permeability of the column.

Column Porogen

% (v/v)

GMA

%

(v/v)

PEGDMA

% (v/v)

Nmax

Plates/m

Ka,

Permeability

10-10

(cm2)

kb

Uracil

Condition

of

opening

epoxy

reaction

A 68 6 26 6000 4.04 0.52 RT, 2 h

B 68 8 24 6800 4.27 0.51 RT, 2 h

C 68 12 20 18000 4.35 0.81 RT, 2 h

D 68 16 16 17000 2.76 0.48 RT, 2 h

E 73 12 15 18000 4.16 0.39 RT, 2 h

F 78 12 10 22000 4.03 0.31 RT, 2 h

G 68 12 20 13000 5.16 0.80 40 °C, 2 h

H 68 12 20 12000 5.26 1.06 60 °C, 2 h

a: K= (µL/Δp)η, where η is the viscosity of acetonitrile [21], L is the column length (15

cm in this case), µ is the sol ent linear elocit , and Δp is the column back pressure.

b: Mobile phase is 98% acetonitrile.

The ratio of the porogen to the monomer content was also evaluated. When

the porogen weight fraction increased from 68% to 78%, the column efficiency

increased from ca.18,000 to 22,000 theoretical plates per meter. In common monolith

polymer column, the increase in porogen will increase the permeability of monolith,

however on the present monolith the permeability is slightly decreased with increasing

ratio of the porogens. The reason for this fact is not certain, unfortunately.

24

The effect of the reaction temperature for opening epoxy groups using

sulfuric acid was also investigated. Higher temperature i.e. 40 ˚C and 60

˚C, could

improve the permeability of column, but the efficiency became worse. It may be due to

the fact that higher temperature is not suitable for reaction of epoxy-ring-opening on

this column then the diols on surface of monolith became less. Therefore, 68% porogen

weight fraction and 12% GMA were chosen as the polymerization conditions, while

room temperature was chosen as the epoxy-opening reaction in further experiments.

Fig 2-2. Back pressure on column C (12% GMA) as a function of flow rate of

acetonitrile-water (98 : 2) as the eluent.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

1.5 2 2.5 3 3.5 4 4.5 5 5.5 6

Bac

k p

ress

ure

/ M

Pa

Flow rate / µL min-1

25

Figure 2-2 shows the back pressure of the column C as a function of the

flow rate of the mobile phase. Linear relationships between the back pressure and the

flow rate are clearly demonstrated, showing good mechanical stability of the monolith.

It should be noted that higher flow rate could be applied to the column. When the flow

rate was 6 µl/min, the back pressure was 1.9 MPa.

2.3.2. Chromatographic properties

In order to investigate the HILIC mode properties of present column, uracil

and toluene were used as test compounds where acetonitrile with variant concentration

from 50% to 98% were used as mobile phase. Figure 2-3 illustrates the retention time of

uracil and toluene as a function of acetonitrile concentration in the mobile phase for the

column C (150 × 0.32 mm i.d.). The retention time of uracil, which is a polar compound,

increased when the concentration of acetonitrile increased from 50% to 98% in mobile

phase, whereas the retention time of toluene, a non-polar compound, decreased with

increasing concentration of acetonitrile. At the lower concentration of acetonitrile, uracil

elutes before toluene whereas it elutes after toluene at higher concentration of

acetonitrile, showing dual retention capability works for polar and non-polar

26

compounds. Commonly, the critical compositions of the mobile phases corresponding

to the transition from the HILIC to the RP mode were around 70% acetonitrile in water.

The separations of polar analytes, such as phenols and amides, were commonly

performed with more than 90% acetonitrile content in the mobile phase to achieve good

retention [20]. The critical value of the poly(GMA-PEGDMA) monoliths stationary

phase was gained when the content of acetonitrile in the mobile phase exceeded 90%,

and for separation of phenols we used 98% acetonitrile as mobile phase. This result

indicates that this monolith still has low hydrophilicity.

On the hydrophilic mode, the retention factor (k) of uracil was influenced by

the composition of GMA. When the GMA content increased from 6 to 12% (v/v), k

increased from 0.52 to 0.81, and decreased for 16% GMA (Table 2-1). Since the

poly(GMA-PEGDMA) monolith possesses diol groups at the surface of the polymer

skeleton, it is expected to show separation ability towards polar compounds due to the

specific interactions such as dipole–dipole and hydrogen bonding interactions.

27

Fig. 2-3. Retention times of uracil and toluene as a function of acetonitrile concentration

in mobile phase. Column, monolithic poly(GMA-PEGDMA), 12% GMA (150 × 0.32

mm i.d.); mobile phase, acetonitrile-water mixtures; flow rate, 3 µl/min; wavelength of

UV detection, 254 nm; analytes, 0.1 % (w/v) each of uracil and toluene; injection

volume, 0.2 µl.

The prepared monolithic column was evaluated by the separation of polar

compounds which are neutral and weakly acidic aromatic compounds. Phenols are one

of large category of environmental pollutants, and therefore, the separation of three

phenols was investigated on the 12% GMA monolith under an isocratic elution

2.00

4.00

6.00

8.00

10.00

12.00

30 40 50 60 70 80 90 100

Ret

enti

on t

ime

/ m

in

Conc. of acetonitrile / %

Toluene

Uracil

28

condition. A typical chromatogram is shown in Figure 2-4. It can be seen that the

separation of three compounds was completed in 8 min, and it is shown that

pyrocathecol has better separation efficiency compared to the others.

Fig. 2-4. Separation of phenols. Column, monolithic poly(GMA-PEGDMA), 12% GMA

(150 × 0.32 mm i.d.); mobile phase acetonitrile-water (98 : 2); flow rate, 3 µl/min;

wavelength of UV detection, 254 nm; analytes, 0.1 % (w/v) each of phenol (1),

pyrocathecol (2) and pyrogallol (3); injection volume, 0.2 µl.

Weak organic acids such as salicylic acid, phthalic acid and isophthalic acid

could also be separated using the same column. The chromatogram is shown in Figure

0 2 4 6 8 10 12 14

0.001 Abs1

2

3

Time / min

29

2-5. Using 98% acetonitrile as the mobile phase, three compounds could be separated

completely.

Fig. 2-5. Chromatogram of aromatic acids. Column, monolithic poly(GMA-PEGDMA),

12% GMA (150 × 0.32 mm i.d.); mobile phase, acetonitrile-water (98 : 2); flow rate, 3

µl/min; wavelength of UV detection, 220 nm; analytes, 0.1 % (w/v) each of salicylic

acid (1), phthalic acid (2) and isophthalic acid (3); injection volume, 0.2 µl.

Since the poly(GMA-PEGDMA) monolith 12% GMA showing dual

retention capability works for polar and non-polar compounds, depending on the

concentration of acetonitrile contained in mobile phase, the separation of PAHs was

0 2 4 6 8 10 12

0.001 Abs

1

2

3

Time / min

30

investigated on the present column. Figure 2-6 shows the separation of six typical PAHs

by using the developed monolithic capillary column with isocratic elution using 70%

acetonitrile as the mobile phase. Six PAHs were separated completely in reasonable

time.

Fig. 2-6. Chromatogram of the separation of PAHs. Column, monolithic poly(GMA-

PEGDMA), 12% GMA (150 × 0.32 mm i.d.); mobile phase, acetonitrile-water (70 : 30);

flow rate, 3 µl/min; wavelength of UV detection, 254 nm; analytes 0.1 % (w/v) each of

naphthalene (1), fluorene (2), anthracene (3), fluoranthene (4), pyrene (5),

benzo[a]pyrene (6); injection volume, 0.2 µl.

0 2 4 6 8 10 12 14 16 18 20 22 24

0.005 Abs

1

2

3

4 5

6

Time / min

31

On the other hand, the monoliths prepared from 2% GMA showed

hydrophobic property. Polar compounds could not be retained on the 2% GMA column.

It is expected that low GMA content in monolith causes less diol groups at the surface

of the polymer skeleton. The hydrophobicity of monoliths was also expected due to the

high methyl groups content in the alkyl end-groups of the PEGDMA. The poly(GMA-

PEGDMA) monolith with 2% GMA was evaluated by using the separation of PAHs.

Using acetonitrile-water as the mobile phase, the six PAHs were separated completely.

Effect of the acetonitrile concentration in mobile phase on the retention of PAHs was

investigated. As shown in Figure 2-7, decreasing acetonitrile content in the mobile

phase increases the retention time of PAHs. It is also found that the poly(GMA-

PEGDMA) monolith with 2% GMA shows good mechanical stability. The back

pressure of the latter column as a function of the flow rate of the mobile phase has a

linear relationship. When the flow rate was 6 µl/min, the back pressure was 2.6 MPa for

the column with 27 cm in length. It shows that higher flow rates can be applied to the

column.

32

Fig. 2-7. Effect of the acetonitrile concentration in mobile phase on the retention of

PAHs. Column, monolithic poly(GMA-PEGDMA), 2% GMA (270 × 0.32 mm i.d.);

mobile phase, acetonitrile-water; wavelength of UV detection, 254 nm; analytes 0.1 %

(w/v) each of naphthalene (1), fluorene (2), anthracene (3), fluoranthene (4), pyrene (5),

benzo[a]pyrene (6); injection volume, 0.2 µl.

2.4. Conclusions

A polymethacrylate-based diol monolithic column based on the

copolymerization of GMA and PEGDMA in the presence of porogens was prepared and

successfully used as stationary phase in HILIC mode. The poly(GMA-PEGDMA)

60 65 700

0.2

0.4

0.6

0.8

1

1.2

12

3

45

6

Conc. of acetonitrile / %

Logk

33

monoliths possess diol groups at the surface of the porous monolith that ascribed to

hydrophilicity of the monolith. Increasing the GMA content in the polymerization

mixture increased hydrophilicity. Phenols, phthalic acid and PAHs were retained on the

stationary phases using acetonitrile-water mixtures as the mobile phase.

However, the higher hydrophilicity of the monolith still remains as a

challenging task due to the fact that separation of polar compounds still needs higher

concentration of acetonitrile as mobile phase.

2.5. References

[1] T. B. Tennikova, B. G. Belenkii, and F. Svec, J. Liq. Chromatogr., 13,(1990), 63.

[2] F. Svec and J. M. J. Fréchet, Anal. Chem., 64, (1992), 820.

[3] F. Svec and J. M. J. Fréchet, Chem. Mater., 7, (1995), 707.

[4] A. J. Alpert, J. Chromatogr., 499, (1990), 177.

[5] Z. J. Jiang , N. W. Smith , P. D. Ferguson, and M. R. Taylor, Anal. Chem., 79,

(2007), 1243.

[6] T. Ikegami, K. Horie, N. Saad, K. Hosoya, O. Fiehn, and N. Tanaka, Anal. Bioanal.

Chem., 391, (2008), 2533

34

[7] J. Lin, S. F. Liu, X. C. Lin, and Z. H. Xie, J. Chromatogr. A, 1218, (2011), 4671.

[8] Y. Li, H. D. Tolley, and M. L. Lee, J. Chromatogr. A, 1217, (2010), 8181.

[9] M. A. Jaoude and J. Randon, Anal. Bioanal. Chem., 400, (2011), 1241.

[10] Z. J. Jiang , N. W. Smith , P. D.Ferguson , and M. R. Taylor, J. Sep. Sci., 32,

(2009), 2544.

[11] Z. Liu, Y. Peng, T. Wang, G. Yuan, Q. Zhang, J. Guo, and Z. Jiang, J. Sep. Sci., 36,

(2013), 262.

[12] P. Jandera, J. Urban, V. Skerikova, P. Langmaier, R. Kubickova, and J. Planeta, J.

Chromatogr. A, 1217, (2010), 22.

[13] D. Lee, F. Svec, and J. M. Fréchet. J. Chromatogr. A, 1051, (2004), 53.

[14] Y. Ueki, T. Umemura, Y. Iwashita , T. Odake, H. Haraguchi, and K. Tsunoda, J.

Chromatogr. A, 1106, (2006), 106.

[15] F. Svec, E. C. Peters, D. Sykora, and J. M. J. Fréchet, J. Chromatogr. A, 887,

(2000), 3.

[16] M. M. Dittmann, K. Masuch, and G. Rozing, J. Chromatogr. A, 887, (2000), 209.

[17] Y. Li, H. D. Tolley, and M. L. Lee, J. Chromatogr. A, 1217, (2010), 4934.

[18] Y. Wen and Y. Q. Feng, J. Chromatogr. A, 1160, (2007), 90.

35

[19] J. Urban and P. Jandera, J. Sep. Sci., 31, (2008), 2521.

[20] X. Wang, H. Lu¨, X. Lin, and Z. Xie, J. Chromatogr. A, 1190, (2008), 365.

[21] L. R. Snyder, J. J. Kirkland, and J. W. Dolan, “Introduction to Modern Liquid

Chromatography”, 3rd ed., 2010, Willey, New Jersey, 884.

36

Chapter 3

Poly(ethylene oxide)-bonded Stationary Phase for Separation of

Inorganic Anions in Capillary Ion Chromatography

3.1. Introduction

Ion chromatography (IC) has been a well-established analytical technique

and the preferred method for the analysis of inorganic or small organic anions and

cations. Various types of stationary phases and separation modes have been developed

in IC since its introduction in 1975 [1]. IC stationary phases are mainly polymer-based

and silica-based. Although silica-based packings cannot be used in strongly acidic or

alkaline mobile phases, and have residual silanols which affect separation of biological

samples, they have specific advantages such as resistance to organic solvents, high

rigidity, and fast mass transfer. In addition, the rich silanol groups on the surface of

silica gel allow easy introduction of many functional groups onto the gel. Thereby,

many researchers have been interested in developing silica-based stationary phases for

IC [1-3]. Liu et al. prepared ammonium anion-exchange stationary phases by chemical

bonding, deactivation, and quaternization on silica. Anions in oil field water were

37

determined successfully on this column [4]. Phenylpropanolamine modified silica for

separation of acidic, basic, and uncharged drugs, and inorganic anions by reversed-

phase and anion-exchange interactions was prepared by Wongyai et al. Modifying the

pH of the mobile phase manipulated the retention of anions [5]. The preparation and

characterization of a novel multi-interaction stationary phase based on 4,4´-dipyridine

modified silica were described [6]. Different series of analytes including PAHs, phenols,

inorganic and organic anions were successfully separated on this multi-interaction

stationary phase, respectively. The multi-interaction mechanism including π–π,

hydrophobic, electrostatic and anion-exchange interactions is involved in the

chromatographic separation.

A poly(ethylene glycol) (PEG) stationary phase was used as the stationary

phase in reversed-phase LC by Guo et al. for separation of phenyl compounds and

Chinese medicine [7]. Besides the hydrophobic interaction, PEG moieties can provide

some other interaction such as hydrogen bonding and dipole-dipole interaction. The

PEG moiety also could form a helix-like conformation in the organic aqueous media.

PEG-bonded silica-based columns e.g., Discovery HS PEG column (Supelco;

Bellefonte, PA) are now commercially available and they have been applied to analysis

of natural products [8-9].

38

Most stationary phases employed in IC have functional groups with charged

or chargeable moieties, where ionic analytes experience electrostatic attractive or

repulsive forces. Inorganic anions could also be separated even if the stationary phase

possesses no ion exchange site. Lamb and Smith prepared a crown ether-bonded

stationary phase and a cryptand-bonded stationary phase for the separation of alkali

metal cations as well as anions. It was reported that the permanently coated alkylated

macrocycles such as n-tetradecyl-18-crown-6 ether (18C6) or n-decylcryptands onto a

reversed-phase column formed positively charged anion-exchange sites when they

combined with eluent cations. Using 18C6 as the eluent additive, it was found that

anion retention increased with increasing eluent strength and organic modifier content

[10]. Rong et al. developed and examined PEG or poly (ethylene oxide) (PEO) as the

stationary phase for IC by physically coating or chemically bonding [11-15]. For

physically coated or chemically bonded PEG/PEO on C30, partitioning was dominantly

involved for the retention of analyte anions [11-13]. On the other hand, the chemically

bonding on aminopropylsilica improved the selectivity for anions, where it was

expected that anions retained in the ion exchange mode. It was suggested that the

cations of eluent coordinated among the multiple PEO chains via ion-dipole interaction,

work as the anion exchange sites [14-15]. The present work examines a new

39

PEGylation reagent for the preparation of the PEO-bonded stationary phase for the

separation of inorganic anions in capillary IC. The tosylated-poly(ethylene oxide)

reagent was cheaper compared with previous PEGylation reagent such as methyl-PEO-

NHS (N-hydroxysuccinimide) ester which is very expensive.

3.2. Experimental

3.2.1. Apparatus

The chromatographic measurements were carried out by using a μLC

system. The eluent was supplied by a pump L.TEX-8301 Micro Feeder (L. TEX

Corporation, Tokyo, Japan) equipped with an MS-GAN 050 gas-tight syringe (0.5 mL;

Ito, Fuji, Japan). A model M435 micro injection valve (Upchurch Scientific, Oak Harbor,

WA, USA) with an injection volume of 0.2 µL, was used as the injector. A UV-970 UV-

Vis detector (Jasco, Tokyo, Japan) was operated at 210 nm and all of the data were

collected by a CDS data processor (LASOFT, Chiba, Japan). The inlet pressure was

monitored by an L.TEX-8150 Pressure sensor (L.TEX). Elemental analysis of the

packing materials was carried out by using an MT-6 CHN Corder (Yanaco, Kyoto,

Japan).

40

3.2.2. Reagents and materials

Reagents employed were of guaranteed reagent grade and were obtained

from Wako Pure Chemical Industries (Osaka, Japan), unless otherwise noted.

Polyethylene glycol monomethyl ether p-toluenesulfonate (Tosylated-PEO, M.W. 1000,

n ≈ 18.5) was obtained from Aldrich (Rockford, IL, USA). Purified water was produced

in the laboratory by using a GS-590 water distillation system (Advantec, Tokyo, Japan).

All solutions used in this work were prepared using the purified water. Porous 3-

aminopropylsilica, TSKgel NH2-60 (5 μm particle diameter, 60 Å mean pore diameter)

was taken from the packed column obtained from TOSOH (Tokyo, Japan).

3.2.3. Preparation of PEO-bonded stationary phase

A 0.21 g of TSKgel NH2-60 and 0.35 g of tosylated-PEO added into 5 mL

of acetonitrile in a 20-mL vial, and the solution was then poured into the stainless steel

tubing with 4.6 mm i.d. The reaction was taken place in oven at 75 ºC for 24 h, followed

by washing with methanol. Figure 3-1 shows the expected scheme of the reaction. The

separation column was prepared from a fused silica capillary tube (0.32mm i.d.×100

mm) using a slurry packing method previously reported [16], followed by washing with

a phosphate buffer (pH 7.5) for 1 h.

41

Fig. 3-1. The structure of the PEO reagent employed as well as the expected reaction

scheme.

3.2.4. Preparation of water and saliva samples

For saliva sample, a 0.4 mL of saliva diluted in 2 mL deionized water, and

centrifuged at 3000 rpm for 5 minute, followed by filtration with a 0.45µm membrane

filter. The saliva sample was then stored in the refrigerator. Tap water samples were

centrifuged at 3000 rpm for 3 minute, followed by filtration using a 0.45µm membrane

filter.

42

3.3. Results and Discussion

3.3.1. Column characterization

The aminopropylsilica and the PEO-bonded stationary phase were

characterized by elemental analysis. The percentages by weight for carbon and

hydrogen that contained in aminopropylsilica (1.1 mmol/g) were 5.18 and 1.55%,

respectively, while the percentages by weight for the latter stationary phase were 10.08

and 2.30% for carbon and hydrogen, respectively. The increase of the carbon and

hydrogen content for the latter demonstrated that PEO was bonded to aminopropylsilica

successfully. According to the results, carbon and hydrogen contained in the PEO-

bonded stationary phase were 8.40 and 22.8 mmol/g, respectively. The surface coverage

of the PEO on the present stationary phase was 0.12 mmol/g, which was calculated

from the difference in the carbon content between the aminopropylsilica and the PEO-

bonded stationary phase. In other words, 11% of amino groups of the aminopropylsilica

were bonded with PEO.

3.3.2. Retention behaviors of anions on PEO-bonded stationary phase

The prepared stationary phase could retain six anions satisfactorily. The

separation was achieved in reasonable time. It was suggested that the cations of eluent

43

coordinated with the oxygen atoms of multiple POE chains via ion-dipole interaction,

work as the anion exchange sites. The eluent and analyte anions can compete for the

trapped cation [14-15]. The proposed coordinating of cation structure is shown in

Figure3-2. Inorganic anions can therefore be separated on the POE-bonded phases in the

ion-exchange mode. The elution order was iodate, bromate, bromide, nitrate, iodide, and

thiocyanate. The elution order of the anions observed was similar as that observed in

common IC. It was also found that bromide and nitrite could not be separated on the

prepared PEO-bonded stationary phase; such retention behavior was also observed in

other works using PEO-bonded stationary phases [14-15].

Fig. 3-2. The proposed structure for coordinating of eluent cation.

44

Aminopropylsilica as based material has amino group that will be an anion

exchange site, but the prepared stationary phase provided better selectivity and strong

retention for the six anions, compared with the unmodified NH2-60 stationary phase.

Using 10 mM sodium chloride as the eluent at a flow rate of 3 µL/min, the six anions

could not be separated completely on the unmodified NH2-60 stationary phase, while

the prepared PEO-bonded stationary phase achieved better separation. Figure 3-3

demonstrates the separation of the six anions on the PEO-bonded aminopropylsilica and

unmodified NH2-60 stationary phase. The PEO chains which are bonded into the

aminopropylsilica seem to play as main role as anion exchange by coordination of the

cations of eluent, leading to the amino group derivatization on the prepared stationary

phase. It should be noted that separation columns were washed with phosphate buffer

(pH 7.5) before use, as described on the preparation of stationary phase. In addition, as

for the derivatized amino groups, it is expected that they do not retain analyte anions

under neutral conditions. The repeatability of the retention time was evaluated under the

operating conditions in Figure 3-3. The relative standard deviations of the retention time

for six measurements were 1.1-4.3%.

45

Fig. 3-3. Separation of six anions on PEO-bonded aminopropyl silica (A) and

unmodified NH2-60 (B) stationary phase. Column, (100 × 0.32 mm i.d.); eluent, 10 mM

sodium chloride; flow-rate, 3.0 μL/min; analytes, iodate (1), bromate (2), bromide (3),

nitrate (4), iodide (5), thiocyanate (6); concentration of analytes, 0.5 mM each; injection

volume, 0.2 μL; wavelength of UV detection, 210 nm.

We also found that the reaction time for the chemically bonding of the PEO

influenced the stability of the prepared stationary phase. The reaction time was varied

from 8 to 24 h, and the stability of the prepared stationary phase increased with the

0 5 10 15 20 25 30

1

2

3

45

6

1,2,3,4

5,6

0.003 Abs

A

B

Time/min

46

increasing reaction time according to the relative standard deviations of the retention

time for measurements. In this work, 24 h was chosen as the optimum reaction time in

preparing PEO-bonded stationary phases since the result showed better stability, which

means lower relative standard deviations of the retention time compared with the other

reaction time.

3.3.3. Effect of eluent cation and eluent anion

Since eluent cations are expected to be coordinated among multiple PEO

chains and work as the anion exchange sites [15,17], the eluent cations could affect the

retention of anions on the prepared stationary phase. The different eluent cations

showed different retention behavior of analyte. Compared with the crown ether which is

a cyclic PEO and its well-known ability to selectively complex with various cations of

alkali and alkaline earth metal ions, the coordinating of eluent cation on the PEO

bonded stationary phase is similar with complexing the cations on the crown ether but

more flexible [17]. It seems the size of the cations may not be very significant for

coordinating the eluent cations. The decrease in the retention of anions was observed

when the CsCl solution used as eluent. It seems that the amount of coordinated cesium

cation on the stationary phase was smaller than those of other monovalent cations when

47

the eluent concentrations were the same.

Fig. 3-4. Separation of six anions on PEO-bonded stationary phase using 10 mM

aqueous solutions of various chlorides. PEO-bonded aminopropyl silica (100 × 0.32 mm

i.d.); eluents, as indicated; other operating conditions as in Fig. 3-3.

0 5 10 15 20 25 30

0.005Abs

Time/min

LiCl

NaCl

KCl

RbCl

CsCl

MgCl2

NH4Cl

1

23

4 5

6

48

The largest retention time was achieved using ammonium chloride solution

for eluent, the reason for this fact is not certain, unfortunately. Figure 3-4 shows the

separation of six anions on PEO-bonded stationary phase using 10 mM aqueous

solution of various chlorides. The elution order for all of the eluent examined was same.

Among the eluents examined, since sodium chloride gave the best resolution, sodium

chloride was used as the eluent in the following experiments.

The effect of the eluent anion was examined using 10 mM aqueous solutions

of sodium salt. The eluent anion affected the retention of analyte anions, which is

reasonable because the eluent anion competed with the analyte anion for the anion-

exchange site. It can be seen that sodium chloride gave the best separation with the

longest retention, followed by sodium perchlorate, sodium dihydrogenphosphate and

sodium sulfate, as demonstrated in Figure 3-5. The poor chromatographic performance

was observed while sodium dihydrogenphosphate as the eluent. Compared with the

other eluents which are formally neutral, dihydrogenphosphate is weakly acidic and it

seems reasonable to assume that poor chromatographic performance is associated with

the protonation of the amino groups on the stationary phase.

49

Fig. 3-5. Separation of six anions on PEO-bonded stationary phase using 10 mM of

various sodium aqueous solutions. Eluents, as indicated; other operating conditions as in

Fig. 3-4.

0 5 10 15 20 25 30

0.005 Abs1

23

4 5

6

Na2SO4

NaClO4

NaH2PO4

NaCl

Time/min

50

3.3.4. Effect of salt concentration

The effect of salt concentration as the eluent on the retention of analyte

anions was examined using sodium chloride as the eluent. As found in common IC, the

retention of analyte anions decreased with increasing eluent concentration. It is also

known that the plots of the logarithm of the retention factor (log k) of analyte anions as

a function of the logarithm of the eluent concentration are linear. Figure 3-6

demonstrates the log k of analyte anions versus the logarithm of sodium chloride

concentration.

Fig. 3-6. The logarithm of retention factor (log k) of analyte anions as a function of the

logarithm of the eluent concentration. Operating conditions as in Fig. 3-4 except for the

eluent as indicated.

51

It can be seen that the plots are almost linear for all analyte anions, except

on the higher concentration of eluent. The slopes of lines are in range -0.42 to-0.47.

Theoretically, if ion exchange is involved alone in the retention, the slopes of lines

should be -1.0 because the analyte anions and the chloride are monovalent in this case.

Contrarily, it was expected in our case that the amount of the coordinated cation

increases with increasing eluent concentration, leading to the increase in the retention of

analyte anions. This may be a reason for observed lower slopes than expected.

3.3.5. Application to real sample

Fig.3-7. Separation of inorganic anions contained in saliva. Eluent, 10 mM sodium

chloride; other operating conditions as in Fig. 3-4.

0 5 10 15 20 25 30

0.003 Abs

1

2 3

4

5

6

4

5

6

Saliva

Standard

Time/min

52

Fig. 3-8. Separation of inorganic anions contained in tap water. Operating condition as

in Fig. 3-4.

The separations of inorganic anions contained in saliva and tap water on a

10 cm prepared column are demonstrated in Figures 3-7 and 3-8, respectively. The

saliva was taken from a healthy non-smoker. Nitrate, iodide and thiocyanate in the

saliva sample could be determined to be 2.6, 9.1 and 3.4 mg/L, respectively. Figure 3-7

shows the retention of the analytes are slightly different for the saliva and the standard

samples, especially for thiocyanate, however it has been proved by using the spiked

samples. On the other hand, nitrate contained in the tap water could be determined to be

2.6 mg/L. The signal of the anions could be enhanced by detecting at 210 nm.

0 5 10 15 20 25 30

0.003 Abs

Time/min

4

1

2 3

4

5

6

Tap water

Standard

53

3.4. Conclusions

The poly(ethylene oxide) stationary phase could be used for the ion

exchange separation of inorganic anions. Cations and anions of the eluent affected the

retention of the analyte anions. The retention of the analyte anions decreased with

increasing eluent concentration.

However, the long-term stability of the stationary phase still remains as a

challenging task due to the fact that the retention time of the anions decreased with

increasing analysis time.

3.5. References

[1] H. Small, T. S. Stevens, and W. C. Bauman, Anal. Chem., 47, (1975), 1801.

[2] Y. Liu, Q. Du, B. Yang, F. Zhang, C. Chu, and X. Liang, Analyst, 137, (2012), 1624.

[3] H. Qiu, X. Liang, M. Sun, and S. Jiang, Anal.Bioanal. Chem., 399, (2011), 3307.

[4] X. Liu, S. X. Jiang, L. R. Chen, Y. Q. Xu, and P. Ma, J. Chromatogr. A, 789, (1997),

569.

[5] S. Wongyai, Chromatographia, 38, (1994), 485.

[6] M. Sun, J. Feng, S. Liu, C. Xiong, X. Liu, and S. Jiang, J. Chromatogr. A,1218,

(2011), 3743.

54

[7] Z. Guo, Y. Liu, J. Xu, Q. Xu, X. Xue, F. Zhang, Y. Ke, X. Liang, and A. Lei. J.

Chromatogr. A, 1191, (2008), 78.

[8] F. Pellati, S. Benvenuti, and M. Melegari, J. Chromatogr. A, 1088, (2005), 205.

[9] F. Cacciola, P. Jandera, Z. Hajdu, P. Cesla, and L. Mondello, J. Chromatogr. A,

1149, (2007), 73.

[10] J. D. Lamb, R. G. Smith, and J. Jagodzinski, J. Chromatogr. A, 640, (1993), 33.

[11] L. Rong and T. Takeuchi, J. Chromatogr. A, 1042, (2004), 131.

[12] L. Rong, L. W. Lim, and T. Takeuchi, Chromatographia, 61, (2005), 371.

[13] L. Rong, L. W. Lim, and T. Takeuchi, J. Chromatogr. A,1128, (2006), 68.

[14] T. Takeuchi, B. Oktavia, and L. W. Lim, Anal. Bioanal. Chem., 393, (2009), 1267.

[15] T. Takeuchi and L. W. Lim, Anal. Sci., 26, (2010), 937.

[16] T. Takeuchi and D. Ishii, J. Chromatogr., 213, (1981), 25.

[17] L. W. Lim, L. Rong, and T. Takeuchi, Anal. Sci., 28, (2012), 205.

55

Chapter 4

Tetraethylene oxide-bonded Stationary Phase for Separation of

Inorganic Anions and Polar Compounds in Capillary Liquid

Chromatography

4.1. Introduction

Benefit of liquid chromatography (LC) mostly comes from plentiful

stationary phases with good performance, and it exhibits excellent advantages in the

separation and analysis of complex samples. Chromatographic performance is

influenced directly by separation mechanism of the stationary phase. For traditional

stationary phases, the main separation mechanism is often single, such as hydrophobic

interaction for separating nonpolar compounds on reversed-phase packing, and ion-

exchange interaction for separating ions on ion-exchange packing [1].

Various types of stationary phases and separation modes have been

developed in IC since its introduction in 1975 [2]. Researchers have been developing

silica-based stationary phases for IC [3-17]. Silica has specific advantages such as

resistance to organic solvents, high rigidity, and fast mass transfer. In addition, the rich

56

silanol groups on the surface of silica gel allow easy introduction of many functional

groups onto the gel, but it also has disadvantages that cannot be used in strongly acidic

or alkaline mobile phases, and have residual silanols which affect separation of

biological samples. Wongyai [7] prepared phenylpropanolamine bonded silica which

displayed both anion-exchange and reversed-phase characteristic, and quantitative

determination of vitamin B12 in multivitamin tablets was performed by this stationary

phase. Liu et al. was successfully determined anions in oil field water using their

ammonium anion-exchange stationary phases which are prepared by chemical bonding,

deactivation, and quaternization on silica [8]. A novel multi-interaction stationary phase

based on 4,4´-dipyridine modified silica were prepared and characterized by Sun et al.

[9]. Inorganic and organic anions were separated individually in anion-exchange

chromatography, while polycyclic aromatic hydrocarbons and phenols were also

successfully separated respectively in reversed-phase chromatography by using the

same column.

In most cases in ion chromatography (IC), ions are separated based on the

difference in electrostatic attraction or repulsion between analyte and the stationary

phase. Therefore, the stationary phase employed for IC has functional groups with

charged or chargeable moieties. Beside various ion exchanger, inorganic anions could

57

also be separated even if the stationary phase possesses no ion exchange site. A few

groups have studied and prepared a stationary phase for IC with non charge or

chargeable moieties. Lamb and Smith prepared a crown ether-bonded stationary phase

and a cryptand-bonded stationary phase for the separation of alkali metal cations as well

as anions [10,11]. Rong et al. developed and examined poly ethylene glycol (PEG) as

the stationary phase for IC in determination of iodide and thiocyanate in seawater

samples by physically or chemically coating [12-14]. Takeuchi et al. have developed the

chemically bonding of polyoxyethylene on aminopropylsilica stationary phase for

determination of inorganic anions in saliva sample. It was expected that anions retained

in the ion exchange mode, which was suggested that the cations of eluent coordinated

among the multiple PEO chains via ion-dipole interaction, work as the anion exchange

sites [15-16]. Takeuchi also separated inorganic anions on a chemically bonded 18-

crown-6 ether (18C6E) stationary phase in capillary IC [17].

In the previous work, we have examined a new PEGylation reagent for the

preparation of the PEO-bonded stationary phase for the separation of inorganic

anions in capillary IC. The tosylated-poly(ethylene oxide) reagent was cheaper

compared with PEGylation reagent such as methyl-PEO-NHS (N-hydroxysuccinimide)

ester which is very expensive. In this work, the shorter chain of ethyleneoxide;

58

tetraethylene glycol monomethyl ether or tetraethylene glycol modified silica were

synthesized and investigated as stationary phase for IC. The prepared stationary phase

also examined for separation of polar compounds such as phenol compounds. There is a

slight difference in performance on both columns in separation of inorganic anions and

phenols. In addition, the present reagents commercially available are not expensive

compared with the two previous reagents.

4.2. Experimental

4.2.1. Apparatus

The chromatographic measurements were carried out by using a μLC

system. The eluent was supplied by a pump L.TEX-8301 Micro Feeder (L. TEX

Corporation, Tokyo, Japan) equipped with an MS-GAN 050 gas-tight syringe (0.5 mL;

Ito, Fuji, Japan) at flow rate 3 μL min-1

. A model M435 micro injection valve (Upchurch

Scientific, Oak Harbor, WA, USA) with an injection volume of 0.2 µL, was used as the

injector. A UV-970 UV-Vis detector (Jasco, Tokyo, Japan) was operated at 210 nm or

254 nm. All of the data were collected by a CDS data processor (LASOFT, Chiba,

Japan). The inlet pressure was monitored by an L. TEX-8150 Pressure sensor (L. TEX).

59

Elemental analysis of the packing materials was carried out by using an MT-6 CHN

Corder (Yanaco, Kyoto, Japan).

4.2.2. Reagents and materials

Tetraethylene glycol monomethyl ether (TEGMM) and 3-

glycidyloxypropyltrimethoxysilane (GPTMS) were obtained from Tokyo Chemical

Industry (Tokyo, Japan), tetraethylene glycol (TEG), pyrocathecol, uracil and toluene

were purchased from Nacalai Tesque (Kyoto, Japan). Phenol and pyrogallol were

obtained from Wako Pure Chemical Industries (Osaka, Japan). All other reagents and

solvents were of commercially available highest grade and were used without further

purification. Purified water was produced in the laboratory by using a GS-590 water

distillation system (Advantec, Tokyo, Japan). Inorganic sample solutions and eluents for

IC used in this work were prepared using the purified water. Toluene was dried over

molec lar sie es 3A (1/16” pellets). The a erage particle diameter of the silica gel

emplo ed was 5 μm with mean pore diameter of 12 nm (Chemical Evaluation and

Research Institute, Japan), and the silica gel was dried at 110°C for 4 h before use.

Stainless-steel tubing with 250 × 4.6 mm i.d. was used as the reaction vessel.

60

4.2.3. Preparation of TEGMM-bonded and TEG-bonded stationary phases

A 0.2-g amount of the dried porous silica gel, 3.5 mL dry toluene and 0.2

mL of GPTMS was placed in a 20-mL vial and the solution was then poured into the

stainless steel tubing. The reaction was carried out at 110°C for 20 h. The vessel was

sometimes manually vibrated during the reaction. After the reaction, the reacted silica

gel was washed with dry toluene and dried at 75°C for 4 h. The dried reacted silica was

added with 3.5 mL of N,N-dimethyl formamide (DMF) and 0.2 mL of TEG or TEGMM,

and then was poured into the reaction vessel. The reaction was taken place in oven at

120 ºC for 24 h for TEG and at 75ºC for 24 h for TEGMM, followed by washing with

methanol. Figure 4-1 shows the expected scheme of the reaction. The separation column

was prepared from a fused silica capillary tube (0.32 mm i.d. × 100 mm) using a slurry

packing method previously reported [18].

61

Fig. 4-1. The scheme of the expected reactions. TEGMM, R = CH3; TEG, R = H

4.3. Results and Discussion

4.3.1. Elemental analysis of the stationary phases

The TEGMM and TEG bonded stationary phases were characterized by

elemental analysis. The percentages by weight for carbon and hydrogen contained in the

Si-GPTMS-TEGMM were 7.87 and 1.72 %, while the percentages by weight for the Si-

GPTMS-TEG were 7.45 and 1.64%, respectively. According to the results, carbon and

hydrogen contained in the Si-GPTMS-TEGMM were 6.6 and 17.1 mmol/g, while

62

carbon and hydrogen contained in the Si-GPTMS-TEG were 6.2 and 16.3 mmol/g,

respectively.

4.3.2. Retention behaviors of anions on TEGMM-bonded stationary phase and TEG-

bonded stationary phase

Both of the prepared stationary phases could retain five anions satisfactorily.

Actually we used sample solutions that contain six anions, unfortunately both of the

prepared stationary phases could not separate bromide and nitrate completely. However,

the separation was achieved in reasonable time. Based on the previous work which used

longer chain of ethylene oxide, i.e. poly (ethylene oxide)bonded into silica, it was

believed that the cations of eluent coordinated with the oxygen atoms of multiple POE

chains via ion-dipole interaction, and then they work as the anion exchange sites. The

eluent and analyte anions can compete for the trapped cation [14-15]. The elution order

of the six inorganic anion observed was similar as that observed in common IC, it was

iodate, bromate, bromide and nitrate, iodide, and thiocyanate.

63

Fig.4-2. Separation of six anions on TEGMM-bonded silica and TEG-bonded silica

stationary phase. Column, (100 × 0.32 mm i.d.); eluent, 2 mM sodium chloride; flow-

rate, 3.0 μL/min; analytes, iodate (1), bromate (2), bromide (3), nitrate (4), iodide (5),

thiocyanate (6); concentration of analytes, 0.1 mM each; injection volume, 0.2 μL;

wavelength of UV detection, 210 nm.

Figure 4-2 demonstrates the separation of the six anions on the present

columns, using 2 mM sodium chloride as mobile phase. It can be seen that both of the

stationary phases have nearly the same profile in separation of the six anions, but TEG

0 5 10 15 20 25 30 35

Si-GPTMS-TEGMM

SI-GPTMS-TEG

Time/min

IO3- BrO3

-

Br-, NO3

I-

SCN-

0.01 Abs

64

bonded silica stationary phase gave better selectivity and stronger retention for the six

anions, compared with the TEGMM bonded silica stationary phase. It may be because

the hydroxylgroup on the end of TEG chains have contributed in coordination of eluent

cations. Under the operating conditions in Figure 4-2, the repeatability of the retention

time was evaluated. The relative standard deviations of the retention time of six anions

were 2.00-2.16% and 0.68-3.21% for separation on TEGMM bonded silica and TEG

bonded silica stationary phase, respectively.

We have optimized the reaction time and the temperature time in chemically

bonding of the tetraethylene oxide to silica in order to obtain better stationary phase

performance. The reaction time was varied from 24 to 72 h and the temperature reaction

was varied from 50°C to 120°C. The increasing reaction time in chemically bonding,

resulted in the decrease in the retention time of anions on the both stationary phase. The

reaction temperature of chemically bonding gave a different result for two prepared

stationary phases. For TEG bonded stationary phase, increasing temperature caused

increase in selectivity and stronger retention for anions. On the other hand, increasing

temperature slightly decreased selectivity and retention for anions on the TEGMM

bonded stationary phase. In this work, 24 h was chosen as the optimum reaction time in

preparing both of ethylene oxide bonded stationary phases and the reaction temperature

65

was 120°C and 75°C for the TEG bonded stationary phase and the TEGMM bonded

stationary phase, respectively.

4.3.3. Effect of eluent cation

The eluent cations could affect the retention of anions on the prepared

stationary phase because eluent cations are expected to be coordinated among multiple

ethylene oxide chains and work as the anion exchange sites [13-14]. The different eluent

cations showed different retention behavior of analyte. Same with the poly(ethylene

oxide) bonding stationary phase on the previous work, it was shown that the size of the

cations may not be very significant for coordinating the eluent cations. The largest

retention time was achieved using sodium sulphate and sodium chloride solution for the

eluent. Figure 4-3 shows the separation of six anions on TEG-bonded stationary phase

using 10 mM aqueous solution of various chlorides. The elution order for all of the

eluent examined was same. Among the eluents examined, since sodium chloride gave

the best resolution, sodium chloride was used as the eluent in the following experiments.

66

Fig. 4-3. Separation of six anions on TEG-bonded stationary phase using 10 mM

aqueous solutions of various chlorides. TEG-bonded silica (100 × 0.32 mm i.d.); eluents,

as indicated; flow-rate, 3.0 μL/min; analytes, iodate (1), bromate (2), bromide (3),

nitrate (4), iodide (5), thiocyanate (6); concentration of analytes, 0.5 mM each; injection

volume, 0.2 μL; wavelength of UV detection, 210 nm.

0 2 4 6 8

Time/min

NaCl

LiCl

KCl

RbCl

CsCl

MgCl2

CaCl2

Na2SO4

12

3,4

5

6

0.05 Abs

67

4.3.4. Effect of salt concentration

Sodium chloride was used as the eluent for examination of the effect of salt

concentration on the retention of analyte anions. As found in common IC, the retention

of analyte anions decreased with increasing eluent concentration. Figure 4-4

demonstrates the retention time of analyte anions versus the concentration of sodium

chloride.

A B

Fig. 4-4. The retention time of analyte anions as a function of eluent concentration; on

TEG bonded stationary phase (A); TEGMM bonded stationary phase (B). Operating

conditions as in Fig. 4-2 except for the eluent as indicated.

0

5

10

15

20

25

30

35

0 5 10 15

Ret

enti

on

Tim

e (m

in)

Con.of NaCl (mM)

1

2

3,4

5

6

0

5

10

15

20

25

30

35

0 5 10 15

Ret

enti

on

Tim

e (m

in)

Conc.of NaCl (mM)

1

2

3,4

5

6

68

4.3.5. The HILIC mode properties of TEG-bonded stationary phase

Since the TEG bonded stationary phase possesses hydroxyl groups at its

chains, it is expected to show separation ability towards polar compounds due to the

specific interactions such as dipole–dipole and hydrogen bonding interactions. In order

to investigate the HILIC mode properties of present column, uracil and toluene were

used as test compounds where acetonitrile with variant concentration from 50% to 98%

were used as mobile phase.

Fig. 4-5. Retention times of uracil and toluene as a function of acetonitrile concentration

in mobile phase. Column, TEG bonded stationary phase (100 × 0.32 mm I.D.); mobile

phase, acetonitrile-water mixtures; flow rate, 3 µL/min; wavelength of UV detection,

254 nm; analytes, 0.002 % (w/v) each of uracil and toluene; injection volume, 0.2 µL.

0

1

2

3

4

5

6

40 50 60 70 80 90 100

Ret

enti

on

Tim

e, m

in

Conc. of Acetonitrile (%)

Uracil

69

Figure 4-5 shows the retention time of uracil and toluene as a function of

acetonitrile concentration in the mobile phase for the column TEG bonded stationary

phase. The retention time of uracil, which is a polar compound, increased when the

concentration of acetonitrile increased from 50% to 98% in mobile phase, whereas the

retention time of toluene, a non-polar compound, was not influenced with increasing

concentration of acetonitrile.

The prepared TEG bonded stationary phase was evaluated by the separation

of polar compounds phenols, one of large category of environmental pollutants, under

an isocratic elution condition. A typical chromatogram is shown in Figure 4-6. It can be

seen that the separation of three compounds was completed less than 7 minutes.

70

Fig. 4-6. Separation of phenols. Column, TEG-bonded stationary phase (100 × 0.32

mm i.d.); mobile phase acetonitrile-water (98 : 2); flow rate, 3 µL/min; wavelength of

UV detection, 254 nm; analytes, 0.01 % (w/v) each of phenol (1), pyrocathecol (2) and

pyrogallol (3); injection volume, 0.2 µL.

4.4. Conclusions

The tetraethylene oxide bonded stationary phases were prepared by

chemically bonding of the tetraethylene glycol monomethyl ether (TEGMM) and

0 2 4 6 8 10

0.01 Abs

1

2

3

Time/min

71

tetraethylene glycol (TEG) on silica via reaction with 3-

glycidyloxypropyltrimethoxysilane. The TEG bonded stationary phase has better

selectivity and strong retention for the five anions, compared with the TEGMM bonded

stationary phase, and it also has hydrophilic property. The present stationary phases

were successfully used as stationary phase for separation of inorganic anions and polar

compounds.

4.5. References

[1] T. H. Dean and J. R. Jezorek, J. Chromatogr. A, 1028, (2004), 239.

[2] H. Small, T. S. Stevens, and W. C. Bauman, Anal. Chem., 47, (1975), 1801.

[3] M. W. Läubi and B. Campus, J. Chromatogr. A, 706, (1995), 103.

[4] K. Ohta, K. Kusumoto, Y. Takao, A. Towata, S. Kawakami, M. Ohashi, and T.

Takeuchi, Chromatographia, 58, (2003),171.

[5] Y. Liu, Q. Du, B. Yang, F. Zhang, C. Chu, and X. Liang, Analyst, 137, (2012), 1624.

[6] H. Qiu, X. Liang, M. Sun, and S. Jiang, Anal. Bioanal. Chem., 399, (2011), 3307.

[7] S. Wongyai, Chromatographia, 38, (1994), 485.

[8] X. Liu, S. X. Jiang, L.R. Chen, Y. Q. Xu, and P. Ma, J. Chromatogr. A, 789, (1997),

569.

72

[9] M. Sun, J. Feng, S. Liu, C. Xiong, X. Liu, and S. Jiang, J. Chromatogr. A, 1218,

(2011), 3743.

[10] J. D. Lamb and R. G. Smith, J. Chromatogr. A, 546, (1991), 73.

[11] J. D. Lamb, R. G. Smith, and J. Jagodzinski, J. Chromatogr. A, 640, (1993), 33

[12] L. Rong and T. Takeuchi, J. Chromatogr. A, 1042, (2004), 131.

[13] L. Rong, L. W. Lim, and T. Takeuchi, Chromatographia, 61, (2005), 371.

[14] L. Rong, L. W. Lim, and T. Takeuchi, J. Chromatogr. A, 1128, (2006), 68.

[15] T. Takeuchi, B. Oktavia, and L. W. Lim, Anal. Bioanal. Chem., 393, (2009), 1267.

[16] T. Takeuchi and L. W. Lim, Anal. Sci., 26, (2010), 937.

[17] T. Takeuchi, K. Tokunaga and L. W. Lim, Anal. Sci., 29, (2013), 423

[18] T. Takeuchi and D. Ishii, J. Chromatogr., 213, (1981), 25.

73

Conclusions

1. A polymethacrylate-based diol monolithic column based on the copolymerization

of GMA and PEGDMA in the presence of porogens was prepared and successfully

used as stationary phase in HILIC mode. The poly(GMA-PEGDMA) monoliths

possess diol groups at the surface of the porous monolith that ascribed to

hydrophilicity of the monolith. Increasing the GMA content in the polymerization

mixture increased hydrophilicity. Phenols, phthalic acid and PAHs were retained on

the stationary phases using acetonitrile-water mixtures as the mobile phase.

However, the higher hydrophilicity of the monolith still remains as a challenging

task due to the fact that separation of polar compounds still needs higher

concentration of acetonitrile as mobile phase.

2. The poly(ethylene oxide) stationary phase could be used for the ion exchange

separation of inorganic anions. Cations and anions of the eluent affected the

retention of the analyte anions. The retention of the analyte anions decreased with

increasing eluent concentration. However, the long-term stability of the stationary

phase still remains as a challenging task due to the fact that the retention time of the

anions decreased with increasing analysis time.

3. The tetraethylene oxide bonded stationary phase was prepared by chemically

bonding of the tetraethylene glycol monomethyl ether (TEGMM) and tetraethylene

glycol (TEG) on silica via reaction with 3-glycidyloxypropyltrimethoxysilane. The

TEG bonded stationary phase has better selectivity and strong retention for the five

74

anions, compared with the TEGMM bonded stationary phase, and it also has

hydrophilic property. The present stationary phases were successfully used as

stationary phase for separation of inorganic anions and polar compounds.

75

Figure list

Chapter 2

Fig. 2-1. Scanning electron microscopy images of the poly(GMA-PEGDMA)

monolithic capillary columns. 6 % GMA (A) and 12% GMA (B).

Fig 2-2. Back pressure on column C (12% GMA) as a function of flow rate of

acetonitrile-water (98 : 2) as the eluent.

Fig. 2-3. Retention times of uracil and toluene as a function of acetonitrile

concentration in mobile phase.

Fig. 2-4. Separation of phenols.

Fig. 2-5. Chromatogram of aromatic acids.

Fig. 2-6. Chromatogram of the separation of PAHs.

Fig. 2-7. Effect of the acetonitrile concentration in mobile phase on the retention of

PAHs.

Chapter 3

Fig. 3-1. The structure of the PEO reagent employed as well as the expected reaction

scheme.

Fig. 3-2. The proposed structure for coordinating of eluent cation.

Fig. 3-3. Separation of six anions on PEO-bonded aminopropyl silica (A) and

unmodified NH2-60 (B) stationary phase.

76

Fig. 3-4. Separation of six anions on PEO-bonded stationary phase using 10 mM

aqueous solutions of various chlorides.

Fig. 3-5. Separation of six anions on PEO-bonded stationary phase using 10 mM of

various sodium aqueous solutions.

Fig. 3-6. The logarithm of retention factor (log k) of analyte anions as a function of the

logarithm of the eluent concentration.

Chapter 4

Fig. 4-1. The scheme of the expected reactions

Fig. 4-2. Separation of six anions on TEGMM-bonded silica and TEG-bonded silica

stationary phase.

Fig. 4-3. Separation of six anions on TEG-bonded stationary phase using 10 mM

aqueous solutions of various chlorides.

Fig. 4-4. The retention time of analyte anions as a function of eluent concentration.

Fig. 4-5. Retention times of uracil and toluene as a function of acetonitrile

concentration in mobile phase.

Fig. 4-6. Separation of phenols.

77

Table list

Chapter 1

Table 1-1. The classification of chromatoraphy

Table 1-2. The development history of capillary based separation methods and their

related technique

Table 1-3. List of several common ion-exchange resins

Chapter 2

Table 2-1. Effect of different compositions of the polymerization mixture on the

efficiency and the permeability of the column.

78

List of publications

1. R. Linda, L. W. Lim, T. Takeuchi

“Methacrylate-based Diol Monolithic Stationary Phase for the Separation of

Polar and Non Polar Compounds in Capillary Liquid Chromatograph ”

Anal. Sci., 29 (6), (2013) 631-635.

2. R. Linda, L. W. Lim, T. Takeuchi

“Pol (eth lene oxide)-bonded Stationary Phase for Separation of Inorganic

Anions in Capillar Ion Chromatograph ”

J. Chromatogr. A, 1294, (2013), 117-121.

79

List of presentations

1. R. Linda, L. W. Lim, T. Takeuchi

“Pol mer monolithic gl cid l methacr late-poly(ethylene glycol) dimethacrylate

col mn for capillar liq id chromatograph ”, Taka ama or ms 2011, Takayama,

November 2011 (Poster Presentation)

2. R. Linda, L. W. Lim, T. Takeuchi

“Pol mer monolithic gl cid l methacr late-poly(ethylene glycol) dimethacrylate

stationar phase for capillar liq id chromatograph ”, IA mposi m with G. D.

Christian, Aichi, 2-3 December 2011 (Oral Presentation)

3. R. Linda, L. W. Lim, T. Takeuchi

“Tos lated-poly(ethylene oxide)-bonded stationary phase in capillary ion

chromatograph ”, eparation ciences 2012, Tok o, 19-20 July 2012 (Oral

Presentation)

4. R. Linda, L. W. Lim, T. Takeuchi

“Tos lated-poly(ethylene oxide)-bonded stationary phase in capillary ion

chromatograph ”, 24th

International Ion Chromatography Symposium, Berlin,

Germany, 17-20 September 2012 (Oral & Poster Presentation)

5. R. Linda, L. W. Lim, T. Takeuchi

“Pol mer monolithic glycidyl methacrylate-poly(ethylene glycol) dimethacrylate

stationar phase for capillar liq id chromatograph ”, The 6th

Asia-Pacific

Symposium on Ion Analysis, Padang, Indonesia, 26-28 November 2012 (Poster

Presentation)

80

Curriculum vitae

Roza Linda

Born on January 16, 1974 in Pekanbaru, Riau, Indonesia

Married with Alwis Nazir

Mother of M. Anugrah Syauqi Alzazirka

1989 – 1992 : Senior High School at SMA Negeri 1 Pekanbaru, Riau, Indonesia

1992 – 1996 : Bachelor at Department of Chemistry, Faculty of Mathematics and

Natural Science, Andalas University, Padang, Indonesia

1997 – 1999 : Master Degree at Post Graduate Program Andalas University, Padang,

Indonesia. (URGE scholarship batch 5, World Bank)

2000 – now : Lecturer at Chemistry Education Department, Faculty of Education,

Riau University, Pekanbaru, Indonesia.

2010 – 2013 : Doctoral Degree at Material Engineering Division, Graduate School of

Engineering, Gifu University, Gifu, Japan. (DIKTI scholarship, Indonesian

Government)

81

Acknowledgements

Thanks and praise to God (Allah Almighty), the merciful and compassionate,

for blessing me in all my life and providing me the great opportunity in my education.

I would like to express my sincerest gratitude and appreciation to my

advisor Professor Toyohide Takeuchi, for great support to me throughout my study with

his wide knowledge, patience and understanding. His precious suggestions,

encouragement and effort have provided a good basis for my dissertation.

I would like to express my heartfelt gratitude to Dr. Lee Wah Lim for each

inspirational, supportive, and helped me in all the time of research and writing

dissertation. She is not only mentors but dear friend.

My deep gratitude to Indonesian go ernment thro gh DIKTI’s scholarship

which is funding my study and my life during the study.

incere thanks to all members in Take chi’s laborator for their kind

support, and also to all my friends in Indonesian Student Association Gifu (PPI Gifu)

for friendship, and being family during I stayed in Gifu.

Last but not least, I would like to thank my deceased father and mother, my

father and mother in law, my sisters and brothers. Special word of gratitude to my

husband Alwis Nazir and my son M. Anugrah Syauqi Alzazirka for their patience, deep

understanding, great support, help and their unconditional love. This dissertation is

dedicated to both of them.

Roza Linda