Artaches A. Kazarian

Online sample pre-concentration via dynamic pH junction in capillary and microchip electrophoresis.

Various analytical techniques have been developed over the years to analyse a large diversity of biomolecules with a constant push towards ultra-sensitive detection. CE is at the forefront of the most powerful analytical tools available to date when considering its superior efficiency and resolution; however, the technique suffers from poor sensitivity as a result of the short path length at the detection site and small injection volumes (typically <1% capillary length). One of the approaches to abate the inherent problem is to employ clever chemistry using sample focusing techniques whereby a large sample plug can be injected, preconcentrated and separated, producing excellent sensitivity and efficiency at the detector. This particular review will focus on the use of dynamic pH junction as a means of improving sensitivity in CE and focuses on the use of a change in analyte ionisation due to different pHs between the sample and electrolyte. The review provides a fundamental discussion of the mechanisms, buffer and sample conditions required to concentrate various analytes and a comprehensive list of published works in tabular format for easy identification of suitable conditions for new applications. The review further encompasses the use of dynamic pH junction in CE and its involvement in combination with other preconcentrations techniques to produce high sensitivity enhancements recorded between the years 1990-2010.

Utilisation of pH stacking in conjunction with a highly absorbing chromophore, 5-aminofluorescein, to improve the sensitivity of capillary electrophoresis for carbohydrate analysis

This study explores the use of pH stacking in conjunction with 5-aminofluorescein as a derivatization agent for the sensitive analysis of simple sugars such as glucose, lactose and maltotriose by capillary electrophoresis (CE). The derivatization agent was selected on the basis of its extremely high molar absorptivity, its compatibility with a 488nm light-emitting diode (LED) and the fact that it has two ionizable groups making it compatible with on-line stacking using a dynamic pH junction. The influence of both acetic and formic acids at concentrations of 0.19, 0.019 and 0.0019molL(-1) were investigated with regard to both derivatization efficiency and the ability to stack using a dynamic pH junction. Superior sensitivity and resolution was obtained in formic acid over acetic acid. Substantially lower peaks were obtained with 0.19molL(-1) formic acid when compared to 0.019 and 0.0019molL(-1) concentrations, which was confirmed by computer simulation studies to be due to the inadequate movement of the pH boundary for stacking. Further simulation studies combined with experimental data showed the separation with the best resolution and greatest sensitivity when the carbohydrates were derivatized with the 0.095molL(-1) formic acid. Utilisation of stacking via dynamic pH junction mode in conjunction with LED detection enabled efficiencies of 150,000 plates and detection limits in the order of 8.5x10(-8)molL(-1) for simple sugars such as glucose, lactose and maltotriose hydrate. The current system also demonstrates a 515 times improvement in sensitivity when compared to using a normal deuterium lamp, and 16 times improvement over other systems using LEDs.

Ion-exchange and hydrophobic interactions affecting selectivity for neutral and charged solutes on three structurally similar agglomerated ion-exchange and mixed-mode stationary phases

The nature and extent of mixed-mode retention mechanisms evident for three structurally related, agglomerated, particle-based stationary phases were evaluated. These three agglomerated phases were Thermo Fisher ScientificIon PacAS11-HC - strong anion exchange, Thermo Fisher Scientific IonPac CS10--strong cation-exchange PS-DVB, and the Thermo Fisher Scientific Acclaim Trinity P1silica-based substrate, which is commercially marketed as a mixed-mode stationary phase. All studied phases can exhibit zwitterionic and hydrophobic properties, which contribute to the retention of charged organic analytes. A systematic approach was devised to investigate the relative ion-exchange capacities and hydrophobicities for each of the three phases, together with the effect of eluent pH upon selectivity, using a specifically selected range of anionic, cationic and neutral aromatic compounds. Investigation of the strong anion-exchange column and the Trinity P1 mixed-mode substrate, in relation to ion-exchange capacity and pH effects, demonstrated similar retention behaviour for both the anionic and ampholytic solutes, as expected from the structurally related phases. Further evaluation revealed that the ion-exchange selectivity of the mixed-mode phase exhibited properties similar to that of the strong anion-exchange column, with secondary cation-exchange selectivity, albeit with medium to high anion-exchange and cation-exchange capacities, allowing selective retention for each of the anionic, cationic and ampholytic solutes. Observed mixed-mode retention upon the examined phases was found to be a sum of anion- and cation-exchange interactions, secondary ion-exchange and hydrophobic interactions, with possible additional hydrogen bonding. Hydrophobic evaluation of the three phases revealed logP values of 0.38-0.48, suggesting low to medium hydrophobicity. These stationary phases were also benchmarked against traditional reversed-phase substrates namely, octadecylsilica YMC-Pac Pro C18 and neutral μPS-DVB resin IonPac NS1-5u, yielding logP values of 0.57 and 0.52, respectively.

Comprehensive analysis of pharmaceutical products using simultaneous mixed-mode (ion-exchange/reversed-phase) and hydrophilic interaction liquid chromatography

Liquid chromatographic assays were developed using a mixed-mode column coupled in sequence with a hydrophilic interaction liquid chromatography column to allow the simultaneous comprehensive analysis of inorganic/organic anions and cations, active pharmaceutical ingredients, and excipients (carbohydrates). The approach utilized dual sample injection and valve-mediated column switching and was based upon a single high-performance liquid chromatography gradient pump. The separation consisted of three distinct sequential separation mechanisms, namely, (i) ion-exchange, (ii) mixed-mode interactions under an applied dual gradient (reversed-phase/ion-exchange), and (iii) hydrophilic interaction chromatography. Upon first injection, the Scherzo SS C18 column (Imtakt) provided resolution of inorganic anions and cations under isocratic conditions, followed by a dual organic/salt gradient to elute active pharmaceutical ingredients and their respective organic counterions and potential degradants. At the top of the mixed-mode gradient (high acetonitrile content), the mobile phase flow was switched to a preconditioned hydrophilic interaction liquid chromatography column, and the standard/sample was reinjected for the separation of hydrophilic carbohydrates, some of which are commonly known excipients in drug formulations. The approach afforded reproducible separation and resolution of up to 23 chemically diverse solutes in a single run. The method was applied to investigate the composition of commercial cough syrups (Robitussin®), allowing resolution and determination of inorganic ions, active pharmaceutical ingredients, excipients, and numerous well-resolved unknown peaks.

Capillary electrophoresis for the analysis of paralytic shellfish poisoning toxins in shellfish: Comparison of detection methods

Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and accumulate in shellfish including mussels, oysters, and scallops, causing possible fatalities when inadvertently consumed. Monitoring of PST content of shellfish is therefore important for food safety, with currently approved methods based on HPLC, using pre‐ or postcolumn oxidation for fluorescence detection (HPLC‐FLD). CE is an attractive alternative for screening and detection of PSTs as it is compatible with miniaturization and could be implemented in portable instrumentation for on‐site monitoring. In this study, CE methods were developed for C4D, FLD, UV absorption detection, and MS—making this first report of C4D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used in combination with FLD. The developed CZE‐UV and CZE‐C4D methods provide better resolution, selectivity, and separation efficiency compared to CZE‐MS and MEKC‐FLD. The sensitivity of the CZE‐C4D and MEKC‐FLD methods was superior to UV and MS, with LOD values ranging from 140 to 715 ng/mL for CZE‐C4D and 60.9 to 104 ng/mL for MEKC‐FLD. With the regulatory limit for shellfish samples of 800 ng/mL, the CZE‐C4D and MEKC‐FLD methods were evaluated for the screening and detection of PSTs in shellfish samples. While the CZE‐C4D method suffered from significant interferences from the shellfish matrix, MEKC‐FLD was successfully used for PST screening of a periodate‐oxidized mussel sample, with results confirmed by HPLC‐FLD. This confirms the potential of MEKC‐FLD for screening of PSTs in shellfish samples.

Development of a novel fluorescent tag O-2-[aminoethyl]fluorescein for the electrophoretic separation of oligosaccharides

This study describes the development of a novel fluorescent tag, O-2-[aminoethyl]fluorescein, for the separation of sugars by capillary electrophoresis with fluorescence detection using an argon ion laser. The tag was synthesised using three consecutive steps namely: esterification, alkylation and hydrolysis, specifically designed to offer a flexible way in which to make an assortment of fluorescent tags from cheap and readily available starting reagents (typically less than

Single column comprehensive analysis of pharmaceutical preparations using dual-injection mixed-mode (ion-exchange and reversed-phase) and hydrophilic interaction liquid chromatography

1 per g of fluorescent tag). Via this flexible synthetic pathway, O-2-[aminoethyl]fluorescein was designed and synthesised with a spacer group to lower steric effects between the fluorescein backbone and the reducing end of the carbohydrate which were anticipated to improve the reactivity of the tag. The newly synthesised tag, O-2-[aminoethyl]fluorescein was evaluated against structurally similar commercial fluorescent motifs namely fluorescent 5-aminomethylfluorescein and non-fluorescent 5-aminofluorescein. Kinetic studies indicated that O-2-[aminoethyl]fluorescein showed similar labeling efficiencies as 5-aminomethylfluorescein, but were achieved in only 30 min, supporting the notion of improved reactivity of the spacer group. The sensitivity of O-2-[aminoethyl]fluorescein was evaluated using maltoheptaose with a detection limit of 1 nM obtained, which was slightly higher than that of 0.3 nM obtained with 5-aminomethylfluorescein, and was due to its lower quantum yield (0.24) when conjugated to the sugar. The separation performance of the tag was also benchmarked with the two commercial reagents using a range of corn syrup oligosaccharides, from 4 to 10 glucose units, typically found in rice starch. Separations were performed using an electrolyte containing 100 mM boric acid, tris at pH 8.65 as background electrolyte, 30 kV applied voltage, 50 microm I.D. x 40 cm (30 cm effective length) capillary. The novel tag showed better resolution of small oligosaccharides, G3 and G4, than the other two reagents, but slightly worse resolution for the longer oligosaccharides, most likely due to the monovalent charge state of the O-2-[aminoethyl]fluorescein compared to the divalent charge of the other two tags.

Separation and characterisation of detonation nanodiamond by capillary zone electrophoresis.

The comprehensive separation and detection of hydrophobic and hydrophilic active pharmaceutical ingredients (APIs), their counter-ions (organic, inorganic) and excipients, using a single mixed-mode chromatographic column, and a dual injection approach is presented. Using a mixed-mode Thermo Fisher Acclaim Trinity P1 column, APIs, their counter-ions and possible degradants were first separated using a combination of anion-exchange, cation-exchange and hydrophobic interactions, using a mobile phase consisting of a dual organic modifier/salt concentration gradient. A complementary method was also developed using the same column for the separation of hydrophilic bulk excipients, using hydrophilic interaction liquid chromatography (HILIC) under high organic solvent mobile phase conditions. These two methods were then combined within a single gradient run using dual sample injection, with the first injection at the start of the applied gradient (mixed-mode retention of solutes), followed by a second sample injection at the end of the gradient (HILIC retention of solutes). Detection using both ultraviolet absorbance and refractive index enabled the sensitive detection of APIs and UV-absorbing counter-ions, together with quantitative determination of bulk excipients. The developed approach was applied successfully to the analysis of a dry powder inhalers (Flixotide(®), Spiriva(®)), enabling comprehensive quantification of all APIs and excipients in the sample.

Postcolumn derivatization of amino acids using reaction flow chromatography columns with fluorescence detection: a fast new approach to selective derivatization techniques

A new method for the characterisation of purified detonation nanodiamond (DND) using CZE has been developed. The influence of BGE conditions on electrophoretic mobility, peak shape and particle aggregation was investigated, with resultant observations supported by zeta potential approximations and particle size measurements. Sodium tetraborate (pH 9.3), Tris (pH 9.3) and sodium phosphate (pH 7) were used in studying the BGE concentration effect on a commercial source of chemically stabilised DND. The BGE concentration had a strong effect on the stability of DND in suspension. The formation of aggregates of various sizes was observed as BGE concentration increased. The effect of pH on the electromigration of DND was examined using sodium phosphate (pH 8 and 10). The CZE method was subsequently applied to four different DND samples, which had undergone different routes of purification following detonation synthesis. Each sample produced a unique electrophoretic peak or profile in sodium tetraborate buffer (pH 9.3), such that the actual separation of DND samples from different sources could be achieved.

Fluorophores and Chromophores for the Separation of Carbohydrates by Capillary Electrophoresis

ABSTRACT Reaction flow (RF) chromatography with fluorescamine reagent and fluorescence detection (FLD) was used for the analysis of amino acids. The performance of RF chromatography was tested against several optimized conventional postcolumn derivatization (PCD) methods. RF columns achieved greater sensitivity compared to conventional PCD methods, without the need for reaction loops, which resulted in more efficient separations. The RF-PCD method also achieved limits of detection (LOD) from the low picomole to subnanomole range. The calibration data of the RF-PCD technique yielded R2 ≥ 0.99 and % relative standard deviation in peak areas ranging from 0.34% to 5%. Through reaction flow chromatography, multiplexed detection was also achieved allowing the monitoring and analysis of derivatized and nonderivatized flow streams simultaneously. GRAPHICAL ABSTRACT