Synthesis of the reversed stationary phase for solid phase extraction using trimethoxyoctadecyl silane

Octadecyl grafting silica was synthesized using trimethoxyoctadecyl silane. Reaction conditions were optimized so that the carbon percentage of resulted material was similar to that of commercial products. Temperature and catalyst played very important roles in the reaction. The material was also undergone the end-capping process to reduce unreacted silanol groups. Final product owned 14.1 % of carbon content and 75 % of the unreacted silanols. Retention behavior of product was tested and compared to the same noes available on the market. Results showed that both resulted material and commercial had the same retention properties.


INTRODUCTION
Solid phase extraction (SPE) is a well-established sample pretreatment technique because it demands less organic solvents and can remove interferences simultaneously.In SPE chemical structure of sorbent materials play a decisive role [1].Currently, the most popular sorbent material is conventional reversed phase (CRP) on silica support.
CRP is synthesized by directly bonding alkoxyoctadecyl silane or chlorooctadecyl silane onto silica surface [2][3][4][5].While chlorolsilane is very reactive, alkoxysilane is one of the least reactive reagents [3].However, during the reaction with silica, chlorosilane produces HCl by-product which can damage the main product.For alkoxysilane, the byproduct is alcohol which is harmless and easy to remove in distilled conditions.Besides, the less reactivity of alkoxysilane can be overcome by employing an appropriate catalyst [6].
In the current study, CRP sorbent was synthesized using alkoxyoctadecyl silane and its retention properties were compared with commercial CRPs.

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Preparation of CRP Silica gel was dried at 200 °C for 24 h.After cooling to room temperature, 2 g of silica were added to a flask, then 20 mL of toluene, 0.5 mL of TMOS and 150 mg p-toluenesulfonic aicd were added.The mixture was heated to 100 °C under reflux and paddle stirred condition for 4 h.The product was filtered and washed three times successively with 30 mL portions of toluene, diethylether, methanol, acetone and finally dried at 50 °C for 2 h.

End-capping of CRP
To a 100 mL flask, add 1 g of CRP, 15 mL of toluene, 1 mL of chlorotrimethyl silane and 0.5 g of imidazole.The mixture was heated to 80 °C under reflux and paddle stirred condition for 4 h.The final product was filtered and washed three times successively with 30 mL portions of toluene, methanol, acetone and finally dried at 50 °C for 2 h.

Sorbent material characterizations
The carbon content was analyzed by Alison wetoxidizing method [7].Briefly, amixture of K2CrO7

Evaluation of retention
To evaluate retention properties of resulted CRP, five analytes with different chemical properties were used (Table 1).The mixture of the five compounds (10 µg/mL) in water were loaded onto the homemade amide-RP cartridges and other three commercial C18 cartridges (Agilent SampliQ C18, Strata C18-E and Isolute C18), the bounded analytes were then eluted with 5 mL mixture of methanol and water.The collected eluents were analyzed by HPLC to calculate the recoveries.

Chromatography conditions
HPLC separations were performed with Agilent 1100 system, UV detector, AscentisTM C18 Supelco separation column (25cm  4.6 mm, 5µm).Detection wavelength was of 254 nm, temperature of 40 °C.Aqueous 0.1 % formic acid (phase A) and acetonitrile 0.1 % formic acid (phase B) were used as mobile phase at 1 mL.min -1 .The mobile phase gradient program was started with 15 % of phase B, increased to 40 % phase B for 14 min and to 100 % for 22 min.

Effect of catalyst
Catalyst is a very important element in silanization.Reaction of octadecyltrimethoxy silane and silica gave only 3.5 % in carbon content without catalyst.The reaction of surface silanols with alkoxysilanes can be catalyzed by either acids or bases [6].As the mechanism showed in figure 2, before removing water to form the linkage to silica surfaces at high temperature, the alkoxy groups of alkoxysilanes are hydrolyzed to form intermediate silanols which are very reactive.Catalyst accelerates the hydrolysis (1) and condensation (2).Fig 3 shows that bases as triethylamine, pyridine and diethylamine had weak effect on the silanization, and the carbon content of the obtained product was from 6 to 8 %.This could be due to their weak basic property and low boiling point (Table 2).Imidazole was a bether catalyst with 12.5 % in carbon amount.However, the best was p-toluenesulfonic acid with 13.2% of carbon loading, corresponding to the reaction yield of 90 %.This was similar to results of the previous publication [6].As a result, ptoluenesulfonic acid was selected for subsequent investigations.As can be seen from Fig. 4, good yield (90 %, corresponding to % C of 13.2 %) was obtained when the number of moles of p-toluenesulfonic acid was a half compared to that of silane.Consequently the mole ratio of 0.5 was selected for the following investigations.

Effect of solvent and temperature
As can be seen from step 4 of silanization mechanism (Fig. 2), the temperature is a very essential factor and need to be optimized.Besides, solvent also needs to be investigated because it affects the solubility of the reagent and the permeability of silica.Fig. 5 shows that the carbon percentage of CRP raises as the reaction temperature increases.However, solvents possessing too low or high polarity such as benzene or 1,2-dichlorobenzene gave low reaction yield.Toluene and xylene had the same trend at temperature lower, than 110 °C, and highest carbon amount of product with these solventsreach up to 13.2 %.Since toluene is less toxic than xylene, it was consequently chosen for subsequent experiments.Fig. 6 shows that the reaction of octadecyltrimethoxy silane and silica happened very fast at the first two hours and then gradually slow down.After 4 hours the carbon loading almost reached the maximum with of 13.2 %.As a result, the reaction should be carried out for 4 h.

End-capping
The surface coverage of silica with octadecyl silane could not have high density due to steric hindrance.In an effort to reduce unreacted silanol groups, significantly smaller silanes were used, socalled end-capping process [8,9].In this current work, the end-capping process was conducted as in Patent EP1361915 B1 with chlorotrimethyl silane without further investigations.Table 4 shows that after the end-capping process completed, a slight increase of the carbon content of CRP (14.1) was observed.

Retention behavior
The five analytes with different aeidic or basi properties (Table 1) were used in testing the retention behavior of the CRP sorbent in comparison to the commercial C18 sorbents.
Elution profiles show that resulted CRP had similar retention behavior as the three other commercial CRPs for all acid and base compounds.Therefore, the ability of purifying and concentrating analytes of resulted CRP were the same as the others.

CONCLUSION
Octadecyl modified silica was synthesized using trialkoxyoctadecyl silane reagent.The of reaction yield between silica and the silane reagent largely depended on the temperature and catalysts.Resulted material was end-capped and possessed 14.1 % of the carbon content.The retention behaving of the oltained product was similar to those of the same commercial products.Therefore, it was able to purify and concentrate analytes as much as other available product.

Fig. 1 .
Fig. 1.Reaction of silica and trimethoxyoctadecyl silane (4 g), H2SO4 (concd):H3PO4 (concd) (3:2, v/v)) (30 mL) were used to oxidize the sorbent material (0.3 g) at high temperature (approximately 200 °C) and produce CO2.The resulted CO2 was absorbed to 20 mL of 0.25 N NaOH solution.Blank sample was also performed by substituting silica for sorbent material.By titrating the NaOH solutions of blank and material samples with HCl 0.25 N and phenolphthalein as indicator, carbon content can be calculated as the following formula: VBl: Volume of HCl solution to titrate absorption solution of blank sample (mL) VS: Volume of HCl solution to titrate absorption solution of sample (mL) CHCl: Concentration of HCl solution (M) m: Weight of sample (g)

Fig. 4 .
Fig. 4. Effect of catalyst amount on reaction of silica and trimethoxyoctadecyl silane

Fig. 5 .
Fig. 5. Effect of temperature and solvent on reaction of silica and trimethoxyoctadecyl silane

Fig. 6 .
Fig. 6.Effect of time on reaction of silica and trimethoxyoctadecyl silane

Fig. 7 .
Fig. 7. Elution profiles of tested analytes with resulted material and three commercial CRPs sorbents.

Table 1 .
Base-acid characteristics of five standards used for testing retention of amide-RP

Table 2 .
Properties of some organic acids and bases.
Fig. 3. Effect of catalyst on reaction of silica and trimethoxyoctadecyl silane

Table 3 .
Properties of some solvents