Effects of Cross-Linking and Spacer Groups on beta-Cyclodextrin Bonded Silica Particles in a Liquid Chromatographic Separation

Manhoe Kim

Department of Chemistry, Air Force Academy (R.O.K.), Cheong-ju, Chungbuk 363-849, Korea

manhoe@hanmail.net

액체 크로마토그래피에서 베타 싸이크로 덱스트린 담지된 실리카입자에 대한 상호연결체 및 중간연결체의 영향들

김만회

363-849 충북 청원군 남일면 쌍수리 공군사관학교 화학과

INTRODUCTION

The most unique character of □-CD is its ability to include selectively various organic and inorganic molecules within hydrophobic cavities. Thus, CDs are good candidates as highly selective bonded phase materials for chromatographic separation.

Even though the difficulty of the immobilization of large size cyclodextrin to inorganic surface, Armstrong et al. made a success in immobilization of CD to silica gel by means of a specific silane linkage (1-3).

Although a variety of linking spacers and synthesis procedures are well known, the effects of spacer groups between CDs and inorganic supports on the formation of inclusion complexes were rarely known. Armstrong noted about the length of the linkage chains between silica gel and CDs. With chains as short as three carbon atoms in the silane, coupling CD is more difficult to accomplish because of the relative size of the CD. With chain as long as eighteen to twenty carbon atoms there is a danger of losing resolution in the separation process (3).

In this study another possible reaction between end-capping agents, trimethylchlorosilane and hydroxyl groups will be presented through a simple chromatographic separation based on inclusion complex formation. Similarly, the effects of spacer groups between CD and solid surface, and cross-linking agents such as 1,8-trichlorosilyloctane on the formation of inclusion complexes are studied. The separation efficiency of the CD-modified silica particles were tested using column chromatography to separate a model mixture of naphthalene isomers, 1- and 2-hydroxynaphthalene (naphthols) and ortho, meta, and para-xylenes.

EXPERIMENTAL SECTION

Silica (particle size 5□, pore size 8.0nm, and surface area 200m2/g, Altech) and glass beads (particle size 50-100□, pore size 31.6nm and surface area 97m2/g, Trisoperl, Schuller) were used as supports. Beta-CD (Wacker Biochem) was attached to the supports by a direct bonding method, using epoxy spacers, and cross-linking agent. 3-Glycidoxypropyl-trimethoxysilane (Aldrich) was used as a spacer group. 1,8-Trichlorosilyloctane (Petrarch Systems) and epichlorohydrin (Aldrich) were used as cross-linking agents. HPLC grade of dimethylformamide (Aldrich), toluene (Aldrich), methanol (Aldrich), de-ionized water were used as solvents and washing agents. A10mL burette (diameter 0.55cm, Pyrex Brand, Fisher) was used as a chromatography column. The bed height was 37cm and the free volume percentage of the packed □-CD bonded glass beads was 53%. The flow rate was 0.25mL/min.

Epoxy spacer cyclodextrin bonded glass beads were prepared according to the method described by Armstrong (3). The amounts of organic loaded on the supports were estimated using a thermal gravimetric analyzer (TGA, Seiko) and a linear heating rate of 10℃/min from room temperature to 600℃. In the direct bonding method, 350mL of dried dimethylformamide, 25g of dried □-CD salt, and 25g of dried glass beads or 10g of silica were kept at 144℃ for 3hr with stirring. The resulting slurry was put aside for a few minutes in order to allow the □-CD bonded-glass beads/silica to be settled. The upper portion of the slurry was removed and the modified glass beads/silica were filtered with a filter paper, and washed with toluene and methyl alcohol. The □-CD bonded-glass beads/silica were dried in a vacuum oven at 80℃ overnight.  A typical □-CD weight loading on the glass beads and silica were 15.6 % and 23.8%, respectively.

In order to stabilize the directly bonded cyclodextrin on the surfaces, Twenty grams of directly bonded □-CD glass beads and 60mL of saturated □-CD salt solution was cross-linked with 1.5g of epichlorohydrin (or 2.5g of 1,8-trichlorosilyloctane) in 120mL of toluene. The mixture was maintained at 90℃ for 50minutes with stirring. The resulting slurry was put aside for a few minutes. The upper portion of the slurry was removed and the modified glass beads/silica were filtered with a filter paper, and washed with toluene and methyl alcohol.

In order to measure the amounts of xylene isomers adsorbed on □-CD, silica, and glass beads, these adsorbents were cleaned in a flow of helium gas at 200℃ for 1.5hour. The adsorbents were cooled down to room temperature 22℃ in a flow of helium gas. The flowing gas was switched to helium containing an isomer of xylene vapor. The weight gains were measured with a balance (Cahn TG-131, Cahn Instruments, Inc.) for 5hr.

Packing materials were packed into the column by slurry packing or dry powder packing. The column chromatographic experiments were carried out at the room temperature (22℃). Xylene and naphthol isomers were tested as elute compounds.  Pure toluene and methyl alcohol were used isocratic mobile phases.  Eluting solvent compositions were analyzed with a HP GC-MS (model 5890/5970).

RESULTS AND DISCUSSION

In the packed column chromatography experiments, two major controlling factors on the separations were observed.  The first was the possibility of formation of inclusion complexes with □-CD pores or the pore structures induced by linking between □-CD and supports, and the other observed from the unmodified silica or glass beads beds was the steric hindrance effect between eluting molecules and the stationary phases. Where molecule having smaller cross sectional area has less steric hindrance in the packed column (elution orders: ortho>meta>para-xylene, 1-naphthol>2-naphthol).

The □-CD bonded silica showed the same elution order of xylene para>ortho>meta-xylene as the xylene adsorption capacity order, and 2-naphthol>1-naphthol. This trend can be interpreted that the □-CD bonded silica has strong binding forces to para-xylene and 2-naphthol due to strong inclusion complex formation with □-CD pores. The separation between meta and ortho-xylene are determined by inclusion complex formation and steric hindrance differences. However, a different elution order, ortho>para>meta-xylene, was resulted from the column packed with the □-CD bonded glass beads due to the large pore and particle sizes of glass beads. It is known that separation selectivity is determined by bonded phase and support material as well. The separation selectivity of the □-CD bonded glass beads between naphthol isomers was much larger than that of □-CD bonded silica.

The column packed with the 3-glycidoxypropyltrimethoxysilane spacer linked □-CD silica showed a different elution order, ortho>para>meta-xylene due to pore narrowing and pore blocking by the spacer group and the large pores induced by spacer groups and the surface of silica. Thus, spacer group decreased the binding force to para-xylene and increased the binding force to ortho-xylene. Similarly, pore narrowing and pore blocking by the spacer group decreased the degree of inclusion complex formation of 2-naphthol when the spacer □-CD glass beads were used.

The cross-linked □-CD directly bonded glass beads by 1,8-trichlorosilyloctane lose the selectivity between naphthol isomers due to pore narrowing and blocking by the cross-linking agent. In contrast, the selectivity between xylene isomers is clearly increased by cross-linking maintaining the same elution order (ortho>para>meta-xylene). Cross-linking narrows the □-CD pore openings, thus, inclusion complex formation of p-xylene is limited. Conversely, large pores induced by cross-linking agent among □-CDs and glass beads give a better binding force for ortho-xylene.

LITERATURE CITED

1. Armstrong, D. W.; DeMond, W., Cyclodextrin Bonded Phases For the Liquid Chromatographic Separation of Optical, Geometrical, and Structural Isomers, J. Chromatogr. Sci, 22, 411-415(1984)

2. Armstrong, D. W., Bonded Phase Material For Chromatographic Separation, U.S. Patent No.4,539,399 (1985)

3. Armstrong, D. W.; DeMond, W.; Alak, A., Liquid Chromatographic Separation of Diastereomers and Structural Isomers on Cyclodextrin-Bonded Phases, Anal. Chem. 57, 234-237(1985)