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A mixed-mode chromatographic method based on surface electrostatic exclusion and reversed-phase chromatography was established for the determination of alkaloids present in Coptis chinensis. The effects of two mobile phase additives, formic acid and acetic acid, on retention, peak shape and selectivity of the alkaloids in Coptis chinensis were investigated using the self-made C18HCE column. Acetic acid (0.1%, v/v) used as the additive was found to be optimum for effective separation of the main alkaloids present in Coptis chinensis. The main chromatographic peaks of Coptis chinensis were recognized by the established method and references, which were coptisine, epiberberine, columbamine, jatrorrhizine, berberine and palmatine, respectively. With reference to the content determination method of Coptis chinensis in the 2015 edition of pharmacopoeia, the linear relationship of berberine in the range of 0.5-100 mg/L was good, the correlation coefficient was 0.9996, and the average recovery was 93.74%. The contents of alkaloids in Coptis chinensis in different batches of Hubei and Chongqing were determined. The method is simple, reliable and accurate, and can be used as reference for separation and analysis of other basic compounds.

A HILIC stationary phase was synthesized through bonding maltose on silica particles via click chemistry. A set of representative polar compounds including sugars, amino acids and small peptides were employed to evaluate chromatographic properties of Click Maltose. The results illustrated the surface adsorption mechanism from the direct hydrogen bonding interaction between sugars and Click Maltose played an important role on retention behavior of sugars under HILIC mode. The chromatographic behavior of anions on Click Maltose was consistent with HILIC/WAX mechanism, while the retention of cations was depended on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mechanism. The Click Maltose stationary phase was successfully used in the separation of standard samples of polar compounds and complex samples of oligosaccharides from the natural product. The Click Maltose also exhibited good reproducibility and stability, which were helpful to further application. Our study confirmed the advantages and application potential of bonded oligosaccharides for HILIC separation.

Two different offline 2-D hydrophilic interaction chromatography (2D-HILIC/HILIC) systems have been developed. In the two systems, a click maltose column was used in the first dimension, an amide column or a click beta-CD column was used in the second dimension, respectively. Both of the systems were used for the analysis of very polar components in Carthamus tinctorius Linn., which is a traditional Chinese medicine. Excellent orthogonality and separation results were obtained in both 2D-HILIC/HILIC systems, while the peak capacity of the system based on click maltose and amide column was higher for its adoption of stationary phase with smaller particle size in the second dimension.

A novel two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography (2D-RPLC/HILIC) system is developed with the introduction of the click beta-cyclodextrin (beta-CD) stationary phase. The offline system shows excellent abilities for the separation of polar and medium-polarity components in traditional Chinese medicine (TCM). It facilitates us not only to separate components that cannot be resolved by uni-dimensional chromatography, but also to achieve much more efficient detection of components with low abundance. The orthogonality of this new system is excellent. A simple geometric approach is developed to characterize the practical orthogonality of 2D-LC in the analysis of complex unknown samples. The mathematical characterization results are in good accordance with the experimental findings, and both demonstrate that the proposed 2D-RPLC/HILIC system is a powerful tool for the separation of polar and medium-polarity complex samples. This system may have a great potential for the separation of peptides and metabolomic compounds with similar polarity.

A novel click oligo(ethylene glycol) (Click OEG) stationary phase was proposed for constructing two-dimensional reversed-phase/reversed-phase liquid chromatography system (2D-RP/RPLC) combined with C18. The new stationary phase was prepared by immobilization of oligo(ethylene glycol) on silica support via click chemistry. And the resulting material was characterized by FT-IR, solid state CP/MAS 13C NMR and elemental analysis. The investigation on orthogonality between Click OEG and C18 was performed by geometric approach with a set of test solutes. The orthogonality reached 68.94%, which is much higher than other combinations. An offline 2DLC system consisted of Click OEG and C18 was constructed and applied to the analysis of a kind of traditional Chinese medicine Lignum Dalbergiae Odoriferae. Excellent separation results validated the high orthogonality between the Click OEG and C18 in the practical separation of complex samples.

Hydrophilic interaction liquid chromatography (HILIC) is an effective technique for separating polar compounds. But its retention equation has not been studied systematically yet. In this study, an appropriate retention equation was established by using eight nucleosides as model analytes and by comparing four retention models on six different HILIC columns. As a result, retention equation Ink = a + b x InC(B) + cxC(B) could be quantitatively described the retention factors with good accuracy in HILIC mode. Based on this equation, the retention times of eight nucleosides under the conditions by other mobile phase can be predicted on each column. All of the predicted relative errors of retention times were smaller than 5%. The established retention model was also successfully applied to predict retention times of the real Traditional Chinese Medicine-Carthaus tinctorius L. sample.

Oligo(ethylene glycol) (OEG) covalently bonded silica was prepared by using click chemistry and employed as a stationary phase for reversed-phase high-performance liquid chromatography. The column efficiency and effect of organic modifier content on retention were investigated. The separation selectivity was also studied with phenyl compounds and an actual sample of natural products. The results indicated that the stationary phase possessed good separation efficiency and separation selectivity in RP-HPLC mode. Moreover, the stationary phase showed good complementary separation selectivity to the C18 stationary phase. The OEG stationary phase had "clustering" function for "homologous component" in the separation of natural products.

Hydrophilic interaction chromatography (HILIC) was employed to separate the co-eluted flavonoids from licorice extract under RP-HPLC mode. HILIC separations were carried out with the Atalantis HILIC Silica column and the CD-based column. The co-eluted flavonoids were well retained and separated on the two HILIC columns under HILIC mode. Similar results were obtained in the separation of another isoflavones sample, from kudzu extract under HILIC mode.

Hemoglobin (Hb) and myoglobin (Mb) were immobilized at the didodecyldimethylammonium bromide (DDAB)-modified powder microelectrode (PME) to fabricate Hb-DDAB-PME and Mb-DDAB-PME. Direct electrochemistry of Hb and Mb were achieved on the DDAB-modified PME. The formal potential was -0.224 V for Hb and -0.212 V for Mb (vs. SCE). The apparent surface concentration of Hb and Mb at the electrode surface was 2.83 x 10(-8) and 9.94 x 10(-8) mol cm(-2). The Hb-DDAB-PME and Mb-DDAB-PME were successfully applied for measurement of NO in vitro. The anodic current peaks for NO oxidation at +0.7 V and the cathodic current peaks for NO reduction at -0.85 V on the CV curves were obtained on the modified electrodes. For detection of NO at +0.7 V, the sensitivity is 3.31 mA microM(-1) cm(-2) for Hb-DDAB-PME and 0.6 mA microM(-1) cm(-2) for Mb-DDAB-PME. The detection limit is 5 nM for Hb-DDAB-PME and 9 nM for Mb-DDAB-PME. The linear response range is 9-100 and 28-330 nM for Hb- and Mb-modified PME, respectively. For the electrochemical detection of NO at -0.85 V by using Hb-DDAB-PME, the detection sensitivity is 39.56 microA microM(-1) cm(-2); the detection limit is as low as 0.2 microM; and the linear response range is 1.90-28.08 microM.

Chromatography is categorized after the type of mobile phase used, liquid, gas, or supercritical chromatography. Liquid chromatography is a very important separation method used in practically all chemistry fields. This chapter focuses on the basic theory necessary for a deeper understanding of the separation process. It covers new trends in liquid chromatography today. Analytical (linear) chromatography can be described by relatively simple models: injection of an n component mixture will give n Gaussian-shaped peaks, which are more or less separated in the chromatogram. The chapter also focuses on analytical (linear) models assuming Gaussian peaks. It also talks about basic retention models and kinetics, band broadening and the plate height concept, sources of zone broadening, and dependence of zone broadening on flow rate. The new technique using small particles was given the name high-performance liquid chromatography (HPLC). Keywords: analytical chromatography; band broadening; Gaussian peaks; high-performance liquid chromatography; hybrid phases; retention models; separation process; zone broadening

A non-derivative method was developed for the qualitative and quantitative analysis of isosteroidal alkaloids from Fritillaria thunbergii. During method development the performance of two universal detectors, the charged aerosol detector (CAD) and evaporative light scattering detector (ELSD), were evaluated. The CAD was found to be 30 to 55 times more sensitive than ELSD enabling the measurement of low levels of reference compound impurities that could not be detected by ELSD. The peak area percent of the reference compound, peimisine, obtained by CAD was 50.10%, but 91.66% by ELSD showing that CAD is suitable to estimate the presence of impurities. The CAD showed good reproducibility with overall intra- and inter-day peak area RSD values of less than 1.8% and 2.7%, respectively and had a linear dynamic range of up to 4 orders of magnitude (0.06-44mg/L) for peimine and peiminine. The optimized method was used for the quantitative analysis of peimine and peiminine from F. thunbergii.

The separation of basic macrolide antibiotics suffers from peak tailing and poor efficiency on traditional silica-based reversed-phase liquid chromatography columns. In this work, a C18HCE column with positively charged surface was applied to the separation of macrolides. Compared with an Acquity BEH C18 column, the C18HCE column exhibited superior performance in the aspect of peak shape and separation efficiency. The screening of mobile phase additives including formic acid, acetic acid and ammonium formate indicated that formic acid was preferable for providing symmetrical peak shapes. Moreover, the influence of formic acid content was investigated. Analysis speed and mass spectrometry compatibility were also taken into account when optimizing the separation conditions for liquid chromatography coupled with tandem mass spectrometry. The developed method was successfully utilized for the determination of macrolide residues in a honey sample. Azithromycin was chosen as the internal standard for the quantitation of spiramycin and tilmicosin, while roxithromycin was used for erythromycin, tylosin, clarithromycin, josamycin and acetylisovaleryltylosin. Good correlation coefficients (r(2)0.9938) for all macrolides were obtained. The intra-day and inter-day recoveries were 73.7-134.7% and 80.7-119.7% with relative standard deviations of 2.5-8.0% and 3.9-16.1%, respectively. Outstanding sensitivity with limits of quantitation (S/N ≥ 10) of 0.02-1 μg/kg and limits of detection (S/N ≥ 3) of 0.01-0.5 μg/kg were achieved.

Bufadienolides possess various bioactivities especially antitumor. Due to the high structural diversity, the separation of bufadienolides often suffers from coelution problem on conventional RP columns. In this work, an off-line two-dimensional normal-phase liquid chromatography×reversed-phase liquid chromatography (2D-NPLC×RPLC) method was developed to separate and characterize bufadienolides in toad skin. Several RP and NP columns were evaluated with five reference bufadienlides. The XUnion C18 and XAmide columns exhibited superior chromatographic performances for bufadienlide separation, and were selected in RPLC and NPLC, respectively. RPLC was used in the second-dimension for the good compatibility with MS, while NPLC was adopted in the first-dimension. The orthogonality of the 2D-NPLC×RPLC system was investigated by the geometric approach using fifteen bufadienolide mixtures. The result was 49.6%, demonstrating reasonable orthogonality of this 2D-LC system. By combining the 2D-LC system with MS, 64 bufadienlides including 33 minor ones and 11 pairs of isomers in toad skin were identified. This off-line 2D-NPLC×RPLC allowed to solve the coelution problem of bufadienlides in one-dimension RPLC, and thus facilitated the identification significantly.

A sensitive non-derivatization method was developed for the analysis of apramycin and impurites using hydrophilic interaction liquid chromatography (HILIC) and charged aerosol detection (CAD). Sample was pretreated with an effective SPE method (recovery >90%) to remove interference with apramycin impurities from sulfate, then analyzed with direct injection. Different chromatography modes of separation and choices of HILIC column were investigated in search of a direct analysis method. The HILIC-CAD method was optimized using a cysteine-bonded zwitterionic HILIC column and compared to the strong cation exchange-ultraviolet (SCX-UV) method with post-column derivatization recommended by the Chinese Pharmacopoeia (veterinary) 2010. The improved chromatographic resolution and peak shape with the HILIC-charged aerosol detection method allows for increase of sample load to 48.9 µg from only 2.8 µg with the SCX-UV approach. More than 16 impurities were detected with this method with improved resolution, and four were identified with MS, while only 7 impurities were detected with the SCX-UV method. Moreover, the current method has a good precision and reproducibility. The intra-day and inter-day of peak area variability was less than or equal to 4.760% RSD and 9.950%, respectively. The average limit of detection and quantization was 80 ng and 200 ng injected on the column, respectively. The overall results demonstrated that the presented method can be used as an alternative to SCX-UV method in the analysis of apramycin and impurities.

An interesting and novel method for the selective and sequential enrichment of singly- and multiply-phosphorylated peptides with a zwitterionic material "Click TE-Cys" is presented. Retention mechanisms between phosphopeptides and Click TE-Cys are systematically investigated by checking the influence of acetonitrile content, pH value, and buffer concentration on the retention of phosphopeptides. Both hydrophilic interaction and electrostatic interaction are involved in retention between phosphopeptides and Click TE-Cys. Based on these results, an optimized method is established for selective enrichment of phosphopeptides using Click TE-Cys. This method not only exhibits high selectivity for phosphopeptides, but also fractionates singly- and multiply-phosphorylated peptides into two fractions. This method was evaluated using relatively complex samples, including peptide mixtures of α-casein and bovine serum albumin (BSA) at a molar ratio of 1:10 and skim milk. This efficient and optimized protocol has great potential for enriching multiply-phosphorylated peptides and could be a valuable tool for specific enrichment of phosphopeptides in phosphoproteome analysis.

Peptides from scorpion venom represent one of the most promising drug sources for drug discovery for some specific diseases. Current challenges in their separation include high complexity, high homologies and the huge range of peptides. In this paper, a modified strong cation exchange material, named MEX, was utilised for the two-dimensional separation of peptides from complex scorpion venom. The silica-based MEX column was bonded with two functional groups; benzenesulfonic acid and cyanopropyl. To better understand its separation mechanisms, seven standard peptides with different properties were employed in an evaluation study, the results of which showed that two interactions were involved in the MEX column: electrostatic interactions based on benzenesulfonic acid groups dominated the separation of peptides; weak hydrophobic interactions introduced by cyanopropyl groups increased the column's selectivity for peptides with the same charge. This characteristic allowed the MEX column to overcome some of the drawbacks of traditional strong cation exchange (SCX) columns. Furthermore, the study showed the great effects of the acetonitrile (ACN) content, the sodium perchlorate (NaClO4) concentration and the buffer pH in the mobile phase on the peptides’ retention and separation selectivity on the MEX column. Subsequently, the MEX column was combined with a C18 column to establish an off-line 2D-MEX × C18 system to separate peptides from scorpion Buthus martensi Karsch (BmK) venom. Due to complementary separation mechanisms in each dimension, a high orthogonality of 47.62% was achieved. Moreover, a good loading capacity, excellent stability and repeatability were exhibited by the MEX column, which are beneficial for its use in future preparation experiments. Therefore, the MEX column could be an alternative to the traditional SCX columns for the separation of peptides from scorpion venom.

Nowadays more and more hydrophilic interaction liquid chromatography (HILIC) columns with diverse functional groups have become commercially available, which pose a challenge to select an appropriate one. However, there is no universal model to provide guidance for selecting HILIC columns. To handle this problem, a retention model named "hydrophilic-subtraction model" was developed to characterize and compare HILIC columns. The hydrophilic-subtraction model, which was designed based on the widely recognized HILIC retention mechanisms including hydrophilic partitioning, hydrogen-bonding and electrostatic interactions, was established by the retention of 41 solutes with various properties on 8 representative HILIC columns. High correlation coefficients (R-2 >= 0.990) and small standard deviations (SD

Cytochrome c was immobilized at didodecyldimethylammonium bromide (DDAB)‐modified powder microelectrode and presented quasi‐reversible electrochemistry. The apparent surface coverage of cytochrome c is greatly enhanced by using powder microelectrode technique, which is 1.21×10−8 mol/cm2, more than one to three orders of magnitude larger than that obtained with thiol and DNA‐modified Au electrode. The cytochrome c modified powder microelectrode was applied for the amperometric determination of superoxide generated by the reaction of hypoxanthine with xanthine oxidase (XOD) in the presence of dioxygen. The detection sensitivity of the modified powder microelectrode is 0.74 µA/cm2 µM, which is larger than that reported in previous publications. The detection limit of the modified powder microelectrode (PME) is 0.5 µM, and the linear detection range is 0.86∼5.93 µM (values of the concentration are all in terms of hypoxanthine concentration in the solution).

The determination of antioxidants continues to be interested, since the oxidative damage is thought to be one of the main mechanisms involved in nearly all chronic renal pathologies. A highly sensitive high performance liquid chromatography-electrochemical detection (HPLC-ECD) method was developed for evaluating the antioxidant properties of Salvia miltiorrhiza (Dan Shen). The method was optimized with respect to selectivity and sensitivity. Chromatographic conditions, including mobile phase pH value, buffer concentration, buffer type, organic solvent type, gradient profile and flow rate, were systematically investigated. Low pH value (2.8), low buffer concentration (20 mmol/L NaH2PO4), a shallow water-acetonitrile gradient, and a flow rate of 0.2 mL/min were the determined optimal conditions for the quantitative analysis of aimed five antioxidants from 14 batches of Dan Shen samples. The described method provided a good recovery (>95%), a very wide linear range (up to 104 for all analytes), a good precision (RSDs

This study investigated the influence of organic sample solvents on separation efficiency of basic compounds under strong cation exchange (SCX) mode. The mixtures of acidic aqueous solution and organic solvent such as acetonitrile, ethanol, methanol and dimethyl sulfoxide (DMSO) were tested as sample solvents. For later-eluting analytes, the increase of sample solvent elution strength was responsible for the decrease of separation efficiency. Thus, sample solvents with weak elution strength could provide high separation efficiencies. For earlier-eluting analytes, the retention of organic sample solvents was the main factor affecting separation efficiency. Weakly retained solvents could provide high separation efficiency. In addition, an optimized approach was proposed to reduce the effect of organic sample solvent, in which low ionic solvent was employed as initial mobile phase in the gradient. At last, the analysis of impurities in hydrophobic drug berberine was performed. The results showed that using acidic aqueous methanol as sample solvents could provide high separation efficiency and good resolution (R > 1.5). (C) 2015 Elsevier B.V. All rights reserved.

Characterization of reverse-phase column selectivity is helpful for chromatographers to select an optimal column. A novel method, based on linear solvation energy relationships (LSERs) combined with fundamental retention equations, was developed to characterize and compare reversed-phase column selectivity. The retention times of 25 elaborately selected solutes on 12 reversed-phase columns were determined in three linear gradient elutions. Using these retention times, fundamental retention equations were acquired by a complex sample analysis software system (CSASS). When 0%, 10%, 20%, 30%, 40% and 50% acetonitrile were introduced into the fundamental retention equations, the corresponding retention factors were predicted and used to obtain LSER equations by multiple linear regression. In the gradient elution, the retention times of solutes could be accurately determined and the excessively long or short analysis time could be avoided. As the retention factor (ln k(w)) at a hypothetical 0% organic modifier closely reflected properties of columns, coefficients of LSERs equations obtained based on In kw were employed to discuss the properties of different stationary phases. An angle and a spider diagram based on solvation energy vectors were used to compare selectivity differences between stationary phases, which provided a visual means for users to select appropriate columns with orthogonal or similar selectivity. These results of column selectivities were compared with those obtained by geometric orthogonality approach, and a consistent result was acquired. Finally, Click TE-CD and XCharge C18PN with highest difference in column selectivity were applied to the separation of Psoralea cotylifolia extraction. (C) 2014 Elsevier B.V. All rights reserved.

Ethnopharmacological relevance The need for an efficacious analgesic without unwanted side effects is urgent. κ-opioid receptor agonists are known to exhibit potent analgesic effects and elicited fewer side effects than other opioid agonists. Thus in this study we chose the κ-opioid receptor as the target to identify the active components from traditional Chinese medicines (TCMs). Materials and methods The κ-opioid receptor was expressed in human embryonic kidney-293 T cells (HEK293T). Fluorometric Imaging Plate Reader (FLIPR) assay was used for the determination of Ca 2+ response when κ-opioid receptor was activated. A novel 2D separation system employing C18HCE as the first dimension and a strong cation exchange column (SCX) as the second dimension was conducted for the purification of the active principles. Results With the aid of HPLC-based activity profiling, activities could be linked to two peaks from Corydalis yanhusuo W. T. Wang ( C. yanhusuo ) extract. Two N-methyltetrahydroprotoberberines with κ-opioid receptor agonist activities were isolated for the first time from C. yanhusuo by using 2D-LC. Conclusions Our study suggests that N-methyltetrahydroprotoberberines may serve as a new scaffold for κ-opioid receptor ligands. The strategy that we adopted can be applied to other naturally-occurring active alkaloids acting at different receptors.

Protein retention is very sensitive to the change of solvent composition in reversed-phase liquid chromatography for so called "on-off" mechanism, leading to difficulty in mobile phase optimization. In this study, a novel 3-chloropropyl trichlorosilane ligand bonded column was prepared for protein separation. The differences in retention characteristics between the 3-chloropropyl trichlorosilane ligand bonded column and n-alkyl chain modified (C2, C4, C8) stationary phases were elucidated by the retention equation l nk=a+cC(B). Retention parameters (a and c) of nine standard proteins with different molecular weights were calculated by using homemade software. Results showed that retention times of nine proteins were similar on four columns, but the 3-chloropropyl trichlorosilane ligand bonded column obtained the lowest retention parameter values of larger proteins. It meant that their retention behavior affected by acetonitrile concentration would be different due to lower |c| values. More specifically, protein elution windows were broader, and retentions were less sensitive to the change of acetonitrile concentration on the 3-chloropropyl trichlorosilane ligand bonded column than that on other columns. Meanwhile, the 3-chloropropyl trichlorosilane ligand bonded column displayed distinctive selectivity for some proteins. Our results indicated that stationary phase with polar ligand provided potential solutions to the "on-off" problem and optimization in protein separation.

N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo Hf), respectively, prior to LC-MS/MS analysis. On the basis of the workflow, a total of 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo Hf deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG.

Hydrophilic interaction liquid chromatography has emerged as a valuable alternative approach to ion-pair chromatography for the separation of aminoglycoside antibiotics in recent years. However, the resolution of structurally related aminoglycosides is a great challenge owing to the limited selectivity. In this work, a cysteine-based zwitterionic stationary phase (named Click TE-Cys) was utilized and compared with five commonly used hydrophilic interaction liquid chromatography columns. Click TE-Cys displayed much better selectivity for structurally similar aminoglycosides. The retention behaviors of aminoglycosides were investigated in detail, revealing that low pH (2.7 or 3.0) and high buffer concentration (≥50 mM) were preferable for achieving good peak shape and selectivity. Effective resolution of ten aminoglycosides including spectinomycin, dihydrostreptomycin, streptomycin, gentamicin C1, gentamicin C2/C2a, gentamicin C1a, kanamycin, paromonycin, tobramycin, and neomycin was realized at optimized conditions. Additionally, spectinomycin and its related impurities were successfully resolved. The results indicated the great potential of the Click TE-Cys column in the separation of aminoglycoside mixtures and related impurities.

Background Current pain management is limited, in particular, with regard to chronic pain. In an attempt to discover novel analgesics, we combined the approach developed to characterize traditional Chinese medicine (TCM), as part of the "herbalome" project, with the reverse pharmacology approach aimed at discovering new endogenous transmitters and hormones. Results In a plant used for centuries for its analgesic properties, we identify a compound, dehydrocorybulbine (DHCB), that is effective at alleviating thermally induced acute pain. We synthesize DHCB and show that it displays moderate dopamine receptor antagonist activities. By using selective pharmacological compounds and dopamine receptor knockout (KO) mice, we show that DHCB antinociceptive effect is primarily due to its interaction with D2 receptors, at least at low doses. We further show that DHCB is effective against inflammatory pain and injury-induced neuropathic pain and furthermore causes no antinociceptive tolerance. Conclusions Our study casts DHCB as a different type of analgesic compound and as a promising lead in pain management. Video Abstract © 2014 Elsevier Ltd.

As a kind of promising anticancer compounds, the preparation of bufadienolides is a hot study spot. However, due to the complexity of biological sample, the purification of bufadienolides from a crude sample (toad skin) is a tough work. In this paper, we reported a new way based on positively charged C18 material (XCharge C18) to quickly separate and purify bufadienolides from toad skin. By this method, the different ionic feature of the amino acid conjugated bufadienolides (AACBs) and the free form bufadienolides (AAUBs) was firstly utilized to obtain distinct separation selectivity on the XCharge C18 column. Additionally, the peak tailing problem of AACBs on conventional C18 was resolved and better resolutions were achieved on the XCharge C18, thus, two kinds of bufadienolides on one column were successfully purified respectively. Taking F13 as an example, the method was validated by liquid chromatography-mass spectrometry (LC-MS), and then 4 AACBs as well as 4 AAUBs were simultaneously purified by preparative XCharge C18. In addition, the application of this method in other fractions was also validated. The results suggested that the developed method is a practical and promising tool for efficient separation and purification of bufadienolides from toad skin.

An effective method aiming at enrichment and analysis of micro-components in traditional Chinese medicine (TCM) was developed. One fraction (fraction E) from the extract of Hedyotis diffusa Willd. was selected as test sample, which was isolated by using the XAD-4 macroporous resin. To study the micro-components, a two-dimensional reverse-phase liquid chromatography (2D-RPLC/RPLC) method was developed, comprising Click OEG and C18 stationary phases as the first and second dimensions, respectively. Of the eight sub-fractions isolated from the first dimension, three sub-fractions (fractions II-IV) containing micro-components were further separated with the second dimension. The 2D-RPLC/RPLC system was proved to possess high orthogonality. Furthermore, the micro-components were characterized by using ultra-performance liquid chromatography-diode array detector/quadrupole time-of-flight mass spectrometry (UPLC-DAD/Q-TOF MS) with electrospray ionization (ESI) source. With the optimized separation and characterization method, a large number (>400) of micro-components were enriched and detected from the extracts of H. diffusa Willd., the majority of which has not been isolated from the herb before. Among these isolated micro-components, 38 compounds involving 24 phenylpropanoids, 7 flavonoids and 7 iridoid glucosides (IGs), were identified or tentatively identified from the H. diffusa extracts on the basis of spectral data of the authentic standards and the fragmentation characteristics information available in literatures. The proposed method made it possible to effectively screen and analyze the micro-components in TCMs or other complex natural medicines. (C) 2014 Elsevier B.V. All rights reserved.

Thiol‐ene/yne radical addition reactions, which possess the classical characteristics of click chemistry, have been emerging as facile and efficient coupling procedures in various fields. Different from the typical Huisgen azide‐yne 1,3‐dipolar cycloaddition, thiol radical click chemistry can be performed without the use of metal catalysts, which is superior in biological application. Additionally, the utilization of metal‐free thiol radical click reactions in the synthesis of chromatographic stationary phases can avoid having residual metal ions in the materials which is detrimental in chromatographic separation, especially for analytes that can form chelates with metal ions. In this chapter, the application of the radical‐mediated thiol‐ene/yne click reaction in surface modification and functionalization iss introduced. Thiol click chemistry has been successfully utilized in surface engineering and patterning, the modification of solid surface with alternative properties, the immobilization of macromolecules, etc. In addition, thiol radical click chemistry has been efficiently used in the preparation of chromatographic stationary phases, including chiral (ion‐exchange) materials, reversed‐phase stationary phases, mixed‐mode stationary phases and hydrophilic interaction liquid chromatography (HILIC) stationary phases.

Peak tailing and nonalkaloid coelution usually hinder alkaloid purification. In this study, a 2DLC, strong cation exchange (SCX) coupled with positively charged RP (PGRP) LC, was developed to overcome these problems. Ten compounds including basic and nonbasic compounds were analyzed. Nonbasic compounds, which are coeluted with basic compounds on RP or PGRP columns, were weakly retained on the SCX column. In addition, a symmetrical peak shape (tailing factors

As a naturally hydrophilic peptide, glutathione was facilely immobilized onto silica surface to obtain a novel hydrophilic interaction/cation-exchange mixed-mode chromatographic stationary phase (Click TE-GSH) via copper-free "thiol-ene" click chemistry. The resulting material was characterized by solid state (13)C/CP MAS NMR and elemental analysis. The measurement of ζ-potential indicated the cation-exchange characteristics and adjustable surface charge density of Click TE-GSH material. The influence of acetonitrile content and pH value on the retention of ionic compounds was investigated for understanding the chromatographic behaviors. The results demonstrated that Click TE-GSH column could provide both hydrophilic and cation-exchange interaction. Taking advantage of the good hydrophilicity and inherent cation-exchange characteristics of Click TE-GSH material, the resolution of neutral fructosan with high degree of polymerization (DP), basic chitooligosaccharides and strongly acidic carrageenan oligosaccharides was successfully realized in hydrophilic interaction chromatography (HILIC), hydrophilic interaction/cation-exchange mixed-mode chromatography (HILIC/CEX), cation-exchange chromatography (CEX) and electrostatic repulsion/hydrophilic interaction chromatography (ERLIC). On the other hand, the separation of standard peptides varying in hydrophobicity/hydrophilicity and charge was achieved in both CEX and HILIC/CEX mode with high efficiency and distinct selectivity. To further demonstrate the versatility and applicability of Click TE-GSH stationary phase, the separation of a human serum albumin (HSA) tryptic digest was performed in HILIC/CEX mode. Peptides were adequately resolved and up to 86 HSA peptides were identified with sequence coverage of 85%. The results indicated the good potential of Click TE-GSH material in glycomics and proteomics.

A method based on a Click XIon zwitterionic stationary phase was developed to separate isomeric saponins efficiently in hydrophilic interaction chromatography (HILIC) mode using methanol as a weak eluent. The retention times of a set of isomeric saponins, ginsenoside Rc (S1), ginsenoside Rb2 (S2), and ginsenoside Rb3 (S3) on five kinds of HILIC columns were compared using acetonitrile/water or methanol/water as mobile phase. All results indicated that the Click XIon column showed the highest retention times of all these isomeric saponins. Then the retention behaviors of these isomeric saponins on the Click XIon column were investigated. The results demonstrated that the retention behaviors of saponins on Click XIon in methanol/water mobile phase were different from that in acetonitrile/water. Methanol/water binary eluent offered enhanced selectivity to these isomeric saponins (αS2/S3(MeOH)=1.16>αS3/S2(ACN)=1.04), and the elution order of ginsenoside Rb2 (S2) and ginsenoside Rb3 (S3) was reversed compared with acetonitrile/water. Moreover, methanol/water solvent has better solubility for saponins than acetonitrile/water, resulting in improved preparative peak shape, which is greatly beneficial for saponin purification. Application to the preparative separation of saponins from leaves of P. notoginseng was also investigated, and eleven saponins, including three sets of isomeric saponins with one new saponin were isolated and identified. All these results indicated that this method was efficient for analytical and preparative separation of saponins, especially for isomeric saponins, containing xylopyranosyl, arabinofuranosyl or arabinopyranosyl units.

Glycoproteins are involved in various cellular activities, including inter- and extracellular signaling. However, glycopeptide signals are significantly suppressed by coeluting non-glycosylated peptides in mass spectrometry-based analysis. For detailed elucidation of the biological functions of glycoproteins, selective enrichment of glycopeptides from non-glycosylated peptides is crucial. In the present study, a SAX material, XCharge SAX, was used in a column in the ERLIC mode with the aim of specifically enriching glycopeptides. Enrichment conditions were initially optimized, and selectivity, glycosylation heterogeneity coverage and detection sensitivity of XCharge SAX were subsequently assessed. In the selectivity assessment, glycopeptides were effectively isolated from a peptide mixture (human serum immunoglobulin G (IgG) and human serum albumin digests) and a tryptic digest of human serum using XCharge SAX. In the evaluation of glycosylation heterogeneity coverage, five glycosites and eleven glycopeptides from horseradish peroxidase were identified after enrichment with XCharge SAX. In detection sensitivity assessment, glycopeptides within four orders of magnitude were identified after enrichment with XCharge SAX. In addition, volatile solvents were used in the loading and eluting buffers so that desalting was not necessary for ERLIC fractions. Our results collectively support the utility of XCharge SAX as a suitable chromatographic material for global glycosylation site analysis.

Purification of high-purity compounds from traditional Chinese medicines (TCMs) plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of TCMs in chemical composition. In this work, a two-dimensional preparation method was successfully developed for the preparation of high-purity compounds from the stem of Lonicera japonica Thunb, based on two novel polar copolymerized RP stationary phases, XAqua C3 and XAqua C18. An XAqua C3 prep column was used to separate the sample in the first-dimensional preparation, and 14 g of sample was fractionated into eight fractions with a recovery of 82%. An XAqua C18 prep column was selected to prepare high-purity compounds in the second-dimensional preparation for its good orthogonality with the XAqua C3 stationary phase. As a result, major compounds in the sample were isolated with more than 99% purity. This method is a potent method to realize the efficient purification of compounds with high purity from the stem of L. japonica Thunb and it shows great potential in the separation of high-purity compounds from complex samples.

Chiral separation of benzoxazine enantiomers was studied on click chemistry based beta-cyclodextrin stationary phases under reversed-phase high performance liquid chromatographic mode. The effects of the type and percentage of the organic modifier, the concentration of triethylammonium acetate buffer (TEAA), and pH on enantioselective separation were examined and studied. It was demonstrated that acetonitrile was better for the chiral separation of benzoxazine enantiomers than methanol. It was observed that the retention time and the resolution of benzoxazine enantiomers decreased with the increase of the volume ratio of TEAA from 0.1% to 1.0%. The separation of benzoxazine enantiomers was of the maximum resolution at pH 4.1. With the optimized mobile phase of the mixture of acetonitrile and 0. 1% TEAA (pH 4.1), all the enantiomers were separated at the baseline. The chiral recognition mechanism is also discussed. The separation was probably based on the inclusion complex interaction and the hydrogen bonding between enantiomers and chiral stationary phases. This work provided the experience for the intensive study of click beta-cyclodextrin bonded stationary phases, and it also illustrated the potential of click chemistry in the preparation of beta-cyclodextrin based chiral stationary phase.

Peptide components of scorpion venom have been employed as useful pharmacological tools in the study of ion channel function. The isolation of individual components is necessary for determination of their biological significance. Here, we have described a novel reversed phase (RP)/ion exchange stationary phase, Click oligo ethylene glycol (Click OEG), and the chromatographic efficiency of its mixed-mode sorbent in peptide separation experiments. The Click OEG presents a mixed-mode RP/weak anion-exchange type stationary phase at pH 3.5 and mixed-mode RP/weak cation-exchange type stationary phase at pH 6.0, and it was suitable for separation of long-chain peptides in scorpion venom. Subsequently, a two dimensional mixed-mode RP-RP system based Click OEG and C18 with different pH values in two dimensions was developed for orthogonal separation of scorpion venom. Furthermore, two fractions were analyzed in depth, and 11 long-chain peptides were purified and sequences were identified by using tandem mass spectrometry incorporating the tryptic approach. Among these, we isolated six novel peptides including one peptide with a new sequence and five transcript-level peptides, and speculated on their possible bioactivities.

Desulfation of a fucoidan from Saccharina japonica by treatment with DMSO-MeOH resulted in partial degradation of polymeric molecules by methanolysis giving rise to a mixture of neutral, monosulfated, and disulfated fucooligosaccharides in the form of methyl glycosides. These oligomeric fragments were characterized by ESI-MS and ESI-CID-MS/MS. It was found that oligosaccharide structures coincided with the polysaccharide backbone built up mainly of (1 -> 3)-linked fucose residues sulfated at positions 4 and 2. (C) 2012 Elsevier Ltd. All rights reserved.

While tailing and overloading of basic compounds remain problematic on most RP columns, a new kind of positively charged RP column named XCharge C18 was found to be superior good for the separation of alkaloids in our practical use. In this work, the surface charge property of the XCharge C18 column was evaluated by the retention of NO3- under different pH values and buffer concentrations. A considerable and pH-dependent positive charge was confirmed on the column. Then overloading behaviors of bases were systematically studied using amitriptyline as a basic probe. Good peak shapes (Tf < 1.5) and extra high loadability with a C-0.5 of about 30,000 mg/L were observed on the column, with commonly used 0.1% formic acid as mobile phase additive. However, increasing the ionic strength of buffer with phosphates led to tailing peaks at high sample amount and sharp decline in loadability (C-0.5 of 2000-3000 mg/L), although it brought higher column efficiency at low sample amount. Higher pH also induced worse performance and lower loadability. The overall results demonstrated the importance of an appropriate level of ionic repulsion for the XCharge C18 column to achieve the good performance for bases, which could be explained by the multiple-site adsorption theory as ionic repulsion would shield the solute from occupying high-energy sites deeper in C18 layer. (c) 2013 Elsevier B.V. All rights reserved.

Two-dimensional reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography (2D-RPLC/HILIC) system was successfully applied for comprehensive characterization of steviol glycosides from Stevia rebaudiana. The experiments were performed in offline mode using an XCharge C18 column in first dimension and an XAmide column in second dimension. In first dimension, preliminary separation of Stevia aqueous extract was accomplished and 30 fractions were collected. Then fractions 1-20 were selected for further purification and 13 compounds with high purity were obtained in second dimension. Comprehensive characterization of these compounds was completed by determination of their retention time, accurate molecular weight, diagnostic fragmentation ions, and nuclear magnetic resonance spectroscopy. As a result, all nine known steviol glycosides, as well as other four steviol glycosides were fully purified. The result demonstrated that this procedure is an effective approach for the preparative separation and comprehensive characterization of steviol glycosides in Stevia. This 2D-RPLC/HILIC method will be a promising tool for the purification of low-abundance compounds from natural products.

A simple and efficient method based on a novel solid phase extraction (SPE) cartridge and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) was developed for the determination of clenbuterol residue in pork. The minced pork sample was ultrasonically extracted by 5% (v/v) perchloric acid and centrifuged at 10 000 r/min for 15 min, then the supernatant was purified by an SMCX cartridge, which is a novel SPE column based on homemade silica matrix with two mixed modes of reversed-phase and strong cation exchange, for the selective enrichment and purification of the analyte. The linear range of the method was 0.25 - 50 microg/kg with a correlation coefficient of 0.998 2. The average recoveries ranged from 62.2% to 72.0% at the three spiked levels of 1.25, 12.5 and 50 microg/kg with the relative standard deviations (RSDs) between 4.2% and 6.1%. The limit of detection (S/N = 3) was 0.05 microg/kg. The method is simple, fast and can be extended to enrich and determine beta-agonist drugs.

Scorpion venom contains a considerable variety of neurotoxic peptides that can act on ionic channels. Here, we describe an orthogonal 2D-reversed phase/hydrophilic interaction chromatography system (RPLC/HILIC) and use it to separate short-chain peptides from Asian scorpion Buthus martensi Karsch (BmK) venom in a high throughput format. Due to its high orthogonality and efficiency, 18 homogenous peptides were purified and sequence identified by MS/MS with collision-induced dissociation. Among them, four peptides were discovered, which only have evidence at transcript-level, were first purified from crude venom in this study. Two peptides named BmKK2-b and Martentoxin-b were found the new cleaved chains of known BmKK2 and Martentoxin. In addition, two novel peptides named BmKK12 and BmKK16 in this paper were sequenced by de novo MS/MS, which we predict, are members of potassium channel toxin α-KTx 17 subfamily by homology to other known peptides found in the Swiss-Prot protein database.

Strong cation exchange (SCX) mode, an alternative to reversed-phase (RP) mode, was described in this paper to obtain symmetrical peak shape and high sample loading for the separation of basic compounds. The retention mechanisms on SCX modified silica sorbents have been demonstrated including electrostatic and hydrophobic interactions. The effects of eluent ionic strength, apparent pH on retention were investigated and the cation-exchange capacity was also characterized. Good efficiency (>56,000 plate m(-1)) and excellent peak shape (Tf < 1.2) for various basic probes were obtained on the SCX column. Furthermore, good peak shapes and separation can be maintained under high concentration injections, indicating good potential in preparative use. These results were also compared with those obtained on two conventional RP stationary phases. The overall results demonstrated that SCX stationary phases can be used as alternatives to RP stationary phases in the separation of basic compounds, especially in the purification of basic compounds. (c) 2012 Elsevier B.V. All rights reserved.

A set of 14 model compounds were selected to evaluate the retention properties of two silica-based cyclodextrin (CD) bonded stationary phases with different spacers (Click Alkyl-CD and Click OEG-CD) under reversed-phase liquid chromatographic (RPLC) mode. The gradient calculation method and CSASS software developed in our group were applied to obtain the retention parameters of the 14 solutes on the two CD bonded stationary phases under 3 gradient conditions. The effect of acetonitrile content in the mobile phase on the retention of both CD stationary phases was studied. It was found that the retention of non-polar and medium polar compounds on the both stationary phases was based on RPLC mode, while the retention of some compounds (such as indazole) on Click OEG-CD was influenced by several kinds of interactions and was not based on RPLC mode. The hydrophobic property of the two CD stationary phases in RPLC mode was studied. High correlation value (R = 0.7) between the retention parameter of Click Alkyl-CD and n-octanol-water partition coefficients of the solutes indicated the high hydrophobicity of Click Alkyl-CD. Nevertheless, the low correlation value (R0.3) between the retention parameter of Click OEG-CD and n-octanol-water partition coefficients indicated the presence of influence of other interactions in addition to hydrophobic interaction on the retention in RPLC mode.

A simple, rapid method for the determination of melamine in milk and milk powder was developed using QuEChERS approach coupled with weak cation exchange chromatography (WCX). The samples were extracted and cleaned-up by medicinal alcohol and lipid adsorbent (LAS) simultaneously, then centrifuged and filtered for high performance liquid chromatographic (HPLC) analysis. The separation was performed on a WCX column (150 mm x 4.6 mm, 5 microm) with 2 mmol/L KH2 PO4 (pH 3.8) as mobile phase. The flow rate was 1.5 mL/min and the detection wavelength was 208 nm. The injection volume was 20 microL and the column temperature was 30 degrees C. Under the optimized conditions, good linearity was obtained in the range of 0.02-20 mg/L with a correlation coefficient (r2) of 0.999 9. For samples spiked with melamine standard in the range of 1-50 mg/kg, the average recoveries of standard in the milk and milk powder were 98.9%-105.2% and 86.4%-102.9%, respectively. The relative standard deviations were 0.9%-3.4% and 1.5%-6.7%, respectively. The limits of detection (LOD, S/Nor = 3) were 0.05 mg/kg (milk) and 0.1 mg/kg (milk powder). The present method is green due to not use of the toxic organic solvents.

Desalting peptides before mass spectrometry analysis is important because salts lead to adduct formation, increased chemical noise and ion suppression effect. A high concentration of salt can clog nanoelectrospray ionization (ESI) emitters. The reverse phase C18 material is commonly used to desalt peptides because of its high binding capacity. However, peptides with high hydrophilicity, such as glycopeptides, are not retained well on this material, resulting in the loss of peptide information. To improve the efficiency of glycopeptide desalting, we introduced a hydrophilic interaction chromatography (HILIC)-based material named click maltose. Four glycoproteins, horseradish peroxidase (HRP), human serum immunoglobulin G (IgG), bovine ribonuclease B (RNase B), and α-1 acid glycoprotein (AGP) were chosen as models and their glycopeptides were desalted with click maltose, AQ C18, Empore C18 and ZipTip C18. Click maltose as a HILIC material exhibited better performance than the other three C18 materials for both number of targeted glycopeptides and their corresponding intensities. In addition, accurate glycopeptide profiling was achieved with click maltose desalting regardless of peptide lengths and glycan types.

The interaction of two Chinese medicinal ingredients and human serum albumin (HSA) has been investigated by high performance affinity chromatography (HPAC). HSA bounded silica-based stationary phase was prepared based on the "click chemistry" strategy, and packed in a column (named as HSA column). The drug-HSA binding ratio was calculated from the difference of the drug's retention times on the HSA column and silica column (blank column). The warfarin-HSA binding ratio determined by this method was similar to the reference reported value by ultrafiltration method. The results indicated that the new HSA column and the HPAC method can be used for the detection of binding ratio of drug and HSA. The binding ratios of puerarin and goitrin determined by the HPAC method were 10.26% and 10.20%, respectively. And the binding ratio of puerarin determined by ultrafiltration was 14.25%. All these results showed that HPAC is a useful method to investigate the interaction between drugs and protein.

The herbalome is a project with the objective of globalizing Chinese herbal medicine (CHM) by clarification of its composition, structure, and function; by establishing a standard resource library; and by interpreting the synergistic and complementary mechanisms of multi-components on multi-targets. In phase I, it focuses on the development of systematic separation methodology for resolving and analyzing the complex components in CHM and establishment of a comprehensive resource library. This review summarizes recent advances in the herbalome project with regard to innovative separation techniques and demonstration of a resource library.

A cysteine-bonded zwitterionic hydrophilic interaction chromatography (hilic) stationary phase (click te-cys) was prepared based on the "thiol-ene" click chemistry. the click te-cys material was characterized by solid state c-13 cross polarization/magic-angle spinning (cp/mas) nmr and elemental analysis. the dynamic evaluation for cytosine, cytidine and orotic acid was performed using van deemter plots. the plate height values were no more than 24 mu m for the flow rate between 0.5 and 5.4 mm s(-1) (0.3-3.5 ml min(-1)), which proved the excellent separation efficiency of click te-cys stationary phase. the influences of the content of water, concentration of salt and ph of the buffer solution on the retention of model compounds were investigated. the results demonstrated that the separation of polar analytes was dominated by the partitioning mechanism, while the contribution of electrostatic interaction was minor. the thermodynamic characteristic of click te-cys stationary phase was also studied according to van't hoff plot. an exothermic process for transferring analytes from the mobile phase to the stationary phase was observed and a linear relationship for ink and 1/t was achieved, indicating no change of retention mechanism within the measured temperature range. besides, the zwitterionic stationary phase exhibited good stability. considering the high hydrophilicity of click te-cys stationary phase, the application in the separation of protein tryptic digests was carried out using hydrophilic interaction chromatography-electrospray ionization mass spectrometry (hilic-esi-ms). more peaks were adequately resolved on the click te-cys column comparing with that on the tsk amide-80 column. in addition, the orthogonality between hilic and rplc system was investigated utilizing geometric approach. the xterra ms c-18 and click te-cys column displayed great difference in separation selectivity, with the orthogonality reaching 88.0%. on the other hand, the orthogonality between click te-cys and tsk amide-80 system was 21.4%, i.e. the selectivity was similar but slightly different from each other. the successful separation of protein digests indicated the great potential of click te-cys stationary phase in the separation of complex samples and applicability in two-dimensional liquid chromatography (2d-lc). (c) 2011 elsevier b.v. all rights reserved.

Selection of a first-dimensional separation method is very important in proteomic studies performed with two-dimensional liquid chromatography (2D-LC), since reversed-phase chromatography (RPLC) is usually chosen as the second-dimensional separation method in most studies. In this paper, we assess the potential use of mixed-mode reversed-phase/weak cation-exchange (RP/WCX) chromatography in 2D-LC proteomic studies. First, a new RP/WCX mixed-mode stationary phase (named C18WCX) was synthesized based on the polar-copolymerized approach, and a C18WCX column with separation efficiency comparative to conventional C18 columns was developed. This new mixed-mode column primarily provides hydrophobic interactions under acidic condition, but can offer hydrophobic and cation-exchange interactions under neutral and weak basic conditions. An off-line 2D-RP/WCX–RPLC system was established using the C18WCX column with a mobile phase of pH 6.5 in the first dimension and a C18 column with a mobile phase of pH 3.0 in the second dimension. The orthogonality of this two-dimensional system, evaluated through the separation of 123 tryptic peptides, was shown to be higher than that of the conventional 2D-RP–RPLC approach. Applying this 2D-LC method to rat brain samples, we identified 1031 proteins and 4397 unique peptides. In addition, this new two-dimensional method improved the identification of basic peptides. Therefore, we propose that this novel 2D-RP/WCX–RPLC system can be used as an alternative approach for the two-dimensional separation of peptides.

The retention behavior of inorganic anions on a triazole-based stationary phase was first examined in ion chromatography. It was initially designed for hydrophilic interaction liquid chromatography and was simply prepared by introducing the triazole groups onto the surface of silica gel via click chemistry. Effective separation of common inorganic anions, including iodate, chloride, bromide, nitrate and iodide, was achieved with Na(2)SO(4) eluent. The logarithm of the retention factor of analytes was observed to be linear with the logarithm of the eluent concentration, and the slopes of the plots were almost the same as those of the ideal theoretical value. The eluent pH value in the range of 3.4-7.0 had little effect on the separation. The utility of the column was demonstrated for the determination of UV-absorbing anions in saliva and tap water.