Preparation and application of covalently bonded polysaccharide-modified stationary phase for per aqueous liquid chromatography

Accepted Manuscript Preparation and application of covalently bonded polysaccharide-modified stationary phase for per aqueous liquid chromatography Tong Chen, Ling Zhu, Huiyuan Lu, Guangsan Song, Yuanyuan Li, Hongbin Zhou, Ping Li, Wanning Zhu, Hechen Xu, Lijun Shao PII:

S0003-2670(17)30228-3

DOI:

10.1016/j.aca.2017.02.013

Reference:

ACA 235073

To appear in:

Analytica Chimica Acta

Received Date: 23 December 2016 Revised Date:

9 February 2017

Accepted Date: 10 February 2017

Please cite this article as: T. Chen, L. Zhu, H. Lu, G. Song, Y. Li, H. Zhou, P. Li, W. Zhu, H. Xu, L. Shao, Preparation and application of covalently bonded polysaccharide-modified stationary phase for per aqueous liquid chromatography, Analytica Chimica Acta (2017), doi: 10.1016/j.aca.2017.02.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical abstract

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Preparation and application of covalently bonded polysaccharide-modified stationary phase for per aqueous liquid chromatography

Tong Chen, Ling Zhu, Huiyuan Lu, Guangsan Song, Yuanyuan Li, Hongbin Zhou, Ping Li,

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Wanning Zhu, Hechen Xu, Lijun Shao

The mixed phosphorylated/methacryloyl polysaccharide derivative was prepared and immobilized onto porous silica surface through the radical polymerization. The

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new stationary phase (MCPP-SP) showed both hydrophilic interaction liquid chromatography (HILIC) and per aqueous liquid chromatography (PALC) characteristics. Using MCPP-SP column, separation of polar compounds including synthetic pigments and sulfa compounds in the PALC mode was successfully accomplished. The results demonstrated that MCPP-SP column exhibited stronger

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retention efficiency for various polar compounds.

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Preparation and application of covalently bonded polysaccharide-modified stationary phase for per aqueous liquid

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chromatography

Tong Chena,*,1Ling Zhub, Huiyuan Luc, Guangsan Songa, Yuanyuan Lid, Hongbin Zhoua, Ping Lia, Wanning Zhua, Hechen Xua, Lijun Shaoa

State Key Laboratory of Food Additive and Condiment Testing, Zhenjiang Entry-exit Inspection

Quarantine Bureau, Zhenjiang 212008, China

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a

Research Institute of Lanzhou Petrochemical Corporation，Lanzhou 730060，China

c

Animanl, Plant and Food Inspection Center, Jiangsu Entry-exit Inspection Quarantine Bureau,

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b

Nanjing 210001, China d

Key Laboratory of Energy and Chemical Engineering, Ningxia University, Yinchuan 750021,

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China

Abstract The mixed phosphorylated/methacryloyl polysaccharide derivative was prepared and immobilized onto porous silica surface through the radical

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polymerization. The successful immobilization of polysaccharide on the silica support was confirmed by FT-IR spectra, elemental analysis and transmission electron

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microscopy (TEM), and so on. The new stationary phase (MCPP-SP) showed both hydrophilic interaction liquid chromatography (HILIC) and per aqueous liquid

chromatography (PALC) characteristics. The chromatographic behaviors were evaluated by investigating the effects of water content, column temperature, mobile phase pH and salt concentration, and a typical PALC retention feature of MCPP-SP based column was observed at high percentage of water content. Compared with C18 *

Corresponding author: Tong Chen. Tel.: +86 511 88825130; fax: +86 511 88825612. E-mail address: [email protected] (Tong Chen)

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column, using MCPP-SP column, separation of polar compounds including synthetic pigments and sulfa compounds in the PALC mode was successfully accomplished. The results demonstrated that MCPP-SP column exhibited stronger retention

method was suitable for the replacement of HILIC.

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efficiency for various polar compounds. PALC as a green chromatography analytical

Keywords: Polysaccharide, Stationary phase, Per aqueous liquid chromatography,

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Green chromatography

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1. Introduction

As a branch of high performance liquid chromatography (HPLC), hydrophilic interaction liquid chromatography (HILIC) has found many useful applications for separation of highly polar compounds that show no or little retention in

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reversed-phase liquid chromatography (RPLC) [1-5]. However, a high percentage of acetonitrile (ACN) (70–95%) is often used in the HILIC mode [6-8]. ACN is ranked as hazardous solvent and has negative influence on the environment. To solve the

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problem, the first option can be to use ethanol instead of acetonitrile. Ethanol, which is produced in large amounts and is biodegradable, has about the same elution

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strength as acetonitrile, costs much less and appears an attractive candidate [9]. A second option is to take advantage of the hydrophobic character of siloxane groups at the surface of silica by using water-rich mobile phases in HPLC analysis [10]. This mechanism is called per aqueous liquid chromatography (PALC) by Sandra et al. [10]. PALC is a green LC analytical method [11]. In the PALC mode, mobile phases contain a high percentage of water (70%-100%), and stationary phase is reversed to

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non-polar to separate polar compounds [12]. It can reduce the use of harmful solvents to help the realization of green liquid chromatography, protect the environment and greatly decrease the cost of determination. Gritti et al. [9] found that PALC could be a

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suitable mode of chromatography as a replacement of HILIC processes for the analysis and separation of polar compounds, highly consuming in acetonitrile. Li et al.

[11] prepared a new carbon nanoparticles-silica column, which could provide the

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similar retention for polar compounds in the HILIC and PALC modes. The new column showed good separation selectivity for polar compounds and hydrophilic

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compounds. Pereira et al. [10] also illustrated the features of PALC with the analysis of catecholamines, nucleobases, acids, and amino acids.

In this work, we prepared a new bonded polysaccharide-modified stationary phase, which was used in the PALC mode. Plant polysaccharide isolated from Momordica charantia L. (MCP) with good stability and solubility in water has been

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demonstrated [13, 14]. Cheng reported that MCP was composed of glucose, galactose, arabinose, rhamnose, and mannose with molar ratio of 24.84:27.94:16.47:24.03:6.72 [15]. The main glycosidic bond configuration was β-configuration. Furthermore, we

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synthesized phosphorylated derivative of MCP (MCPP), and phosphorylated modification could improve the water solubility of MCP. Then, MCPP was effectively

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immobilized onto the surface of porous silica particles by the radical co-polymerization reaction, which was stable enough while contacting with water-rich mobile phase for a long time. Due to the existence of hydroxyl and phosphate groups on MCPP, the stationary phase showed both PALC and HILIC characteristics and a weaker ion exchange interaction. The chemical structure of the stationary phase was analyzed by elemental analysis, FI-IR spectra, TEM and scanning electron microscopy (SEM). The water content, column temperature, pH and ionic strength of

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mobile phase were fundamental parameters that need to be investigated in the retention mechanism of polar compounds under the PALC mode. In the PALC mode, highly aqueous eluents with the range of 90-100% were mainly studied. This not only

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helped to reduce the use of hazardous solvents, but also made green LC possibilities.

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2. Experimental

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2.1. Materials and reagents

The crude MCP from Momordica charantia L. was obtained from Shaanxi Lixin Biotechnology Co. (China). Spherical silica (5 µm particle size, 10 nm pore size, 380 m2/g surface area) was purchased from Fuji Silysia Chemical (Aichi, Japan).

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3-(Methacryloyloxy)-propyltrimethoxysilane (MPTMS), methacryloyl chloride and 3-phosphonopropionic acid were obtained from Alfa Aesar (Shanghai, China). (AIBN),

4-dimethylamino

pyridine

(DMAP)

and

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α,αʹ-azodiisobutyronitrile

N,N-dimethylformamide (DMF) were from Aladdin Chemistry Co., Ltd. (Beijing,

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China). Papain was from Beijing Huamei Biotechnology Co., Ltd (China). SephadexG-100 was from Pharmacia Co. (Sweden). Sodium benzoate, potassium sorbate, caffeine, vitamin B3, melamine, thymine,

uracil, cytosine, thymidine, cytidine, and uridine were from Aladdin Chemistry Co., Ltd. (Beijing, China). Nine synthetic pigments were purposed from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Ten sulfa compounds were from Dr. Ehrenstorfer (Augsburg, Germany). ACN of HPLC grade was from Merck (Darmstadt,

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Germany). Deionized water (>18 MΩ cm−1) from a RiOs system was used throughout

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the experiments (Merck Millipore, American).

2.2. Purification of MCP

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The crude MCP was purified as follows: the protein was removed by the Sevage

method [16], combined with papain (150 U/mL). After centrifugation, the supernatant

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was dialyzed with distilled water for 48 h. Through Sephadex G-100 column, the purified MCP was obtained. The molecular weight (Mw) of MCP was 8.5 kDa. The content of MCP was measured by Vitriol–Phenol taking anhydrous glucose as

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standard control.

2.3. Phosphorylated modification of MCP

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3-phosphonopropionic acid (0.5 g) was mixed with 10 mL of anhydrous DMF. Then, 40 mg of DMAP as catalyst was added and stirred for 2 h. 0.5 g of MCP

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dissolved in 10 mL of anhydrous DMF was added dropwise into the reaction mixture. The mixture was stirred continuously for 24 h at room temperature. The products were dialyzed (molecular weight cutoff 3 kDa) by distilled water for 24 h to remove DMF, DMAP, and so on. MCPP was obtained after lyophilizing.

2.4. Preparation of methacryloyl MCPP derivative

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MCPP (0.5 g) was dissolved in anhydrous DMF (35 mL) at room temperature

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with stirring for 30 min, and 0.5 mL of methacryloyl chloride was added into the mixture. The reaction mixture was stirred under N2 atmosphere for 10 h at 60 ℃. Then,

derivative was obtained after lyophilizing in vacuum.

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2.5. Immobilization of MPTMS on silica surfaces

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the mixture was precipitated with ethanol. After centrifugation, methacryloyl MCPP

Spherical silica (2.0 g) was suspended in 30 mL of anhydrous toluene, and 2.0 mL MPTMS was added with a stirring. Then, 0.5 mL of NH3·H2O was added

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dropwise. The reaction mixture was stirred under N2 atmosphere for 20 h at room temperature. After centrifugation, the mixture was washed with dichloromethane, acetone and methanol, respectively. Then the products were dried under vacuum at

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60 ℃ overnight. MPTMS-bonded silica was obtained.

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2.6. Polymerization

Methacryloyl MCPP (0.5 g) was dissolved in anhydrous DMF (30 mL) at room

temperature with stirring for 30 min, and 2.0 g of MPTMS-bonded silica was added into the mixture. Then, the polymerization was performed by the initiating of AIBN (1.0 wt.% of monomer) under N2 atmosphere at 70 ℃ for 24 h. The final product was

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filtered, intensively washed with water and methanol respectively, and then dried under vacuum at 60 ℃ overnight. The new bonded polysaccharide-modified stationary

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phase (MCPP-SP) was obtained. The routes for the synthesis of the new stationary phase were shown in Fig. 1.

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Figure 1

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2.7. Characterization

The IR spectra was recorded on a Nicolet NEXUS-670 FT-IR spectrophotometer (Thermo Fisher Scientific, USA). The P content of MCPP and MCPP-SP were determined on an inductively coupled plasma atomic emission spectrophotometer

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(ICP-AES) IRIS ER/S (TJA Company, USA). Elementary analysis was measured on a Vario EL elemental analysis system (Elementar Co., Germany). The morphology of the samples was recorded by a Tecnai-G2-F30 transmission electron microscopy

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(TEM) (FEI, USA). Field emission scanning electron microscopy (FE-SEM) images were obtained on a JSM-7001F (Japan). Brunauer–Emmett–Teller (BET) surface area,

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SBET, was determined from the linearity of BET equation. Pore size and its

distribution

were

calculated

using

desorption

Barrett–Joyner–Halenda (BJH) formula.

2.8. Column packing and chromatographic evaluation

isotherm

branch

and

the

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A HPLC column of 150 mm×4.6 mm I.D. was prepared by a slurry packing method. MCPP-SP was suspended in methanol and slurry-packed into a stainless steel

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column under a constant pressure of 40 MPa with methanol as the pushing solvent. All the chromatographic evaluation of MCPP-SP based column was performed

on a 1260 Series HPLC system from Agilent Technologies (Waldbronn, Germany)

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equipped with a diode array detector detector (DAD). Column temperature was at room temperature (25 ± 2 ℃). The flow rate was 0.8 mL/min. A set of test probes with

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a concentration of 15 µg/mL were prepared in water/ACN (3/1, v/v). Each measurement was replicated three times. The dead time was 1.4 min, which was determined by injecting 5 µL methanol with water/ACN (1/1, v/v) as the mobile

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phase.

3. Results and discussion

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3.1. Structural characterization of stationary phase

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The resulting stationary phase was characterized using FT-IR, ICP-AES and

elemental analysis, respectively. The FT-IR spectra of the native MCP and MCPP were shown in Fig. 2A and B. Compared with MCP, two new strong absorption peaks appeared at 1710 cm−1 and 1268 cm−1 for MCPP, assigned to the C=O stretching vibration and the P=O asymmetric stretching, respectively. Furthermore, according to the results of ICP-AES (Table 1), the P content of the native MCP was 0, but that of

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MCPP was 2.78%. These results indicated that the phosphorylated modification of MCP had actually occurred.

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Fig 2C was the FI-IR spectra of the methacryloyl MCPP derivative. In comparison with MCPP, a stronger absorption peak was observed at 1710 cm−1 corresponding to the C=O bond stretching vibration, indicating that the methacryloyl

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MCPP derivative was synthesized successfully. The results of the elemental content were shown in Table 1. A decrease of P content and the increase of C and H content in

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the methacryloyl MCPP derivative were also observed due to the introduction of the methacryloyl group.

After immobilization of MPTMS on silica surfaces, its FT-IR spectra was also measured (Fig. 2E). Compared with original silica (Fig. 2D), absorption bands at 1712

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and 1640 cm−1 were attributed to C=O and C=C stretching, and the carbon content of MPTMS-silica was 8.65%, which indicated that MPTMS has been immobilized on silica surfaces. In Fig. 2F, although the characteristic peaks of MCP was buried in the

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strong and broad Si-O-Si band and Si-O band of silica, the peak at 1712 cm−1 became stronger on the MCPP-SP after polymerization, and P content was increased

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obviously in comparison with MPTMS-silica, indicating that MCPP was successfully bonded to MPTMS-silica. Figure 2 Table 1

The morphology of original silica and the MCPP-SP were further illustrated by

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TEM and SEM in Fig. 3. As could be seen from Fig. 3A, B and D, the surface morphology of the original silica was very smooth at different magnifications. Fig. 3C

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and E showed the surface morphology of the MCPP-SP. Obviously, no agglomeration could be observed, the polymer was uniformly distributed on the silica surface.

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Figure 3

The pore properties of stationary phase before and after the modification were

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obtained by N2 adsorption study. Parameters of surface areas and pore sizes of stationary phase were given in Table 2. Compared with original silica, surface area and pore size of MCPP-SP materials were smaller, were a consequence of

Table 2

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modification.

3.2. Chromatographic evaluation of MCPP-SP based column under the HILIC mode

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and the PALC mode

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3.2.1 Retention properties in the HILIC mode

Solvent strength in the eluent was the most significant effect on the retention of

compounds [17-19]. In the HILIC mode, the hydrophilic interaction would enhance and the retention time would increase by decreasing the polarity of the eluent [20, 21]. In order to investigate the HILIC properties of the MCPP-SP column, sodium

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benzoate, caffeine and vitamin B3 were chosen as test probes. The volume percentage of ACN was change from 80% to 98% in Fig. 4. The retention times of all test

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compounds displayed an increasing trend upon the increasing of ACN content in the mobile phase, which indicated a typical HILIC retention mechanism. The stationary phase exhibited pronounced retention for polar compounds in the HILIC mode.

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3.2.2 Retention properties in the PALC mode

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Figure 4

For protecting the environment and reducing the cost of determination, we were more interested in PALC mode. The peak shape of thymine was studied in the PALC

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and HILIC modes. The volume percentage of water was 90% (PALC mode) and 10% (HILIC mode), respectively. The flow rate was 0.8 mL/min. The similar retention times were 3.619 min and 3.755 min, and the peak asymmetry factors were 1.09 and

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1.12 in the PALC and HILIC mode, respectively. Compared with the HILIC mode, the peak shape of thymine in the PALC mode had no significant difference. The results

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showed that PALC was possible as a new retention mode alternative to HILIC for the separation of polar compounds.

3.2.2.1 Effect of water content in mobile phase on retention

In order to understand the chromatographic properties of the MCPP-SP column

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in the PALC mode, firstly, the effect of water content on the retention behavior was investigated. Five polar compounds (thymine, uracil, cytosine, thymidine and cytidine)

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were used as test probes by varying the volume percentage of water from 60% to 100%. As shown in Fig. 5, the retention of all test compounds displayed a slight increase trend upon the increasing of water content from 60% to 90%, and then

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increased dramatically when the water content further increased to 100%. The reason might be that the surfaces of the MCPP-SP column were simultaneously saturated

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with water and ACN, which led to slight increase of the retention factors of compounds with the volume percentage of water from 60% to 90%. But varying the volume percentage of water from 90% to 100%, small variation of ACN content in the mobile phase could lead to the drastic composition change of the adsorbed eluent

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multilayer onto the MCPP-SP column. So, under the condition of high water content, a little change of mobile phase would result in a strong change in the retention factor.

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Figure 5

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3.2.2.2 Effect of column temperature on retention

Column temperature which had significant effect on mobile phase viscosity,

solute diffusion coefficients and the solute–stationary phase interactions, was an important influential factor for retention and selectivity in LC. The contribution of temperature to the retention was mainly expressed by the van’t Hoff equation [22, 23]:

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ln k = −

∆H ° ∆S ° + + ln φ RT R

Where k，∆HO，∆SO，R，T and Φ were the retention factor of the analyte, the

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standard partial molar enthalpy of transfer, the standard partial molar entropy of transfer, the gas constant, the absolute temperature and the phase ratio, respectively. The relationship between ln k and 1/T was explored in the temperature range of

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20–60 ℃. As shown in Fig. 6, due to the k value was strongly dependent on the

column temperature, the retention factors of five polar compounds decreased as

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temperature increases. This was based on the well-known fact that high temperature led to the decrease in viscosity of the mobile phase, the increase of diffusivity and solubility of analyte in the mobile phase [24-26], which would result in the decrease

Figure 6

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of retention factor of analyte.

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3.2.2.3 Effect of mobile phase pH on retention

In the present study, the effect of the mobile phase pH was investigated in the

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PALC mode with six test compounds under eight pH values ranging from 3 to 6.5 on the MCPP-SP column. As shown in Fig. 7, the retention times for potassium sorbate and sodium benzoate decreased significantly when pH changed from 3 to 6.5. The possible reason was that they were dissociated gradually with the increase of pH value, generated electrostatic repulsion with phosphate groups of the MCPP-SP column, leading to a decrease of retention. However, the retention times of the other

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compounds that was the basic compounds, including cytidine, cytosine, caffeine and melamine, increased slightly with increasing the pH value of mobile phase. It was

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possibly that the stationary phase obtained more negative charge with the increase of pH value, which enhanced ion exchange interaction with the above basic compounds,

leading to an increase of retention. Therefore, the change of the retention time was

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due to a change in the charge state and density of the stationary phase and compounds.

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Figure 7

3.2.2.4 Effect of salt concentration on retention

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The salt concentration would affect the peak shape and retention of a solute in the PALC mode [27]. In general, addition of buffer salts into the mobile phase would increase the symmetry of peaks and improve separation efficiency. In the present

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study, the effect of various concentrations of ammonium acetate on retention was investigated from 5 mM to 50 mM. As shown in Fig. 8, by increasing the salt

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concentration, only a slight decrease of the retention time of analytes was observed except for uridine which remained almost unchanged. This could be explained that higher salt concentrations increased the eluting strength of the mobile phase, and the salt ions would suppress electrostatic attraction between the basic compounds and phosphate groups on the MCPP-SP column, which would decrease the retention of basic solutes.

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Figure 8

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3.2.2.5 Stability and reproducibility

The operation stability of the MCPP-SP column was evaluated by a mixture of

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potassium sorbate and sodium benzoate through 15 continuous injections under the

chromatographic conditions of 10 mM ammonium acetate/ACN (95/5, v/v) as the

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mobile phase, 25 ℃ of column temperature, 0.8 mL/min of flow rate and 260 nm of DAD detection. The relative standard deviations (RSDs) of retention times of analytes were 1.02% and 0.77%, respectively. This result demonstrated a good stability of the MCPP-SP column in the PALC mode.

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Additionally, intra-day and inter-day reproducibility of retention times for model compounds (thymine, uracil, cytosine and melamine) in fifteen days were also studied under the PALC mode. The chromatographic conditions were the same to the

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conditions of stability evaluation, except the mobile phase was water/ACN (95/5, v/v). The results showed that the RSDs of all of model compounds were 1.56%, 0.98%,

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0.77% and 1.29%, respectively. They were no more than 2%. Furthermore, no obvious change of the retention times after three months continuous use was observed, which further showed that the stationary phase could be widely and reliably used as separating material.

3.3 Applications

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To demonstrate the potential of the MCPP-SP column to separate polar

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compounds under the PALC mode, some representative polar compounds was investigated. Meanwhile, C18 silica columns were used for comparative studies.

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3.3.1 Separation of nine synthetic pigments under the PALC mode

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In the food industry, the determination of synthetic pigments was important, because more evidence indicated that the abuse of synthetic pigments might cause cancer, and some people were much sensitive to particular food pigments [28]. A mixture of nine synthetic pigments including tartrazine, carmine, sunset yellow, allura

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red, brilliant blue, new red, amaranth, basic orange 2 and rhodamine B2 were selected as model compounds. As could be seen in Fig. 9, under the PALC mode, nine synthetic pigments were separated with good peak shapes and good efficiency within

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30 min on the MCPP-SP column in comparison with that on the C18 column. Nine synthetic pigments could be effectively retained by a lot of the phosphate groups and

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hydroxyl groups of MCPP on the MCPP-SP column. Figure 9

3.3.2 Separation of ten sulfa compounds under the PALC mode

Ten kinds of sulfa compounds were also investigated to demonstrate the

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separation ability of the MCPP-SP column, and the results were shown in Fig. 10. The separation of sulfa compounds on the MCPP-SP column was better than that on the

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C18 column, with more peaks adequately resolved. This further proved the high separation efficiency of the MCPP-SP column for polar compounds. However,

broadened peaks were observed on the new stationary phase. It was possible that too

overloading effects under the PALC mode.

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high water concentration could seriously affect the symmetry of the peaks because of

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Figure 10

4. Conclusion

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In the paper, we described synthesis, characterization and chromatographic evaluation of a novel polysaccharide-modified stationary phase. The MCPP-SP column exhibited both HILIC and PALC properties. PALC could provide similar

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retention capacity as HILIC for polar compounds. Retention behaviors of MCPP-SP column towards the selected analytes were studied by the effects of water content,

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column temperature, pH and salt concentration of mobile phase. The phosphate groups and hydroxyl groups in the structure of MCPP facilitated the stationary phase’s applications in the PALC mode. Compared with C18 column, the resulting MCPP-SP column displayed excellent retention efficiency for various polar compounds, such as synthetic pigments and sulfa compounds. Considering green chromatography and the current shortage of ACN, PALC could be used as a suitable mode alternative to HILIC

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for the separation of polar compounds.

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Acknowledgments

This work was supported by Zhenjiang Social Development Project

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(SH2014021), the Natural Science Foundation of Jiangsu province of China

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(BK20161075).

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[27] Z.M. Guo, Y. Jin, T. Liang, Y.F. Liu, Q. Xu, X.M. Liang, A.W. Lei, J. Chromatogr. A 1216 (2009) 257–263.

[28] F. Feng, Y. Zhao, W. Yong, L. Sun, G. Jiang, X. Chu, J. Chromatogr. B 879 (2011)

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1813-1818.

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Table 1 Elemental analysis and ICP-AES characterization

Mw (kDa)

Elemental analysis (%) C

H

ICP-AES (%) P

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Sample

8.5

23.15

4.27

0

MCPP

8.9

20.77

3.61

2.78

Methacryloyl MCPP

-

21.37

4.02

1.85

MPTMS-silica

-

8.65

1.37

0

MCPP-SP

-

11.68

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MCP

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1.93

23

0.39

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Table 2 Physical properties of the stationary phase

Dporeb (nm)

Original silica

380

10

MCPP-SP materials

311

SBET, surface area. Dpore, pore size.

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b

SBETa (m2/g)

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a

Sample

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Fig. 1 Schematic illustration of the MCPP-SP materials.

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Fig. 2 FT-IR spectra of MCP (A), MCPP (B), methacryloyl MCPP (C), original silica (D),

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MPTMS-bonded silica (E) and MCPP-SP materials (F).

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Fig. 3 TEM image of (A, B) spherical silica; (C) MCPP-SP materials; SEM image of (D) spherical

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silica; (E) MCPP-SP materials.

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Fig. 4 Effect of acetonitrile content in the mobile phase on the retention of analytes. Mobile

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phase: water/ACN; flow rate: 0.8 mL/min, DAD detection: 260 nm.

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Fig. 5 Effect of water content in the mobile phase on the retention of analytes. Mobile phase:

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water/ACN; flow rate: 0.8 mL/min, DAD detection: 260 nm.

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Fig. 6 Effect of column temperature on the retention of analytes. Mobile phase: water/ACN

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(95/5, v/v); flow rate: 0.8 mL/min, DAD detection: 260 nm.

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Fig. 7 Effect of mobile phase pH on the retention of analytes. Mobile phase: water/ACN

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(95/5, v/v) containing 10 mM ammonium acetate; flow rate: 0.8 mL/min, DAD detection: 260 nm.

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Fig. 8 Effect of ammonium acetate concentration in the mobile phase on the retention of

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analytes. Mobile phase: ammonium acetate solution/ACN (95/5, v/v); flow rate: 0.8 mL/min,

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DAD detection: 260 nm.

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Fig. 9 Separation of nine synthetic pigments. A, separation on the MCPP-SP column; B, separation on the C18 column. Mobile phase: gradient: 0–5 min 95%

, ACN;

, 10 mM ammonium acetate solution;

,5–10 min 95%→75% Ⅱ,10–15 min 75% Ⅱ,15–20 min 75% → 95%

Ⅱ,20–25 min 75% → 95% Ⅱ,25–30 min 95% Ⅱ; flow rate: 0.8 mL/min, DAD detection: 254 nm,

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436 nm and 627 nm. Peaks: (1) tartrazine, (2) carmine, (3) sunset yellow, (4) allura red, (5)

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brilliant blue, (6) new red, (7) amaranth, (8) basic orange 2, (9) rhodamine B2.

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Fig. 10 Separation of ten sulfa compounds. A, separation on the MCPP-SP column; B, separation

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on the C18 column. Mobile phase: ACN/10 mM ammonium acetate solution (3/97, v/v); flow rate: 0.8 mL/min, DAD detection: 270 nm. Peaks: (1) sulfacetamide, (2) sulfamethizole, (3) sulfisoxazole, (4) sulfachloropyridazine, (5) sulfamethoxazole, (6) sulfamerazine, (7) sulfadoxin,

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(8) sulfamethoxypyridazine, (9) sulfamethazine, (10) sulfaphenazole.

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Dear Editors, Research highlights was studied as follows: 1. A new bonded plant polysaccharide-modified stationary phase was papered.

studied.

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2. Per aqueous liquid chromatography (PALC) that was a green LC analytical method was

3. The new stationary phase exhibited stronger retention efficiency for various polar

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compounds in the PALC mode.

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Sincerely yours,

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Prof. Tong Chen