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    Chinese Journal of Chromatography
    2013, Vol. 31, No. 4
    Online: 22 April 2013

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    Special column of chromatographic stationary phases
    Foreword for special column of chromatographic stationary phase
    2013, 31 (4):  289-289.  DOI: 10.3724/SP.J.1123.2013.04001
    Abstract ( 807 )   [Full Text(HTML)] () PDF (471KB) ( 199 )  
    Development of intelligent monolithic stationary phase for chromatography
    SHEN Ying,, QI Li*, MAO Lanqun, CHEN Yi
    2013, 31 (4):  290-296.  DOI: 10.3724/SP.J.1123.2013.03001
    Abstract ( 1382 )   HTML ( 159 )   PDF (900KB) ( 204 )  

    In recent years, great attention has been paid to monolith, which is developed as a new generation of chromatographic stationary phase, due to its advantages of in-situ preparation, fast mass transfer and high permeability. Besides, as a class of functional materials, intelligent polymer could response to external environmental stimuli, such as temperature, pH, salt and so on. This intelligent polymer is usually modified onto solid support surface for fabrication of environmental responsive materials, which could be widely applied in drug release, chemical sensing, cell growth, etc. As a result, the construction of intelligent surface on monolith can provide new opportunities for its development in chromatography. This review summarizes the development of intelligent monolith as chromatographic stationary phase during recent years.

    Development and application of separation materials for mixed-mode chromatography
    DONG Xuefang, CAI Xiaoming, SHEN Aijin, WEI Jie, GUO Zhimou*, LIANG Xinmiao*
    2013, 31 (4):  297-302.  DOI: 10.3724/SP.J.1123.2012.12009
    Abstract ( 1673 )   HTML ( 6 )   PDF (823KB) ( 342 )  

    Mixed-mode chromatography has received more and more attention due to its unique chromatographic characteristics recently. In this review, a summary of the development and applications of mixed-mode chromatography is presented. According to the types of hydrophilic interaction/ion-exchange mixed-mode chromatography (HILIC/IEX), reversed-phase/hydrophilic interaction mixed-mode chromatography (RPLC/HILIC) and reversed-phase/ion-exchange mixed-mode chromatography (RPLC/IEX), the preparation and applications of each type are introduced. Compared with single mode chromatography, the selectivity as well as the sample loading capacity of the mixed-mode chromatography can be improved. The researches mostly focus on the design of mixed-mode stationary phases and the applications especially to bio-analysis. The mixed-mode chromatography will be a potent approach for the analysis and separation of complex samples, and it will also be an effective and complementary tool in practical separation work.

    Applications of polymeric monoliths in separation of bio-macromolecules
    BAI Ligai, NIU Wenjing, YANG Gengliang*
    2013, 31 (4):  303-309.  DOI: 10.3724/SP.J.1123.2012.01047
    Abstract ( 1446 )   HTML ( 168 )   PDF (876KB) ( 233 )  

    In recent years, the applications of high performance liquid chromatographic polymeric monoliths in the separation of macromolecules have been developed. In the review, the characters and new developments of bio-macromolecules separation by using the polymeric monoliths, combining with the works in our laboratory are summarized. Moreover, related influential reports are referred.

    Preparation of highly hydrophilic strong cation exchangers and their applications in protein analysis
    LIU Jizhong, HUANG Yanyan, YANG Bo, CHANG Jianhua, LIU Guoquan, ZHAO Rui*
    2013, 31 (4):  310-316.  DOI: 10.3724/SP.J.1123.2012.12005
    Abstract ( 1356 )   HTML ( 7 )   PDF (838KB) ( 277 )  

    Based on the needs of new packing materials for rapid and efficient separation, purification and analysis of biomacromolecules, a novel sulfonic acid-type strong cation exchange resin (SP-G-PGMA SCX resin) was prepared. The porous poly(glycidyl methacrylate) microspheres (PGMA) were selected as the matrix and glucose was used as the hydrophilic modifier to block the hydrophobic domains of PGMA beads. Glucose modification on PGMA beads improved the biocompatibility and reduced the non-specific adsorption so as to increase the recoveries of protein. The PGMA beads possess the porous structure and the relatively high specific surface area, which make the PGMA-based resins good permeability and high loading capacity. The application of such SP-G-PGMA SCX resin for the chromatographic separation of biomacromolecules was explored. Four basic proteins were baseline separated within 6 min with the column size of 100 mm?4.6 mm. The adsorption capacity of lysozyme on SP-G-PGMA SCX resin was determined as 39.5 g/L. The results make the material promising for the separation and purification of biomacromolecules.

    Preparation of a novel polymer monolith using atom transfer radical polymerization method for solid phase extraction
    SHEN Ying, QI Li*, QIAO Juan, MAO Lanqun, CHEN Yi
    2013, 31 (4):  317-321.  DOI: 10.3724/SP.J.1123.2012.10041
    Abstract ( 1088 )   HTML ( 5 )   PDF (856KB) ( 177 )  

    In this study, a novel polymer monolith based solid phase extraction (SPE) material has been prepared by two-step atom transfer radical polymerization (ATRP) method. Firstly, employing ethylene glycol dimethacrylate (EDMA) as a cross-linker, a polymer monolith filled in a filter head has been in-situ prepared quickly under mild conditions. Then, the activators generated by electron transfer ATRP (ARGET ATRP) was used for the modification of poly(2-(dimethylamino)ethyl-methacrylate) (PDMAEMA) on the monolithic surface. Finally, this synthesized monolith for SPE was successfully applied in the extraction and enrichment of steroids. The results revealed that ATRP can be developed as a facile and effective method with mild reaction conditions for monolith construction and has the potential for preparing monolith in diverse devices.

    Preparation and modification of polyhedral oligomeric silsesquioxane-containing hybrid monolithic capillary columns
    OU Junjie*, LIN Hui, LIU Zheyi, ZHANG Zhenbin, DONG Jing, ZOU Hanfa*
    2013, 31 (4):  322-328.  DOI: 10.3724/SP.J.1123.2012.03012
    Abstract ( 1271 )   HTML ( 5 )   PDF (870KB) ( 149 )  

    Two kinds of hybrid monolithic capillary columns were prepared via free radical polymerization. First, the hybrid monolithic column was in situ fabricated by using two polyhedral oligomeric silsesquioxanes (POSS), POSS-methacryl substituted (cage mixture, n=8, 10, 12) (POSS-MA) and POSS-(1-propylmethacrylate)-heptaisobutyl substituted (Bu-POSS), as crosslinker and monomer, respectively, in the presence of porogenic solvents (propanol/PEG400) and initiator (azodisobutyronitrile). Second, the hybrid monolithic column was similarly prepared by using POSS-MA, as sole monomer under the same conditions. The resulting two kinds of hybrid monolithic columns, poly(Bu-POSS-co-POSS-MA) and poly(POSS-MA), were characterized and applied for the separation of alkylbenzenes by capillary liquid chromatography (cLC), showing good mechanical stability and reproducibility. Additionally, the poly(POSS-MA) monolith could be easily modified with other organic monomers, such as stearyl methacrylate (SMA) and benzyl metacrylate (BeMA), with the presence of methacryl groups on the surface of the poly(POSS-MA) monolith. Compared with the raw poly(POSS-MA) monolith, the post-modified poly(POSS-MA) monolith exhibited higher column efficiency and better selectivity. This simple process of using POSS reagents via free radical polymerization is providing a novel way for the preparation of the organic-silica hybrid monolithic columns.

    Preparation and retention mechanism of a mixed-mode reversed-phase/strong-cationic-exchange chromatographic packing
    PENG Xitian, WANG Jue, FENG Yuqi*
    2013, 31 (4):  329-334.  DOI: 10.3724/SP.J.1123.2012.11009
    Abstract ( 1327 )   HTML ( 4 )   PDF (819KB) ( 220 )  

    A simple and efficient method has been proposed for the preparation of octyl-sulfonic co-bonded silica (OSS) packing by the method of “mixed ligand”. The resulting OSS packing was characterized by elemental analysis and ion-exchange capacity to prove the successful immobilization of octyl and sulfonic groups on the surface of silica gel. Then the retention mechanism of several basic analytes on the developed OSS phases was evaluated under the conditions of reversed-phase liquid chromatography (RPLC) mobile phase. The results indicated that the OSS stationary phases demonstrated a mixed-mode reversed-phase/strong-cationic-exchange (RP/SCX) retention mechanism and ion-exchange interaction maybe dominate the retention of the basic analytes. By changing the salt concentration of mobile phase, the one-site and two-site mixed-mode retention models of the several basic analytes on the OSS phases were obtained by investigating the logarithm and reciprocal relationships of retention factor and salt concentration. On the basis of the linear fitting of the two mathematical equations of the retention models, the experimental results demonstrated that the two-site model was more suitable for the description of the retention mechanism of the basic analytes on the OSS phases. Furthermore, the individual RP or SCX contribution to total retention was obtained according to the mathematical equations of the two-site retention mechanism, which can provide some valuable guidance for the separation of complex samples.

    Sub-micrometer mesoporous SBA-15 silica rods as stationary-phase for capillary electrochromatography separation
    ZHANG Lingyi, WANG Zhicong, ZHANG Weibing*
    2013, 31 (4):  335-341.  DOI: 10.3724/SP.J.1123.2012.11016
    Abstract ( 1112 )   HTML ( 3 )   PDF (873KB) ( 238 )  

    Mesoporous SBA-15 silica rods were packed into capillary as stationary phase for capillary electrochromatography (CEC) separation. The attractive properties of the sub-micrometer material are its radical size of 400 nm and highly ordered cylindrical mesopores running along the same direction. The special structure is formed by piling of rod shape particles, which provide good permeability and facilitate the packing procedure of small size CEC column. The modified silica particles were successfully applied for the reversed-phase (RP) and ion-exchange (IE) electrochromatographic separation of non-polar and polar compounds with satisfactory efficiency (140000 plates/m for benzene using 70%(v/v) acetonitrile). The van Deemter plot showed the hindrance to mass transfer because of the existence of pore structure. The lowest plate height of 7.1 mm was obtained at the linear velocity of 3.2 cm/min. The prepared ion exchange column was applied to the separation of Chinese traditional medicines Ginseng and Rhizoma Gastrodiae.

    Synthesis of porous spherical silicon oxynitride material and evaluation of its properties in reversed-phase chromatographic separation
    ZHONG Hongmin, ZHANG Hua, WAN Huihui,*
    2013, 31 (4):  342-347.  DOI: 10.3724/SP.J.1123.2012.11032
    Abstract ( 1058 )   HTML ( 3 )   PDF (818KB) ( 181 )  

    Silica has been widely used as HPLC column packing material. However, the fact that base can attack the silanol and dissolve the silica embarrasses the utilization of silica stationary phase in high pH mobile phases (pH>8). In our previous research, the use of porous spherical silicon oxynitride (sph-SiON) material from high temperature nitridation of silica microspheres as stationary phase for HPLC has been explored, and the sph-SiON is stable to alkaline mobile phases and demonstrates excellent separation of a variety of polar compounds in hydrophilic interaction liquid chromatography (HILIC) mode. Herein, the degree of nitridation was studied as a function of temperature of nitridation at 750-1050 ℃, yielding the silicon oxynitride with 0.40%-12.0% (mass fraction) nitrogen from elemental analysis. At the temperature of 1050 ℃, the nitrogen content increased from 12.0% to 24.5% with the nitridation time increasing from 20 h to 120 h. The sph-SiON is stable when disposed in different pH aqueous solutions for one week. The sph-SiON material can be modified to give hydrophobic surface through the reaction of surface Si-NHx with dimethyloctadecylchlorosilane. Elemental analysis and 13C cross-polarization magic-angle spinning (CP/MAS) NMR spectrum of C18-sph-SiON prove the integration of C18 alkyl groups attached onto the sph-SiON surface. The chromatographic evaluation of C18-sph-SiON in reversed-phase separation mode was performed with alkylbenzenes as hydrophobic probes. Three alkylbenzene compounds can be separated and retained well on C18-sph-SiON even in the mobile phase of methanol/H2O (70/30, v/v) with 78507 plates/m, and an excellent tailing factor (0.95) can be obtained for ethylbenzene. In comparison with C18-SiO2, C18-sph-SiON shows distinct differences with respect to different classes of analytes, i.e. neutral analyte naphthalene, acidic analyte ibuprofen, and basic analyte amitriptyline.

    Preparation of boronate affinity monolithic column with high specific surface using highly structurally symmetrical copolymerizing reagents
    LIU Yunchun, LIU Zhen*
    2013, 31 (4):  348-351.  DOI: 10.3724/SP.J.1123.2012.02014
    Abstract ( 1300 )   HTML ( 4 )   PDF (785KB) ( 338 )  

    Boronate affinity monolithic column is an important chromatographic medium for the selective isolation of the cis-diol containing compounds. However, it is difficult to control the micro-structure of the monolithic capillary during its preparation process. In this article, using melamine and tris(2,3-epoxypropyl) isocyanurate (TEPIC), both are highly symmetrical in molecular structure, as the copolymerizing reagents, a boronate affinity monolithic capillary with high specific surface was prepared by in-situ polymerization according to the “teamed boronate affinity” principle. The pH for binding with cis-diol containing compounds of this monolithic capillary was decreased to neutral range due to the teamed bonorate affinity. The specific surface area was dramatically increased due to the use of the highly symmetrical copolymerizing reagents, which was 80.3 m2/g and apparently higher than those for other boronate affinity monolithic capillaries reported in references.

    Highlight
    Recent applications of graphene-based materials in capillary electrophoresis
    YE Nengsheng*, LI Jian, GU Xuexin
    2013, 31 (4):  352-354.  DOI: 10.3724/SP.J.1123.2012.12045
    Abstract ( 1369 )   HTML ( 4 )   PDF (724KB) ( 256 )  
    Graphene and graphene-based materials have been widely applied in biochemical and chemical analysis because of its unique physical and chemical properties. Recent applications of graphene-based materials in capillary electrophoresis (CE) were highlighted, such as modification of amperometric detection electrode, preparation of monolithic column, modification of capillary inner wall and modification of microchip. The potential application of graphene materials in CE is discussed in this review.
    Articles
    Preparation of a trypsin immobilized reactor on silver wire modified by atom transfer radical polymer and its application in proteome identification
    ZHOU Lianqi, ZHANG Jiao, TIAN Fang, ZHANG Yangjun*, QIAN Xiaohong
    2013, 31 (4):  355-361.  DOI: 10.3724/SP.J.1123.2012.10030
    Abstract ( 1197 )   HTML ( 3 )   PDF (1697KB) ( 245 )  
    The routine proteolysis of proteins is performed in solution, but it suffers from drawbacks such as long incubation time, enzyme autodigestion, and non-reusability. Therefore we here demonstrated that trypsin could be immobilized on silver wire modified by atom transfer radical polymerization to prepare a new kind of enzyme immobilized reactor. The digestion efficiency, repeatability and recovery of the silver wire-trypsin reactor (SW-Trypsin) were evaluated by mass spectrometry (MS) analysis. Highly efficient digestion was achieved by using SW-Trypsin within only 20 min. Standard protein could be almost completely digested with sequence coverage up to 93%, which is higher than that of 79% sequence coverage obtained by in-solution digestion for 16 h. Bovine serum albumin (BSA) was digested eight times within a month by using the SW-Trypsin. The results of sequence coverage were between 89% to 97%, with an average sequence coverage of 94%, which showed that SW-Trypsin had good stability. In addition, the recovery test by using myoglobin (MYO) showed that the recovery rate was 87.67%. At last, the extract from Tengchong thermophilic bacteria was digested by SW-Trypsin in 20 min and in-solution trypsin in 16 h. The results of sequence coverages and the number of identified proteins were similar. Moreover, the ratio of the number of peptides with zero missed cleavages to the number of all identified peptides by using SW-Trypsin was higher than that by in-solution digestion. Also, the SW-Trypsin was easily removed from the digestion solution. Good performances of SW-Trypsin implied that it has a good prospect in the application in future proteomics research.
    Analysis of proteins in the extracts of Physalis alkekengi L. var. franchetii (Mast.) Makino using nanoflow reversed-phase liquid chromatography-tandem mass spectrometry
    YU Haiyang, YAN Jiaze, GUO Ming, JIN Yan*
    2013, 31 (4):  362-366.  DOI: 10.3724/SP.J.1123.2012.11023
    Abstract ( 1266 )   HTML ( 3 )   PDF (899KB) ( 230 )  
    The protein was extracted from Physalis alkekengi L. var. franchetii (Mast.) Makino fruit by using water extraction and acid precipitation methods, and it consisted of 188 mg/g of protein on dry basis of the extract. Among 18 amino acids, eight essential amino acids for human account for 31% were found in this extract. Based on shotgun proteomics method, the protein extracted from Physalis fruit was analysed by nanoflow reversed-phase liquid chromatography-tandem mass spectrometry (nano-RPLC-MS/MS) system. Combined with database searches and bioinformatics analysis, the protein species and six molecular functions were identified, including with catalytic activity, antioxidant activity, enzyme regular activity, nutrient reservoir activity, transporter activity and binding activity. And three antioxidant activity-related proteins were identified. These results may lay the foundation for further study of the functional properties of the proteins in Physalis fruit.
    Ionization suppression effect of phosphopeptides in nano-electrospray ionization quadrupole time-of-flight mass spectrometry
    ZOU Yao, JIANG Wuhui, ZOU Lijuan*, LI Xiuling*, LIANG Xinmiao
    2013, 31 (4):  367-371.  DOI: 10.3724/SP.J.1123.2012.11024
    Abstract ( 1081 )   HTML ( 4 )   PDF (786KB) ( 406 )  
    Protein phosphorylation is an important post-translational modification (PTM), and involves in many cell activities and disease processes. Nowadays, mass spectrometry (MS) is a powerful tool in peptide-based proteomics. But phosphopeptides, especially multiply phosphorylated peptides, are difficult to be detected by MS. One opinion is that the presence of unphosphorylated peptides lead to the ionization suppression of phosphopeptides. But contrary opinion exists. Moreover, the suppression effect caused by different types of phosphopeptides hasn’t been studied yet. In this study, a set of synthetic standard phosphopeptides (singly, doubly and triply phosphorylated peptides) with the same amino acid sequence, and the unphosphorylated peptides were injected into mass spectrometer with or without mixing. Through the ratios of signal intensities of the phosphopeptides before and after mixing, we confirmed that the signals of phosphopeptides were suppressed by those of the unphosphorylated peptides. In addition, the signals of the doubly phosphorylated peptides were slightly suppressed by those of the singly phosphorylated peptides with the same amino acid sequence. The signals of the triply phosphorylated peptides were notably suppressed by the singly and doubly phosphorylated peptides with the same amino acid sequence. Therefore, the singly and multiply phosphorylated peptides should be fractionated and enriched before mass spectrometry analysis.
    Rapid screening and confirmation of 281 pesticide residues in apples, tomatos and cabbages by liquid chromatography/quadrupole time-of-flight mass spectrometry
    ZHAO Zhiyuan, SHI Zhihong, KANG Jian, PENG Xing, CAO Xinyue, FAN Chunlin*, PANG Guofang, Lv Meiling
    2013, 31 (4):  372-379.  DOI: 10.3724/SP.J.1123.2012.01034
    Abstract ( 1679 )   HTML ( 10 )   PDF (1019KB) ( 396 )  

    The application of QuEChERS coupled to liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) for screening 281 pesticide residues in apples, tomatos, and cabbages has been investigated. The established method includes the extraction by acetonitrile/acetic acid (99∶1, v/v), and clean-up by primary secondary amine (PSA) sorbent. The resultant residues were further analyzed by LC-Q-TOF/MS. For apples, tomatos, and cabbages, the percentages of pesticides with the average recoveries (n=5) in the range of 70%-120% were 98.6%, 99.3%, and 98.2%, respectively; and the relative standard deviations (RSDs) for the recoveries were not more than 20%; in the concentration range of 0.25-10 MRL, the percentages of the pesticides with the correlation coefficients (r2) greater than 0.99 were 95.7%, 96.1%, and 98.2%, respectively; the LODs were 0.03-4.47 μg/kg, 0.01-4.49 μg/kg and 0.02-3.61 μg/kg, respectively. Rapid screening and confirmation can be achieved with accurate mass database and library search without the standard compounds. The method has been applied to 30 fruit and vegetable samples randomly purchased from the local markets, and the 13 pesticides were detected. It was found that the level of methamidophos in one cabbage sample exceeded the MRL regulated by China of GB 2763-2012 and the MRL regulated by European Union (EU), and the level of rotenone in one tomato sample exceeded the MRL regulated by EU.

    Determination of carcinogenic aromatic amines derived from azo colorants in textiles and leather by ultra high performance liquid chromatography-tandem mass spectrometry
    WEN Yuyun, OU Yan, HE Mingchao, GONG Zhenbin*
    2013, 31 (4):  380-385.  DOI: 10.3724/SP.J.1123.2012.10037
    Abstract ( 1591 )   HTML ( 4 )   PDF (783KB) ( 278 )  
    A rapid determination method was developed for the quantification and confirmation of 22 carcinogenic aromatic amines derived from azo colorants in textiles and leather by ultra high performance liquid chromatography-tandem electrospray ionization mass spectrometry (UHPLC-MS/MS). The methods of EN 14362-1:2012 (for textiles) and ISO 17234-1:2010 (for leather) were adopted for sample pretreatment, finally diluted with methanol. The target compounds were separated by an Eclipse XDB-C18 RRHD column and eluted with methanol and water in gradient, and then determined by positive electrospray ionization mass spectrometry under multiple reaction monitoring (MRM) mode. The external standard method was used for the quantitative analysis. The separation conditions, fragment voltages, collision energies, etc. were optimized. The limits of quantification (LOQ) were below 0.2 mg/kg for different compounds, matrix spike recoveries ranged from 70% to 120% at the spiked levels of 500, 1000 and 1500 μg/L, and the relative standard deviations (RSDs) were less than 15%. The proposed method is rapid, sensitive, accurate and selective.
    Nano-electrospray ionization-ion mobility spectrometry and its combination with high performance liquid chromatography
    CHEN Chuang, WANG Weiguo, LIANG Xixi, ZHOU Qinghua, PENG Liying, WEN Meng, JU Bangyu, ZHAO Kun, LIU Jun, LI Haiyang*
    2013, 31 (4):  386-391.  DOI: 10.3724/SP.J.1123.2012.12008
    Abstract ( 1083 )   HTML ( 3 )   PDF (1414KB) ( 256 )  
    A nano-electrospray ionization-ion mobility spectrometer (nanoESI-IMS) was built up. Firstly, the effects of the parameters such as drift gas flow rate and solvent flow rate on the desolvation capability were studied and optimized. Then, a series of compounds were used to characterize the nanoESI-IMS system. The results showed that, complete desolvation was achieved for nano-electrospray ion droplets with the nanoESI-IMS apparatus. The limit of detection of this instrument for trioctylamine could reach 10 μg/L. Finally, this instrument was coupled to the high performance liquid chromatography (HPLC) as a detector for amines analysis. A test mixture containing triethylamine, diethylamine and butylamine was successfully separated and determined by the HPLC-nanoESI-IMS system. Linear response ranges of about two orders of magnitude were achieved for triethylamine, diethylamine and butylamine with this system.
    Monolithic molecularly imprinted column-high performance liquid chromatography for enrichment and determination of trace cytokinins in plant samples
    SUN Lin, DU Fuyou*, RUAN Guihua, HUANG Yijia
    2013, 31 (4):  392-394.  DOI: 10.3724/SP.J.1123.2012.10036
    Abstract ( 1427 )   HTML ( 4 )   PDF (808KB) ( 347 )  
    A method based on monolithic molecularly imprinted polymer enrichment combining with high performance liquid chromatography (mMIP-HPLC) detection was developed for the selective determination of trace cytokinins (CTKs) in plant samples. Monolithic molecularly imprinted polymer (mMIP) column was prepared in stainless steel tube by using kinetin as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, and toluene-dodecanol as the porogenic solvents. Compared with non-imprinted polymer (NIP) monolith, the prepared mMIP exhibited selective separation ability, good reproducibility and reusability, and high extraction efficiency in the separation and enrichment of the four CTKs. Under the optimized experimental conditions, mean recoveries were 91.9%、80.0%、87.5% and 50.2% for kinetin (K), kinetin glucoside (KR), trans-zeatin (tZ) and meta-topolin (mT), respectively, with the corresponding RSDs less than 11.8%. The proposed mMIP-HPLC method was successfully applied in the separation and determination of the four cytokinins in different plant samples.