Emily F. Hilder

Separation and sample pre-treatment in bioanalysis using monolithic phases: A review

In order to support drug discovery and development studies within the pharmaceutical industry there has been an increased use of innovative bioanalytical assays and associated analytical technology. Performing quantitative bioanalysis in a variety of biological matrices can also involve the use of sample preparation techniques, complex HLPC column switching and microfluidic systems. Development of assays for diverse therapeutic agents in biomatrices, such as plasma and urine, can be very technically challenging to obtain the sensitivity, speed and specificity required. This challenge focuses on the quantification of drugs and metabolites at very low concentration levels, in an excess of biological matrix and in a high-throughput manner. One area of wide interest is the use and application of monolithic phases where emerging technology has been implemented successfully. This review presents an overview of the application of monolithic phases in a bioanalytical setting, including the bioanalytical challenges that need to be overcome; the synthesis, use and applicability of monolithic phases (with emphasis on polymer-based phases); the currently available bioanalytical techniques and approaches; and future possibilities for these phases.

Review of recent advances in the preparation of organic polymer monoliths for liquid chromatography of large molecules.

In recent years the use of monolithic polymers in separation science has greatly increased due to the advantages these materials present over particle-based stationary phases, such as their relative ease of preparation and good permeability. For these reasons, these materials present high potential as stationary phases for the separation and purification of large molecules such as proteins, peptides, nucleic acids and cells. An example of this is the wide range of commercial available polymer-based monolithic columns now present in the market. This review summarizes recent developments in the synthesis of monolithic polymers for separation science, such as the incorporation of nanostructures in the polymeric scaffold as well as the preparation of hybrid structures. The different methods used in the surface functionalization of monolithic columns are also reviewed. Finally, we critically discuss the recent applications of this column technology in the separation of large molecules under different chromatographic mode.

Recent advances in polymer monoliths for ion-exchange chromatography

The use of polymeric materials in ion-exchange chromatography applications is advantageous because of their typically high mechanical stability and tolerance of a wide range of pH conditions. The possibility of using polymeric monoliths in ion-exchange chromatography is therefore obvious and many of the same strategies developed for polymeric particles have been adapted for use with polymeric monoliths. In this review different strategies for the synthesis of polymeric monoliths with ion-exchange functionality are discussed. The incorporation of ion-exchange functionality by co-polymerization is included, as also are different post-polymerization alterations to the monolith surface such as grafting. The formulations and strategies presented include materials intended for use in analytical separations in ion-exchange chromatography, sample pre-treatment or enrichment applications, and materials for capillary electrochromatography. Finally, examples of the use of polymeric monoliths in ion-exchange chromatography applications are included with examples published in the years 2003 to 2008.

Porous polymer monoliths for extraction: Diverse applications and platforms

Polymer monoliths are becoming increasingly popular as sorbent materials, and, along with silica monoliths, they are sometimes touted as replacements for the particulate stationary phases used in HPLC. This critical and prospective review shows how polymer monoliths are in fact finding numerous extraction roles that do not resemble HPLC. They are showing great promise as extractors in a remarkable range of platforms, formats and hyphenated systems with functions ranging from chromatographic preconcentration to large-scale preparative extraction. Monolith surface chemistry, morphology and the approaches to monolith synthesis are discussed with regards to these emerging roles.

Towards high capacity latex-coated porous polymer monoliths as ion-exchange stationary phases

The preparation of high capacity agglomerated monolithic ion-exchangers for capillary ion chromatography is described. Post-modification of reactive monoliths was investigated as an alternative to co-polymerisation of a suitable functional monomer with an overarching goal of increasing ion-exchange capacity. Direct sulfonation of poly styrene-co-divinyl benzene monoliths using concentrated sulfuric acid or chlorosulfonic acid was unsuccessful even for monoliths containing as low as 8% crosslinker. In contrast, chemical transformation of reactive monoliths containing glycidyl methacrylate was used to increase the ion-exchange capacity by up to more than thirty-fold with ion exchange capacities of 14-29 microequiv g(-1) achieved. Three different reactions were considered, including reaction with 4-hydroxybenenesulfonic acid under basic conditions; reaction with thiobenzoic acid followed by transformation to a reactive thiol and the subsequent oxidation to the sulfonic acid; and direct sulfonation with sodium sulfite. Of these, the reaction with sodium sulfite resulted in the most significant increase in the capacity and the best separation performance. In the isocratic mode separation efficiencies of over 13,500 plates m(-1) were observed (for iodate). The separation of seven inorganic anions was also demonstrated using a hydroxide gradient.

Identification of inorganic ions in post-blast explosive residues using portable CE instrumentation and capacitively coupled contactless conductivity detection.

Novel CE methods have been developed on portable instrumentation adapted to accommodate a capacitively coupled contactless conductivity detector for the separation and sensitive detection of inorganic anions and cations in post‐blast explosive residues from homemade inorganic explosive devices. The methods presented combine sensitivity and speed of analysis for the wide range of inorganic ions used in this study. Separate methods were employed for the separation of anions and cations. The anion separation method utilised a low conductivity 70 mM Tris/70 mM CHES aqueous electrolyte (pH 8.6) with a 90 cm capillary coated with hexadimethrine bromide to reverse the EOF. Fifteen anions could be baseline separated in 7 min with detection limits in the range 27–240 μg/L. A selection of ten anions deemed most important in this application could be separated in 45 s on a shorter capillary (30.6 cm) using the same electrolyte. The cation separation method was performed on a 73 cm length of fused‐silica capillary using an electrolyte system composed of 10 mM histidine and 50 mM acetic acid, at pH 4.2. The addition of the complexants, 1 mM hydroxyisobutyric acid and 0.7 mM 18‐crown‐6 ether, enhanced selectivity and allowed the separation of eleven inorganic cations in under 7 min with detection limits in the range 31–240 μg/L. The developed methods were successfully field tested on post‐blast residues obtained from the controlled detonation of homemade explosive devices. Results were verified using ion chromatographic analyses of the same samples.

Identification of homemade inorganic explosives by ion chromatographic analysis of post-blast residues

Anions and cations of interest for the post-blast identification of homemade inorganic explosives were separated and detected by ion chromatographic (IC) methods. The ionic analytes used for identification of explosives in this study comprised 18 anions (acetate, benzoate, bromate, carbonate, chlorate, chloride, chlorite, chromate, cyanate, fluoride, formate, nitrate, nitrite, perchlorate, phosphate, sulfate, thiocyanate and thiosulfate) and 12 cations (ammonium, barium(II), calcium(II), chromium(III), ethylammonium, magnesium(II), manganese(II), methylammonium, potassium(I), sodium(I), strontium(II), and zinc(II)). Two IC separations are presented, using suppressed IC on a Dionex AS20 column with potassium hydroxide as eluent for anions, and non-suppressed IC for cations using a Dionex SCS 1 column with oxalic acid/acetonitrile as eluent. Conductivity detection was used in both cases. Detection limits for anions were in the range 2-27.4ppb, and for cations were in the range 13-115ppb. These methods allowed the explosive residue ions to be identified and separated from background ions likely to be present in the environment. Linearity (over a calibration range of 0.05-50ppm) was evaluated for both methods, with r(2) values ranging from 0.9889 to 1.000. Reproducibility over 10 consecutive injections of a 5ppm standard ranged from 0.01 to 0.22% relative standard deviation (RSD) for retention time and 0.29 to 2.16%RSD for peak area. The anion and cation separations were performed simultaneously by using two Dionex ICS-2000 chromatographs served by a single autoinjector. The efficacy of the developed methods was demonstrated by analysis of residue samples taken from witness plates and soils collected following the controlled detonation of a series of different inorganic homemade explosives. The results obtained were also confirmed by parallel analysis of the same samples by capillary electrophoresis (CE) with excellent agreement being obtained.

Anion-exchange capillary electrochromatography with indirect UV and direct contactless conductivity detection

Conductivity detection is applied to ion‐exchange capillary electrochromatography (IE‐CEC) with a packed stationary phase, using a capacitively coupled contactless conductivity detector with detection occurring through the packed bed. Columns were packed with a polymeric latex‐agglomerate anion‐exchanger (Dionex AS9‐SC). A systematic approach was used to determine suitable eluants for IE‐CEC separations using simultaneous indirect UV and direct conductivity detection. Salicylate and p‐toluenesulfonate were identified as potential eluant competing anions having sufficient eluotropic strength to induce changes in separation selectivity, but salicylate was found to be unsuitable with regard to baseline stability. It was also found for both indirect UV and direct conductivity detection that homogenous column packing was imperative, and monitoring of the baseline could be used to assess the homogeneity of the packed bed. Using a p‐toluenesulfonate eluant, the separation of eight common anions was achieved in 2.5 min. Direct conductivity detection was found to be superior to indirect UV detection with regard to both baseline stability and detection sensitivity with detection limits of 4–25 μg/L being obtained. However, the calibration for each anion was not linear over more than one order of magnitude. When using conductivity detection, the concentration of the eluant could be varied over a wider range (2.5–50 mM p‐toluenesulfonate) than was the case with indirect UV detection (2.5–10 mM), thereby allowing greater changes in separation selectivity to be achieved. By varying the concentration of p‐toluenesulfonate in the eluant, the separation selectivity could be manipulated from being predominantly ion‐exchange in nature (2.5 mM) to predominantly electrophoretic in nature (50 mM).

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.

On-line simultaneous and rapid separation of anions and cations from a single sample using dual-capillary sequential injection-capillary electrophoresis

A novel capillary electrophoresis (CE) approach has been developed for the simultaneous rapid separation and identification of common environmental inorganic anions and cations from a single sample injection. The method utilised a sequential injection-capillary electrophoresis instrument (SI-CE) with capacitively-coupled contactless conductivity detection (C(4)D) constructed in-house from commercial-off-the-shelf components. Oppositely charged analytes from a single sample plug were simultaneously injected electrokinetically onto two separate capillaries for independent separation and detection. Injection was automated and may occur from a syringe or be directly coupled to an external source in a continuous manner. Software control enabled high sample throughput (17 runs per hour for the target analyte set) and the inclusion of an isolation valve allowed the separation capillaries to be flushed, increasing throughput by removing slow migrating species as well as improving repeatability. Various environmental and industrial samples (subjected only to filtering) were analysed in the laboratory with a 3 min analysis time which allowed the separation of 23 inorganic and small organic anions and cations. Finally, the system was applied to an extended automated analysis of Hobart Southern Water tap water for a period of 48 h. The overall repeatability of the migration times of a 14 analyte standard sample was less than 0.74% under laboratory conditions. LODs ranged from 5 to 61 μg L(-1). The combination of automation, high confidence of peak identification, and low limits of detection make this a useful system for the simultaneous identification of a range of common inorganic anions and cations for discrete or continuous monitoring applications.