Aptamer based hybrid monolithic pipette tips supported by melamine sponge for enrichment of proteins.

BACKGROUND: The convenient preparation and application of functionalized organic-inorganic hybrid monolithic materials have obtained substantial interest in the pretreatment of complex samples by solid-phase extraction (SPE). Compared to the in-tube solid-phase microextraction in fused-silica capillaries, micro SPE in plastic pipette tips have fascinating merits for the easily operated enrichment of trace target analytes from biological samples. However, the poor compatibility of organic-inorganic hybrid monoliths with plastics leads to the rare appearance of commercial hybrid monolithic pipette tips (HMPTs). Therefore, how to synthesize the organic-inorganic hybrid monolithic materials with better extraction performance in plastic pipette tips becomes a challenge. RESULTS: We develop a facile and cheap strategy to immobilize organic-inorganic hybrid monoliths in pipette tips. Melamine sponge was employed as the supporting skeleton to in situ assemble amine- and thiol-bifunctionalized hybrid monolithic material via "one pot" in a pipette tip, and gold nanoparticles (GNPs) and thiol-modified aptamer against human α-thrombin were sequentially attached to the hybrid monolith within the HMPTs. The average coverage density of the aptamer with GNPs as an intermediary reached as high as 818.5 pmol µL-1. The enriched thrombin concentration was determined by a sensitive enzymatic chromogenic assay with the limit of detection of 2 nM. The extraction recovery of thrombin at 10 nM in human serum was 86.1 % with a relative standard deviation of 6.1 %. This proposed protocol has been applied to the enrichment and determination of thrombin in real serum sample with strong anti-interference ability, low limit of detection and high recovery. SIGNIFICANCE: The amine- and thiol-bifunctionalized HMPTs prepared with sponge as the skeleton frame provided a novel substrate material to decorate aptamers for efficient enrichment of proteins. This enlightens us that we can take advantage of the tunability of sponge assisted HMPTs to produce and tailor a variety of micro SPE pipette tips for broader applications on the analysis of trace targets in complex biological, clinic and environmental samples.

A novel zwitterionic magnetic nanocomposite developed for non-invasive speciation analysis of inorganic chromium.

3-(2-Aminoethylamino)propyltriethoxysilane and carboxyethylsilanetriol sodium salt were grafted on silica-coated Fe3O4 nanoparticles via sol-gel process to prepare novel amine- and carboxyl-bifunctionalized magnetic nanocomposites (SMNPs-(NH2 + COOH)). After well characterized, this doubly functionalized material was used as magnetic solid-phase extraction (MSPE) adsorbent to separate and enrich inorganic chromium species followed by inductively coupled plasma-mass spectrometry detection. The optimization of MSPE operation parameters including pH was conducted. It is reasonably elucidated that the adsorption mechanisms of zwitterionic SMNPs-(NH2 + COOH) towards chromium species are electrostatic and/or coordination interactions. Cr(VI) and Cr(III) can be adsorbed around pH 3.0 and around 10.0 respectively with strong anti-interference ability not only from other co-existing ions but also from the two labile species each other, and eluted by dilute nitric acid solution. With a 15-fold enrichment factor, the limits of detection of Cr(VI) and Cr(III) were 0.008 and 0.009 µg L-1, respectively, profiting from the maximum adsorption capacities of 7.52 and 6.11 mg g-1. The just one magnetic extraction matrix based speciation scheme possesses excellent convenience and friendliness to Cr(VI) and Cr(III) without any oxidation or reduction prior to capture of these two species. This protocol has been successfully applied to the speciation analysis of inorganic chromium in real-world environmental water samples.

Effect of ionic liquids as mobile phase additives on retention behaviors of G-quadruplexes in reversed-phase high performance liquid chromatography.

G-quadruplexes (G4s) play an important role in a variety of biological processes and have extensive application prospects. Due to the significance of G4s in physiology and biosensing, studies on G4s have attracted much attention, stimulating the development or improvement of methods for G4 structures and polymorphism analysis. In this work, ionic liquids (ILs) were involved as mobile phase additives in reversed-phase high performance liquid chromatography (RP-HPLC) to analyse G4s with various conformations for the first time. How ILs affected the retention behaviors of G4s was investigated comprehensively. It was found that the addition of ILs markedly enhanced G4 retention, along with obvious amelioration on chromatographic peak shapes and separation. The influence of pH of mobile phase and types of ILs were also included in order to acquire an in-depth understanding. It appeared that the effect of ILs on G4 retention behaviors was the result of a combination of various interactions between G4s with the hydrophobic stationary phase and with the IL-containing mobile phase, where ion pair mechanism and enhanced hydrophobic interaction dominated. The findings of this work revealed that ILs could effectively improve the separation of G4s in RP-HPLC, which was conducive to G4 structural analysis, especially for G4s polymorphism elucidation.

Prediction of the n-Octanol/Water Partition Coefficients of Basic Compounds Using Multi-Parameter QSRR Models Based on IS-RPLC Retention Behavior in a Wide pH Range.

The n-octanol-water partition coefficient (logP) is an important physicochemical parameter which describes the behavior of organic compounds. In this work, the apparent n-octanol/water partition coefficients (logD) of basic compounds were determined using ion-suppression reversed-phase liquid chromatography (IS-RPLC) on a silica-based C18 column. The quantitative structure-retention relationship (QSRR) models between logD and logkw (logarithm of retention factor corresponding to 100% aqueous fraction of mobile phase) were established at pH 7.0-10.0. It was found that logD had a poor linear correlation with logkw at pH 7.0 and pH 8.0 when strongly ionized compounds were included in the model compounds. However, the linearity of the QSRR model was significantly improved, especially at pH 7.0, when molecular structure parameters such as electrostatic charge ne and hydrogen bonding parameters A and B were introduced. External validation experiments further confirmed that the multi-parameter models could accurately predict the logD value of basic compounds not only under strong alkaline conditions, but also under weak alkaline and even neutral conditions. The logD values of basic sample compounds were predicted based on the multi-parameter QSRR models. Compared with previous work, the findings of this study extended the pH range for the determination of the logD values of basic compounds, providing an optional mild pH for IS-RPLC experiments.

Magnetic Ti3C2 MXene Nanosheets Prepared for Enrichment of Phosphopeptides.

MXenes have received lots of attention since discovered and have been applied in various fields. In this work, Ti3C2-Fe3O4 composites with exposed non-modified Ti3C2 MXene nanosheets were designed and prepared by an in situ growth strategy and then applied in the enrichment of phosphopeptides. The two-dimensional composites could interact with the phosphopeptides through a metal oxide affinity chromatography mechanism provided by Ti-O and Fe-O bonds and a hydrophilic interaction chromatography mechanism by surface hydroxyl groups. This magnetic nanomaterial with a specific surface area of 66.1 m2·g-1 had high sensitivity to phosphopeptides (0.5 nmol·L-1) and high selectivity (1:1000 of the molar ratio of ß-casein to bovine serum albumin). Non-fat milk was adopted as a real sample to preliminarily examine the applicability of the Ti3C2-Fe3O4-based protocol. Subsequently, Qingkailing injection, a kind of traditional Chinese medicine injection, was introduced to further explore the suitability of the nanocomposites for phosphopeptide enrichment from more complex matrices and satisfactory results were obtained.

Residue screening and analysis of enrofloxacin and its metabolites in real aquatic products based on ultrahigh-performance liquid chromatography coupled with high resolution mass spectrometry.

The abuse of enrofloxacin (ENR) in aquaculture and the lack of monitoring of other metabolites except ciprofloxacin (CIP) may lead to unknown harmful effects on human health. In this study, ENR metabolites were screened in real fish samples based on ultrahigh-performance liquid chromatography coupled with Q-Orbitrap mass spectrometry combined with Compound Discoverer software, and another metabolite deethylene-ENR besides CIP was detected and identified for the first time. Correspondingly, a method for the determination of ENR and CIP and the semi-quantitative analysis of deethylene-ENR in aquatic products was established. Method validation illustrated that excellent linearity and satisfactory recoveries of analytes were obtained. Limits of detection of ENR and CIP were both 0.1 µg kg-1, and their limits of quantification both 1 µg kg-1. CIP and deethylene-ENR were detected in 12 of 14 ENR-positive fish samples, so deethylene-ENR should be of concern as a possible risk candidate in aquatic products.

Determination of chlorpromazine and its metabolites in animal-derived foods using QuEChERS-based extraction, EMR-Lipid cleanup, and UHPLC-Q-Orbitrap MS analysis.

Chlorpromazine (CPZ) is abused in animal husbandry and can be extensively metabolized in humans and animals. However, the actual monitoring mainly focuses on the parent compound but lacks attention to its metabolites. A method was developed and validated firstly for identification and determination of CPZ and its four major metabolites in animal-derived foods using ultrahigh-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry in combination with QuEChERS preparation method. Satisfactory recoveries of analytes spiked in fish and pork samples ranged from 72 to 117 %, and limits of quantification were 2.0 and 1.0 µg kg-1 for fish and pork samples respectively. Moreover, through the hydrolysis experiments of CPZ, its hydrolysates, such as CPZ-sulfoxide, CPZ-N-oxide and CPZ-sulfoxide-N-oxide, were identified as potential risk compounds. The developed method has been successfully applied to the determination of CPZ and its metabolites in actual commercial samples, as well as to the screening of other CPZ-related risk compounds.

A combination approach using two functionalized magnetic nanoparticles for speciation analysis of inorganic arsenic.

Mercapto- and amino-functionalized magnetic nanoparticles, Fe3O4@SiO2@MPTMS (SMNPs-MPTMS) and Fe3O4@SiO2@APTES (SMNPs-APTES), have been applied as magnetic solid-phase extraction (MSPE) sorbents to directly extract arsenite (As(III)) and arsenate (As(V)) respectively, followed by inductively coupled plasma-mass spectrometry (ICP-MS) detection. Various MSPE parameters were optimized including dose of magnetic adsorbent, pH of sample solution, loading and elution conditions of analytes, adsorption capacity and reusability of SMNPs-MPTMS and SMNPs-APTES for As(III) and As(V) respectively. Under the optimized MSPE conditions, this combined scheme possesses excellent selectivity and strong anti-interference ability without any oxidation or reduction prior to capture of these two species. It is found that with a 25-fold enrichment factor, the limits of detection of As(III) and As(V) were 23.5 and 10.5 ng L-1, respectively. To verify the reliability of the proposed protocol, a certified reference material of environmental water was analyzed, and the results for inorganic arsenic species were in close agreement with the certified values. The applicability of the combination strategy for speciation analysis of inorganic arsenic was evaluated in spiked tap, river, lake and rain water samples. Good recoveries of 89%-96% and 90%-102% were achieved for As(III) and As(V), respectively, with the relative standard deviation ranges of 3.2%-8.0% and 2.5%-7.6%. Through the characterization of functionalized magnetic nanoparticles and the optimization of MSPE experiment, it is confirmed that the existence of mercapto and amino groups on SMNPs-MPTMS and SMNPs-APTES sorbents are responsible for the extraction of As(III) and As(V), respectively, via coordination and electrostatic interactions.

Insight into the hydrophilic interaction liquid chromatographic retention behaviors of hydrophilic compounds on different stationary phases.

In this work, the correlations between retention behavior and lipophilicity of a large set of hydrophilic neutral and ionic analytes were studied based on three hydrophilic interaction liquid chromatography (HILIC) stationary phases, including zwitterionic, crosslinked diol and triazole stationary phases. It was found that HILIC, due to the diversity of retention mechanism, is a more complex chromatography separation mode than reversed-phase liquid chromatography (RPLC) which has been widely accepted for lipophilicity assessment. Because electrostatic interactions contributed to the overall retention of the charged solutes on all three stationary phases, ion-strength of the mobile phase kept the same during the whole experiment. After the correlations between retention factor log k and water volume fraction Φ were investigated, the mixed retention model was revealed to be more suitable for HILIC retention behavior than other single models including partitioning and adsorption model. Moreover, in order to bridge the relationship between HILIC log k and lipophilicity parameter log D, net charge ne and Abraham solvation parameter were introduced in the quantitative structure-retention relationship (QSRR) model. Although the correlation coefficients between log D and log k were still moderate, the significant improvement in correlation has made HILIC a potential choice as the complement of RPLC for log D measurement.

Monolithic alkylsilane column: A promising separation medium for oligonucleotides by ion-pair reversed-phase liquid chromatography.

In this paper, a monolithic octadecylsilane column and particle-packed octadecylsilane columns were used to investigate the retention behaviors of oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC). Results showed that, with same base composition, hairpin oligonucleotides always had weaker retention than corresponding random coil oligonucleotides on the monolithic column, but not on the particle-packed columns. In addition, the linear correlation between the retention factor k of oligonucleotides and the reciprocal of temperature (1/T), especially for hairpins, was relatively weaker on the particle-packed columns, as compared to the correlation on the monolithic column. The correlation between k and 1/T became weaker with decreasing particle size of the particle-packed columns. Moreover, results revealed that the overall retention order on the particle-packed column with small particles (3 µm) differed greatly from that on the monolithic column. In contrast, the retention order on the 10 µm particle-packed column was very close to that on the monolithic column. From the above, we inferred that oligonucleotides could keep their primary conformations unchanged when passing through the monolithic column, attributed to the special pore structures of the monolith. However, the conformations of oligonucleotides were suppressed or even destroyed when oligonucleotides passed through the particle-packed columns. This because the narrow and tortuous channels created by the stacked stationary phase particles could lead to more complex and unequable retention behaviors. Therefore, the monolithic column exhibited better retention regularity for oligonucleotides of secondary structure especially for hairpins than the particle-packed columns. It is noteworthy that the monolith-based IP-RPLC opens an intriguing prospect in accurately elucidating the retention behaviors of oligonucleotides.

A novel strategy for retention prediction of nucleic acids with their sequence information in ion-pair reversed phase liquid chromatography.

In this work, retention behaviors of oligonucleotides and double-stranded deoxyribonucleic acids (dsDNAs) have been investigated in ion-pair reversed-phase liquid chromatography (IP-RPLC). We demonstrated that classic linear solvent strength (LSS) model is applicable for describing isocratic retention of oligonucleotides and dsDNAs, which indicated that nucleic acids share the similar retention mechanism as other common small molecules in IP-RPLC. The separation of nucleic acids in IP-RPLC is driven by both hydrophobic and electrostatic interactions. We defined the parameter lnkw/S obtained from LSS model in IP-RPLC as chromatographic hydrophobic and electrostatic interaction index (CHEI). CHEI of a nucleic acid has been revealed to correlate well with its gradient retention time. Notably, we proposed a strategy for retention prediction based on CHEI and base sequence information of nucleic acids. Corresponding to base locus, each base sequence is converted to a featured locus vector consisting of zeros and ones. CHEI prediction models were established by support vector regression (SVR) algorithm with locus vectors. Predicted CHEI values have been applied to predict retention times under desired gradient elution runs. This protocol is easy to grasp and worth pursuing further development for more precise retention prediction performance of nucleic acids in IP-RPLC.

Study on the polymorphism of G-quadruplexes by reversed-phase HPLC and LC-MS.

Polymorphism is inherent for G-quadruplexes (G4s), and the different structural forms are important for the participation in different biological functions of telomeres. A lot of progress has been made in the exploration of G4 polymorphism. However, quick separation and reliable assessment methods for different conformations of G4 are still very few. In this work, the polymorphism of three sequences d[(G3T)3G3], d[(G3C)3G3] and d[(G3T)4] annealed in six different solutions were investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC), liquid chromatography-mass spectrometry (LC-MS), fluorescence spectroscopy, circular dichroism spectroscopy, together with native-polyacrylamide gel electrophoresis. Different G4 conformations of these three sequences can be separated clearly by RP-HPLC, and further characterized by on-line LC-MS analysis. It is revealed that high-order structures other than intramolecular quadruplexes were favored for d[(G3T)3G3] and d[(G3C)3G3] under the annealing conditions. However, flanking loop impeded d[(G3T)4] to form higher-order structures than dimer. In addition, the nature and concentration of cation, as well as the annealing solution component, all have decent influence on the stability and relative ratios of various G4 building blocks. Based on the above findings, RP-HPLC and LC-MS combined with spectroscopic techniques can be used as a facile and powerful tool for quick separation and identification of G4s in solutions, and for effective assessment of DNA sequences and annealing environments on G4 polymorphism. The established protocol provides a novel strategy for evaluating G4 polymorphism, which will facilitate studies on quadruplex structures and their biophysical properties.

Determination of reversed-phase high performance liquid chromatography based octanol-water partition coefficients for neutral and ionizable compounds: Methodology evaluation.

Reversed-phase liquid chromatography (RPLC) based octanol-water partition coefficient (logP) or distribution coefficient (logD) determination methods were revisited and assessed comprehensively. Classic isocratic and some gradient RPLC methods were conducted and evaluated for neutral, weak acid and basic compounds. Different lipophilicity indexes in logP or logD determination were discussed in detail, including the retention factor logkw corresponding to neat water as mobile phase extrapolated via linear solvent strength (LSS) model from isocratic runs and calculated with software from gradient runs, the chromatographic hydrophobicity index (CHI), apparent gradient capacity factor (kg') and gradient retention time (tg). Among the lipophilicity indexes discussed, logkw from whether isocratic or gradient elution methods best correlated with logP or logD. Therefore logkw is recommended as the preferred lipophilicity index for logP or logD determination. logkw easily calculated from methanol gradient runs might be the main candidate to replace logkw calculated from classic isocratic run as the ideal lipophilicity index. These revisited RPLC methods were not applicable for strongly ionized compounds that are hardly ion-suppressed. A previously reported imperfect ion-pair RPLC method was attempted and further explored for studying distribution coefficients (logD) of sulfonic acids that totally ionized in the mobile phase. Notably, experimental logD values of sulfonic acids were given for the first time. The IP-RPLC method provided a distinct way to explore logD values of ionized compounds.

Retention of nucleic acids in ion-pair reversed-phase high-performance liquid chromatography depends not only on base composition but also on base sequence.

The study on nucleic acid retention in ion-pair reversed-phase high-performance liquid chromatography mainly focuses on size-dependence, however, other factors influencing retention behaviors have not been comprehensively clarified up to date. In this present work, the retention behaviors of oligonucleotides and double-stranded DNAs were investigated on silica-based C18 stationary phase by ion-pair reversed-phase high-performance liquid chromatography. It is found that the retention of oligonucleotides was influenced by base composition and base sequence as well as size, and oligonucleotides prone to self-dimerization have weaker retention than those not prone to self-dimerization but with the same base composition. However, homo-oligonucleotides are suitable for the size-dependent separation as a special case of oligonucleotides. For double-stranded DNAs, the retention is also influenced by base composition and base sequence, as well as size. This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase. In addition, no specific influence of guanine and cytosine content was confirmed on retention of double-stranded DNAs. Notably, the space effect resulted from the stereostructure of nucleic acids also influences the retention behavior in ion-pair reversed-phase high-performance liquid chromatography.

Magnetic solid-phase extraction based on a polydopamine-coated Fe3 O4 nanoparticles absorbent for the determination of bisphenol A, tetrabromobisphenol A, 2,4,6-tribromophenol, and (S)-1,1'-bi-2-naphthol in environmental waters by HPLC.

Polydopamine-coated Fe3 O4 magnetic nanoparticles synthesized through a facile solvothermal reaction and the self-polymerization of dopamine have been employed as a magnetic solid-phase extraction sorbent to enrich four phenolic compounds, bisphenol A, tetrabromobisphenol A, (S)-1,1'-bi-2-naphthol and 2,4,6-tribromophenol, from environmental waters followed by high-performance liquid chromatographic detection. Various parameters of the extraction were optimized, including the pH of the sample matrix, the amount of polydopamine-coated Fe3 O4 sorbent, the adsorption time, the enrichment factor of analytes, the elution solvent, and the reusability of the nanoparticles sorbent. The recoveries of these phenols in spiked water samples were 62.0-112.0% with relative standard deviations of 0.8-7.7%, indicating the good reliability of the magnetic solid-phase extraction with high-performance liquid chromatography method. In addition, the extraction characteristics of the magnetic polydopamine-coated Fe3 O4 nanoparticles were elucidated comprehensively. It is found that there are hydrophobic, π-π stacking and hydrogen bonding interactions between phenols and more dispersible polydopamine-coated Fe3 O4 in water, among which hydrophobic interaction dominates the magnetic solid-phase extraction performance.

Magnetic solid-phase extraction of brominated flame retardants from environmental waters with graphene-doped Fe3O4 nanocomposites.

Graphene-doped Fe3O4 nanocomposites were prepared by a solvothermal reaction of an iron source with graphene. The nanocomposites were characterized by transmission electron microscopy, atomic force microscopy, X-ray diffraction, superconducting quantum interference, Raman spectroscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. This nanomaterial has been used as a magnetic solid-phase extraction sorbent to extract trace brominated flame retardants from environmental waters. Various extraction parameters were optimized including dosage and reusability of the nanocomposites, and pH of sample matrix. The reliability of the magnetic solid-phase extraction protocol based on graphene-doped Fe3O4 nanocomposites was evaluated by investigating the recoveries of 2,4,6-tribromophenol, tetrabromobisphenol A, 4-bromodiphenyl ether, and 4,4'-dibromodiphenyl ether in water samples. Good recoveries (85.0-105.0%) were achieved with the relative standard deviation ranging from 1.1-7.1%. Moreover, it is speculated from characterization and magnetic solid-phase extraction experiment that there is not only π-π stacking but also possible hydrophobic interaction between the graphene-doped Fe3O4 nanocomposites and analytes.

Determination of the n-octanol/water partition coefficients of weakly ionizable basic compounds by reversed-phase high-performance liquid chromatography with neutral model compounds.

A strategy to utilize neutral model compounds for lipophilicity measurement of ionizable basic compounds by reversed-phase high-performance liquid chromatography is proposed in this paper. The applicability of the novel protocol was justified by theoretical derivation. Meanwhile, the linear relationships between logarithm of apparent n-octanol/water partition coefficients (logKow '') and logarithm of retention factors corresponding to the 100% aqueous fraction of mobile phase (logkw ) were established for a basic training set, a neutral training set and a mixed training set of these two. As proved in theory, the good linearity and external validation results indicated that the logKow ''-logkw relationships obtained from a neutral model training set were always reliable regardless of mobile phase pH. Afterwards, the above relationships were adopted to determine the logKow of harmaline, a weakly dissociable alkaloid. As far as we know, this is the first report on experimental logKow data for harmaline (logKow = 2.28 ± 0.08). Introducing neutral compounds into a basic model training set or using neutral model compounds alone is recommended to measure the lipophilicity of weakly ionizable basic compounds especially those with high hydrophobicity for the advantages of more suitable model compound choices and convenient mobile phase pH control.

Solid phase extraction of magnetic carbon doped Fe3O4 nanoparticles.

Carbon decorated Fe3O4 nanoparticles (Fe3O4/C) are promising magnetic solid-phase extraction (MSPE) sorbents in environmental and biological analysis. Fe3O4/C based MSPE method shows advantages of easy operation, rapidness, high sensitivity, and environmental friendliness. In this paper, the MSPE mechanism of Fe3O4/C nanoparticles has been comprehensively investigated, for the first time, through the following three efforts: (1) the comparison of extraction efficiency for polycyclic aromatic hydrocarbons (PAHs) between the Fe3O4/C sorbents and activated carbon; (2) the chromatographic retention behaviors of hydrophobic and hydrophilic compounds on Fe3O4/C nanoparticles as stationary phase; (3) related MSPE experiments for several typical compounds such as pyrene, naphthalene, benzene, phenol, resorcinol, anisole and thioanisole. It can be concluded that there are hybrid hydrophobic interaction and hydrogen bonding interaction or dipole-dipole attraction between Fe3O4/C sorbents and analytes. It is the existence of carbon and oxygen-containing functional groups coated on the surface of Fe3O4/C nanoparticles that is responsible for the effective extraction process.

Preparation of poly(trimethyl-2-methacroyloxyethylammonium chloride-co-ethylene glycol dimethacrylate) monolith and its application in solid phase microextraction of brominated flame retardants.

A capillary poly(trimethyl-2-methacroyloxyethylammonium chloride-co-ethylene glycol dimethacrylate) monolith was in situ synthesized by thermally initiated free radical co-polymerization using trimethyl-2-methacroyloxyethylammonium chloride (MATE) and ethylene glycol dimethacrylate (EGDMA) as functional monomer and cross-linker, respectively. N,N-dimethylformamide and polyethylene glycol 6000 were used as solvent and porogen, respectively. The morphology and porous structure of the resulting monoliths were assessed by scanning electron microscope. In order to prepare practically useful poly(MATE-co-EGDMA) monoliths with low flow resistance and good mechanical strength, some parameters such as PEG-6000 to DMF ratio, total monomer to porogen ratio, and crosslinker to monomer ratio were optimized systematically. Moreover, the extraction mechanism was evaluated using two series of compounds, alkylbenzenes and weak acids, as model compounds on poly(MATE-co-EGDMA) monoliths as liquid chromatographic stationary phase. Finally, the monoliths were applied as the solid phase microextraction medium, and a simple off-line method for simultaneous determination of three brominated flame retardants, 2,4,6-tribromophenol (TBP), tetrabromobisphenol A (TBBPA) and 4,4'-dibrominated diphenyl ether (DBDPE), in environmental waters was developed by coupling the polymer monolith microextraction to HPLC with UV detection. The regression equations for these three brominated flame retardants showed good linearity from their limit of quantification to 5000ng/mL. The limits of detection were 0.20, 0.15 and 0.10ng/mL for TBP, TBBPA and DBDPE, respectively. The recovery of the proposed method was 78.7-106.1% with intra-day relative standard deviation of 1.3-4.4%.

Influence of variation in mobile phase pH and solute pK(a) with the change of organic modifier fraction on QSRRs of hydrophobicity and RP-HPLC retention of weakly acidic compounds.

The variation in mobile phase pH and ionizable solute dissociation constant (pK(a)) with the change of organic modifier fraction in hydroorganic mobile phase has seemingly been a troublesome problem in studies and applications of reversed phase high performance liquid chromatography (RP-HPLC). Most of the early studies regarding the RP-HPLC of acid-base compounds have to measure the actual pH of the mixed mobile phase rigorously, sometimes bringing difficulties in the practices of liquid chromatographic separation. In this paper, the effect of this variation on the apparent n-octanol/water partition coefficient (K(ow)″) and the related quantitative structure-retention relationship (QSRR) of logK(ow)″ vs. logk(w), the logarithm of retention factor of analytes in neat aqueous mobile phases, was investigated for weakly acidic compounds. This QSRR is commonly used as a classical method for K(ow) measurement by RP-HPLC. The theoretical and experimental derivation revealed that the variation in mobile phase pH and solute pK(a) will not affect the QSRRs of acidic compounds. This conclusion is proved to be suitable for various types of ion-suppressors, i.e., strong acid (perchloric acid), weak acid (acetic acid) and buffer salt (potassium dihydrogen phosphate/phosphoric acid, PBS). The QSRRs of logK(ow)″ vs. logk(w) were modeled by 11 substituted benzoic acids using different types of ion-suppressors in a binary methanol-water mobile phase to confirm our deduction. Although different types of ion-suppressor all can be used as mobile phase pH modifiers, the QSRR model obtained by using perchloric acid as the ion-suppressor was found to have the best result, and the slightly inferior QSRRs were obtained by using acetic acid or PBS as the ion-suppressor.