Abstract
Silica, as a stationary phase, has low separation efficiency accompanied by overlapping, broadened, and tailed peaks, so it needs to be modified to improve its efficiency. This study aims to develop a silica-based stationary phase modified by tetraethylene glycol (TEG) to separate phenolic compounds. Silica was modified by a chemical bond between silanol groups on the silica surface and TEG through a 3-glycidyloxypropylmethoxysilane reaction. The modified silica was packed into a capillary column and used to separate simple phenolic compounds consisting of phenol, pyrocatechol, and pyrogallol. A sample of 0.2 µL was injected into the capillary liquid chromatography and the mobile phase employed was acetonitrile 98% with a flow rate of 3 μL min−1. Elution was also done isocratically in this process and detection was carried out at a wavelength of 254 nm. The mixture of simple phenolic compounds was successfully separated in less than 7 min. The asymmetry factor and resolution were 1.43–2.12 and 1.72–5.43 respectively. The number of the theoretical plates ranged from 213 to 7,857. Columns containing Si-TEG stationary phase also separate phenolic compounds, which consist of gallic acid, syringic acid, ferulic acid, and caffeic acid. A sample of 0.2 µL was injected into the capillary liquid chromatography and successfully separated the mixture in less than 12 min. The samples were eluted isocratically using a mixture of methanol and 50 mM phosphate buffer pH 2.5 (8:92) with a flow rate of 3 μL min−1. The phenolic acids compounds were detected at a wavelength of 280 nm. The chromatogram showed four separate peaks. The asymmetry factor and resolution were 1.53–1.63 and 1.14–1.74, respectively, but the number of the theoretical plates was low, ranging from 190 to 796.
Introduction
Phenol is one of the secondary metabolites produced from the reaction between shikimic acid and pentose phosphate through the phenylpropanoid metabolism in plants. Phenol generally consists of a benzene ring and one or more hydroxyl substituents. Based on the phenol structure the compounds may be classified into three types: phenolic acids, flavonoids, and polyphenols [1]. Phenolic acids have a straightforward structure with one benzene ring and one or more carboxylic acid groups constitute just the basic skeleton of phenolic acids. An equally important component is additionally the hydroxyl group [2]. Phenolic acids are abundant in plants. It has a key role in the growth and reproduction of plants and gives colour and taste to vegetables and fruits [3]. Phenolic acid also has the potential as an antioxidant that can inhibit free radicals and prevent diseases caused by oxidative damage, such as coronary heart disease, stroke, and cancer [4].
Tempuyung (Sonchus arvensis) is an Indonesian medicinal plant with high phenolic acid [5]. It also contains other bioactive compounds such as sesquiterpene lactones, flavonoids, triterpenes, and steroids [6]. Several techniques have been developed to separate, identify, and determine the amount of phenolic acids using reversed-phase liquid chromatography.
The reversed-phase liquid chromatography is one of the methods to separate phenolic compounds by using a silica-based stationary phase [7]. The use of silica as a stationary phase has some weaknesses, such as 1) the peak in the base is always tailing and has low efficiency due to ionic interactions or lone pair with an acidic silanol group; 2) the shape of the resulted chromatogram has broad peaks and is sometimes still overlapping; 3) this stationary phase is only stable and works well in 2–8 pH [8].
One way to improve the weaknesses is to modify the silica surface by designing the molecular surface using a silane agent as a precursor [9]. Some researchers have modified this stationary phase for different purposes and used diverse separation methods, such as Torres et al. (2021), who used poly(ethylene glycol) (H(OCH2CH2)nOH) to modify the stationary phase [10]. Moreover, Takeuchi et al. [11, 12] modified the stationary phase using poly(ethylene oxide) (H(OCH2CH2)nOH) and tosylate-poly(ethylene oxide) (CH3(OCH2CH2)nOSO2C6H4CH3) and this resulted in a good separation between inorganic anions. Another study was conducted by Linda et al. [13] using tetraethylene glycol (H(OCH2CH2)4OH) and tetraethylene glycol monomethyl ether (CH3(OCH2CH2)4CH3) through the 3-glycidoxypropyltrimethoxysilane (GPTMS) reaction pathway and also shows good result in the separation of organic anions and phenolic compounds.
This research further modified a stationary phase of silica gel using tetraethylene glycol (TEG) via the glycidoxypropyltrimethoxysilane (GPTMS) reaction pathway. The modifications of silica-tetraethylene glycol involve a sol-gel process by a specific molecular interaction or covalent bonds between silanol groups on the silica surface and organic polymers. During this process, the condensation of silanol groups can form silica complexes that can separate these two phases. It can be prevented by adding glycidoxypropyltrimethoxysilane (GPTMS). GPTMS is an alkoxide encompassing epoxide rings [14]. This GPTMS has high reactivity and is commonly utilised as a coupling agent to fortify interactions between the organic phase and organic polymer [15]. Besides, adding GTPMS can produce products with good texture, morphology, and particle size uniformity [16]. The silanol group within GPTMS and on the silica surface will react to form stable siloxane groups. Meanwhile, the epoxide group at the end of the GPTMS chain will open with the addition of N,N-dimethylformamide (DMF), which then binds to organic polymers [17]. Furthermore, the modified stationary phase was studied to separate compounds derived from phenolic acids (e.g., gallic acid, caffeic acid, syringic acid, and ferulic acid) to determine the optimum conditions for separation. This study aims to develop a silica-based stationary phase modified with tetraethylene glycol as the modifier to separate phenolic acids.
Experimental
The research was conducted at the Takeuchi Laboratory, Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Japan. Materials used include tetraethylene glycol (TEG) (Nacalai Tesque, Kyoto, Japan), GPTMS (Tokyo Chemical Industries, Tokyo, Japan), silica gel with a particle diameter of 5 μm and pore diameter of 60 Å, standard Pyrocatechol was obtained from Nacalai Tesque (Kyoto, Japan), Standard gallic acid 98%, ferulic acid 97% (Combi Blocks, San Diego, United States). Standard phenol and pyrogallol, caffeic acid 98%, standard syringic acid 98%, 100 mM phosphate buffer pH 2.5, N,N-dimethylformamide, acetonitrile, methanol, dry toluene, and acetic acid from Wako Pure Chemical Industries, Osaka, Japan were used. Purified water was produced in the laboratory using a GS-590 water distillation system (Advantec, Tokyo, Japan).
The tools used were stainless steel columns, silica capillary columns (100 × 0.32 mm i.d.), an FTIR spotlight-400 spectrophotometer (PerkinElmer, Kyoto, Japan), a Scanning Electron Microscope (SEM) from Shimadzu, an elemental analyzer (MT-6 CHN Corder, Yanaco, Kyoto, Japan), ultrasonicator (USD-3R; As One, Osaka, Japan), a water bath (IEC61010-2-020, Kubota, Japan), capillary liquid chromatography system consisting of an L.TEX 8301 Micro Feeder from L.TEX (Tokyo, Japan) equipped with an MS-GAN 050 gas fuse syringe (0.5 µL; Ito, Fuji, Japan) as a pump, M-435 microinjection valve from Upchurch Scientific (Oak Harbor, USA) as an injector, a 100 × 0.32 mm i.d. fused silica micro-column from GL Sciences (Tokyo, Japan), and a UV-2705 UV detector from Jasco (Tokyo, Japan). Data were obtained using a Chromatopac C-R7Ae from Shimadzu (Kyoto, Japan).
The procedure involves the preparation of the stationary phase from TEG-modified silica, characterization of the resulting stationary phase, and optimization of the phenolic and phenolic acid separation condition.
Silica-TEG stationary phase preparation
According to Linda et al. [13], the silica-TEG stationary phase preparation can be carried out by mixing 0.20 g of dry silica gel with 3.50 mL of dry toluene and 0.20 mL of GPTMS in a 20.0 mL vial. The solution was poured into a stainless steel column. The reaction was carried out in a vessel oven at 110 °C for 20 h and occasionally stirred manually. The resulting silica product was washed with dry toluene and dried at 75 °C for 4 h. It was then reacted with 3.50 mL of N, N-dimethylformamide (DMF) and 0.20 mL of TEG. The solution was transferred into a vessel and the reaction occurred in an oven at 120 °C for 24 h. The result of the reaction was washed using methanol and packed in a capillary column made of silica (100 × 0.32 mm i.d.) using the slurry method, as reported by Takeuchi and Ishii [17].
Silica-TEG stationary phase characterization
The stationary phase that has been prepared was characterized using FTIR spectroscopy to estimate the functional groups bound to the stationary phase. This silica-TEG stationary phase was mixed with 5–10% of KBr powder and ground with a mortal until it was smooth. Afterward, the mixture was made into KBr pellets with the help of 5,000–10,000 PSI pressure. The pellets were analyzed using FTIR spectroscopy. Furthermore, the stationary phase was also characterized using SEM to understand the morphology of the stationary phase surface and characterized by elemental analysis to see the composition of the stationary phase before and after the reaction.
Sample and standard solution preparation
A stock solution of gallic acid, caffeic acid, syringic acid, and ferulic acid 1,000 μg mL−1 was prepared by dissolving 10 mg of each standard in 10 mL of methanol 40% and water. Each standard solution was diluted to form a concentration ranging from 2 to 20 μg mL−1 and a standard calibration curve was prepared. As much as 1 g of dry tempuyung sample was weighed and dissolved into 5 mL of methanol 40%. The solution was then sonicated for 30 min at room temperature, filtered using a 0.45-μm membrane filter, and diluted to 10 mL with the mixture of methanol and water.
Optimization of chromatographic separation conditions
The optimization process was conducted using a silica capillary column (100 × 0.32 mm i.d.). The parameters analyzed were the type of mobile phase and its composition. The flow rate used was 3.0 μL min−1 [18]. Acetonitrile, methanol, a mixture of methanol and acetic acid [19], and a mixture of methanol and phosphate buffer [13] were the mobile phase compositions used. The elution was done isocratically. Detection was carried out at a wavelength of 280 nm [20]. Optimum separation conditions were obtained when (1) the number of maximum detected peaks has a relatively high peak intensity, (2) a narrow and symmetrical peak, and (3) the resolution of separation for each peak is at least 1.5.
Results and discussion
Stationary phase of tetraethylene glycol-modified silica
The stationary phase of tetraethylene glycol-modified silica was synthesized using silica gel with particle and pore diameters of 5 μm and 60 Å, respectively. The silica was initially activated by heating at 105 °C for 4 h. It aims to remove impurities and water adsorbed in the silica pore. The activated silica gel was reacted with GPTMS, which acts as the coupling agent to intensify interactions between silica and TEG [15]. This reaction occurred in a dry toluene solvent at 110 °C for 20 h. According to Rogers and Zhang [20], the reaction was initiated with the hydrolysis of the methoxylane group in GPTMS to form silanol groups (Si–OH). Hereafter, this group would condense with silanol groups on the silica surface to form siloxane groups (Si–O–Si) and left epoxide groups in the terminal position. The reaction is described in Fig. 1.
Synthesis reaction of Si-GPTMS (1) GPTMS Hydrolysis (2) Si-GPTMS condensation
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01131
The formed Si-GPTMS was washed using dry toluene to remove the remaining GPTMS that were not reacted (Fig. 2). Once the washing process was completed, Si-GPTMS was dried at 75 °C to evaporate the remaining toluene from the washing process. The use of dry toluene as a solvent and washing solution aims to prevent excess water within the reaction. Excessive water can cause an epoxide ring opening in GPTMS to produce diol groups so that GPMTS cannot react with TEG.
GPTMS epoxide ring opening reaction due to water
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01131
The addition of N,N-dimethylformamide (DMF) would open the epoxide ring on Si-GPTMS, forming a secondary alcohol that enables TEG molecules to bind to silica [21]. This reaction occurred at 120 °C for 24 h. According to Linda et al. [13], the reaction of Si-TEG formation is described in Fig. 3. The formed Si-TEG was, hereafter, washed using methanol to remove the remaining DMF and TEG that were not reacted so that pure Si-TEG was obtained.
The reaction of Si-TEG formation
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01131
Material composing elements
The percentage of elements within modified silica can be determined by using a CHN elemental analyzer. The elements contained in the modified silica are presented in Table 1.
The percentage of elements in Si-GPTMS and Si-TEG
| Elements | Synthesis Result | Literature (Linda et al. [13]) | |
| Si-GPTMS | Si-TEG | ||
| %C | 8.79 | 9.39 | 7.45 |
| %H | 1.91 | 2.04 | 1.64 |
| %N | 0.10 | 0.47 | – |
It is clearly seen from the table above that the total of elements C and H in Si-TEG are higher than in Si-GPTMS. The increase of elements C and H is caused by the binding between GPTMS molecules on the silica surface and TEG – which has a longer chain – that more C and H are attached to the silica surface. Stirring is one way to increase the efficiency of the reaction. Constant stirring will trigger more TEG molecules attached to silica. The number of elements C and H from the synthesis result was higher than the results reported by [18]. The appearance of element N is caused by the contaminants in the stainless steel column and the residual DMF that has not been washed or removed from Si-TEG.
Si-TEG functional group
Identification of functional groups in TEG-modified silica was accomplished using FTIR by emitting infrared light at a wavelength of 4,000–400 cm−1. The IR spectrum for the Si-GPTMS and Si-TEG materials is presented in Fig. 4.
Spectrum FTIR of Si-GPTMS and Si-TEG
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01131
Figure 4 shows differences in the indicating a change in functional groups. The IR spectrum of Si-GPTMS shows several main functional groups in silica, such as absorption at wave number 795 and 457 cm−1, which describes the Si–O bond and absorption at wave number 1077 cm−1 depicting siloxane groups (Si–O–Si). The IR spectrum showed no absorption broadening from the OH group at a wavelength of 3,500–3,200 cm−1. It is suspected that silanol groups (Si–OH) on the silica surface have bound to GPTMS. The absorption reinforces it at a wave number 942 cm−1, which describes the epoxide group from GPTMS attached to silica.
The IR spectrum of Si-TEG –the result of the reaction between Si-GPTMS and TEG reaction – shows adsorption peaks that are almost similar to the IR Si-GPMTS spectrum. The formation of Si-TEG was evidenced by the absence of the epoxide absorption peak at wave number 942 cm−1. This is caused by the reaction between the alcohol groups at the end of the TEG chain and the epoxide groups, resulting in the opening of the epoxide ring. Moreover, there was an absorption peak at 2,927 cm−1, indicating the bond of C–H. Another absorption peak also appeared at 1,390 cm−1, denoting the bond of C–O, as well as an absorption peak at 1,390 cm−1, which suggests the presence of a C–O bond, and an absorption peak at wave numbers 1,663 and 660 cm−1, which represents an O–H bond. The IR absorption characteristics of Si-GPTMS and Si-TEG are detailed in Table 2.
Si-TEG particle morphology
The morphology of the surface or size of a material can be observed using a scanning electron microscope (SEM). The result of peak analysis on chromatogram of the separation of the phenolic compounds is shown in Table 3. SEM can provide an image of the surface of a material through high-emission scanning. Silica particles can be observed at a magnification of 2,000 times. The scanning result is shown in Fig. 5.
The result of peak analysis on chromatogram of the separation of the phenolic compounds
| Parameter | Compound | Reference value (CDER 1994) | ||
| Phenol (1) | Pyrocatechol (2) | Pyrogallol (3) | ||
| 2.74 | 3.60 | 4.89 | – | |
| A | 585,736 | 141,535 | 515,160 | – |
| N | 5,075 | 7,857 | 213 | ≥2,000 |
| R | – | 5.43 | 1.72 | ≥1.5 |
| 2.12 | 1.43 | 1.70 | ≤2 | |
The number of theoretical plates (N), resolution (R), the asymmetry factor (
Particles morphology of (1) Si-GPTMS (2) Si-TEG
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01131
The morphology of Si-GPTMS and Si-TEG is spherical, as Cheng et al. (2010) reported. A measurement using Image-J software in Fig. 5 provides information that the average size of Si-GPTMS particle is 5.5920 ± 0.8333 µm, whereas the average Si-TEG particle size is 5.1397 ± 0.5504 µm. As a product of the first stage modification, there are no significant differences in morphology and particle size of Si-TEG compared to the second stage modification. It is assumed that the effect of this unobserved modification is influenced by the location of modification in the silica pore. Therefore, an electron microscope with a better lens is needed to magnify up to the pore.
Optimum condition for phenolic acid separation
The stationary phase of Si-TEG was packed into a 100 mm
This modification process involves the formation of covalent bonds between silanol groups on the silica surface and TEG. A long carbon chain and a hydroxyl group in Si-TEG can provide non-polar and polar characteristics in the stationary phase, thus allowing two modes of separation in the column, namely Reverse Phase Liquid Chromatography (RPLC) and Hydrophilic Liquid Chromatography (HILIC). The RPLC separation typically uses a non-polar stationary phase and a polar mobile phase; the separation also involves hydrophobic interactions [26]. On the other hand, HILIC has the opposed principle to RPLC. HILIC is usually used to separate polar or ionic compounds. The stationary phase used was polar, while the mobile phase contained high organic compounds. The separation process is based on partitioning polar compounds between the mobile phase and the water-rich surface of the stationary phase [27]. Furthermore, the separation in HILIC commonly involves hydrophilic interactions, electrostatic, hydrogen bonding, or dipole-dipole interactions [28].
The mode of separation in the column was evaluated by determining the retention time of the hydrophobic and hydrophilic compounds. Toluene and uracil were the hydrophobic and hydrophilic compounds used. The mobile phase used was acetonitrile 20–80% (acetonitrile: aquabidest) with a flow rate of 3 μL min−1. Elution was done isocratically and detection was carried out at a wavelength of 254 nm. As illustrated in Fig. 6, the retention time of uracil is higher than that of toluene at an acetonitrile concentration of 20–60%. The stationary phase had a greater affinity towards uracil. Hence, uracil eluted more slowly than toluene. This indicates a hydrophilic interaction between the stationary phase and uracil. Thus, the mode of separation in this condition was Hydrophilic Liquid Chromatography (HILIC).
The relationship of uracil and toluene retention time as a function of acetonitrile concentration in the mobile phase
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01131
In contrast, toluene had a stronger affinity for the stationary phase at 80% acetonitrile concentration, eluting more slowly than uracil. It is believed to happen due to hydrophobic interaction between the stationary phase and toluene. Therefore, the mode of separation that occurred in this condition was RPLC. The spike shows the mode change between RPLC and HILIC in the retention time of toluene when the acetonitrile concentration is about 60% [29].
The column with Si-TEG stationary phase was used to separate phenolic compounds consisting of phenol, pyrocatechol, and pyrogallol. The mobile phase employed was acetonitrile 98% and water 2% with a flow rate of 3 μL min−1. Elution was also done isocratically in this process and detection was carried out at a wavelength of 254 nm [13]. Based on the chromatogram, the value of some system suitability parameters was determined, including the asymmetry factor (
The chromatogram in Fig. 7 shows three well-separated peaks. A fairly high-resolution value supports it. The three peaks were identified as phenol, pyrocatechol, and pyrogallol, respectively. The chromatogram depicts tailed phenol and pyrocatechol peaks and a widened pyrogallol peak. The value of the asymmetrical factor of pyrocatechol and pyrogallol peaks is moderately good, with the phenol peak showing the highest asymmetry. The tailed and widened peaks can be caused by the non-homogeneous stationary phase contained in the column and slow mass transfer. Should the asymmetry factor's value be high, the total of theoretical plates obtained decreases. This can be resolved by extending the column size. Compared to the reference value from CDER [31], the value obtained in this study is still in an acceptable range; thus, the column can be used to separate phenolic acids.
Chromatogram of separation of simple phenolic compounds using a Si-TEG stationary phase column
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01131
Phenolic acid compounds comprising gallic acid, syringic acid, caffeic acid, and ferulic acid were separated using a column containing Si-TEG stationary phase. The separation process was completed with isocratic elution and detected at a wavelength of 280 nm. The mobile phase examined was methanol as well as acetonitrile, in various concentrations of each, with a flow rate of 3 μL min−1. Using methanol as the mobile phase in various concentrations (60–90%) indicates that some peaks have not been appropriately separated. Meanwhile, the acetonitrile mobile phase in various concentrations (80–98%) showed one peak, so separation did not occur.
The separation mechanism occurring in the stationary phase can be explicated through a partition method. The partitioning process begins with forming a cavity the size of an analyte in the stationary phase. The contact between the hydrophobic stationary phase and the mixed mobile phase causes the less polar mobile phase to be adsorbed on the stationary phase's surface; hence, polar analytes are contained within the polar mobile phase components. The less polar analyte will be contained in the mobile phase components adsorbed on the surface of the stationary phase. This allows a transfer of analytes from the mobile phase to the stationary phase. Analytes in the stationary phase will interact with the components of the stationary phase which are grouped into carbon chains, silanol groups, and adsorbed organic solvent. The silica modification process with TEG changes silanol groups into nonreactive siloxane so that the interaction of the analyte with the silanol groups can be neglected. The analytes interact with the carbon chains through hydrophobic interaction and are not influenced by the changes in the mobile phase, so they have constant magnitude and this interaction can also be neglected.
In addition, analytes can interact with organic solvents adsorbed on the surface of the stationary phase. The mobile phase used was the mixture of methanol and water and acetonitrile and water; as a result, methanol and acetonitrile are adsorbed on the stationary phase surface. Methanol is a stronger proton acceptor than acetonitrile thus it can form hydrogen bonds with carboxyl groups in phenolic acids that are proton donors. As a result, using methanol provides a more effective result than acetonitrile [32, 33].
The result of the separation was improved by adding a certain amount of acid and buffer into the methanol. Acetic acid and citric acid with a concentration of 1% [33, 34] and 50 mM pH 2.5 phosphate buffer were added. The separation of phenolic acid using mobile phases of methanol and the addition of citric acid shows two peaks that are not appropriately separated with high retention time. In contrast, using a mixture of methanol and acetic acid, and methanol and the phosphate buffer in the mobile phase demonstrates better separation results (Fig. 8).
The comparison of the chromatogram of the phenolic acid separation results with methanol as the mobile phase in various acids and buffers
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01131
The separation of phenolic acid using a mixture of methanol and acetic acid as the mobile phase in various concentrations nevertheless shows some peaks that are not adequately separated. This is caused by the pH value of the mobile phase being larger than the pH of phenolic acid and instigates dissociation. The dissociated phenolic acid forms deprotonated species so that they will be carried away by the more polar components of the mobile phase and decrease the amount of phenolic acid extracted in the stationary phase. Besides, the ability of dissociated phenolic acid as proton donors reduces so that the interaction between phenolic acid and methanol adsorbed on the surface of the stationary phase also diminishes, and so does the selectivity of the separation.
The result of phenolic acid separation using a mobile phase of methanol and phosphate buffer 50 mM pH 2.5 in various compositions shows a better separation result following increased phosphate buffer in methanol (Fig. 9). This happens because the higher the phosphate buffer added to the methanol, the lower the pH value obtained. The pH values of the mobile phase mixture of methanol and phosphate buffer in various compositions have been reported by Subirats et al. [33]. The mobile phase containing phosphate buffer in methanol in the 80–100% range has a lower pH value than the phenolic acid pH value so that the phenolic acid does not dissociate.
Phenolic acid separation results using a mixture of mobile phase methanol and 50 mM phosphate buffer in various compositions
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01131
Phenolic acid will be contained in the less polar mobile phase components and extracted to the stationary phase. Phenolic acid will interact with methanol adsorbed on the surface of the stationary phase. This can increase the selectivity of separation [32].
The results of separating phenolic acid using a mixture of mobile phase methanol and 50 mM phosphate buffer pH 2.5 with the composition (8:92) are considered the optimum mobile phase. The chromatogram of the separation results is described in Fig. 10, showing that the baseline obtained is unstable. An inhomogeneous mobile phase can cause this.
Chromatogram of phenolic acid separation using a mixture of mobile phase methanol and 50 mM phosphate buffer (8:92)
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01131
In addition, the chromatogram shows that there are four peaks separated from one another. A fairly good resolution value supports this. The resulting peak is slightly tailed. This is evidenced by the asymmetry factor value, which is not too large. The tailed peaks are likely to occur due to the inhomogeneity of the stationary phase, creating voids in the column. However, the separation results show a very small number of theoretical plates. Compared with the reference value from CDER (1994), the asymmetry factor value and resolution obtained have met the requirements, while the number of theoretical plates obtained has not met these requirements (Table 4). This can be fixed by extending the column size.
Results of peak analysis on the chromatogram of phenolic acid separation using a mixture of mobile phase methanol and 50 mM phosphate buffer (8:92)
| Parameter | Peak number | Standard values (CDER 1994) | |||
| (1) | (2) | (3) | (4) | ||
| 3.43 | 4.93 | 7.19 | 9.23 | – | |
| A | 1,885,996 | 6,108,616 | 3,012,749 | 2,155,249 | – |
| N | – | 190 | 457 | 796 | ≥2,000 |
| R | – | 1.53 | 1.63 | 1.53 | ≥1.5 |
| 1.14 | 1.54 | 1.23 | 1.74 | ≤2 | |
The number of theoretical plates (N), resolution (R), the asymmetry factor (
Conclusion
The stationary phase of TEG-modified silica has been successfully synthesized and confirmed by characterization using an FTIR spectrophotometer. The success is indicated by the absence of absorption of epoxides at wave number 942 cm−1 and the presence of IR absorption at wave numbers 2,927 cm−1 (C–H), 1,390 cm−1 (C–O), 1,663(O–H), 660 cm−1 (O–H). The modified silica stationary phase can be utilized to separate simple phenolics and phenolic acid compounds. The simple phenolic compounds consist of phenol, pyrocatechol, and pyrogallol. The mobile phase employed was acetonitrile 98% with a flow rate of 3 μL min−1. Elution was done isocratically in this process and detection was carried out at a wavelength of 254 nm. The mixture of simple phenolic compounds was successfully separated in less than 7 min. The asymmetry factor and resolution were 1.43–2.12 and 1.72–5.43 respectively. The number of the theoretical plates ranged from 213 to 7,857. Phenolic acid compounds were separated using a mixture of mobile phase methanol and 50 mM phosphate buffer pH 2,5 (8:92) at a flow rate of 3 μL min−1. Elution was carried out isocratic. Detection was done at a wavelength of 280 nm. This is evidenced by four peaks with a resolution value of 1.53–1.63 and an asymmetry factor of 1.14–1.74. Meanwhile, the number of theoretical plates obtained is still low.
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