The Application Notebook-03-02-2010

Polychlorinated biphenyls (PCBs) are a class of extremely persistent industrial chemicals manufactured for use in electrical transformers, capacitors, inks, paints, pesticides, dust control or insulating fluids. Estimates have put the total global production of PCBs on the order of 1.5 million tons.

There is considerable attention placed on the synthesis and characterization of polypeptoids, a new class of synthetic polypeptide analogues. Because of intense hydrogen-bonding among chains of these polymers, aggressive solvents such as hexafluoroisopropanol (HFIP) must be used as the mobile phase to support solubility.

Pharmaceutical/Drug Discovery
The Application Notebook

March 02, 2010

Ultra fast separations of acetaminophen, caffeine, propofol and parabens are achieved in 30 s using the Thermo Scientific Accela UHPLC system with the 80 Hz PDA detector. The performance of the Accela PDA detector is highlighted by its low noise level, low drift, four orders of magnitude linear working range and high sensitivity.

Medical/Biological
The Application Notebook

March 02, 2010

Recent technological advances in macromolecular crystallography led to focus on the study of more and more sophisticated biological systems, such as protein–protein complexes. Nevertheless, they represent one of the most challenging areas in modern structural biology.

The procedure involves a rapid and efficient pretreatment with SampliQ QuEChERS kits. The homogenized liver sample was initially extracted in a buffered aqueous/1% acetic acid acetonitrile system with an extraction and partitioning step after the addition of salts. Finally, the sample was cleaned up using dispersive solid-phase extraction (dispersive-SPE). The final extracts were analysed by the sensitive and selective determination of all compounds in a single run using LC–ESI-MS–MS operating in positive ion multiple reaction monitoring (MRM) mode. Sulphapyridine was selected as the internal standard. The accuracy of the method, expressed as recovery, was between 53 and 93%. The precision, expressed as RSD, was between 2.1 and 16.8%. The established 5 ng/g limits of quantification (LOQ) were much lower than the respective maximum residue limit (MRL) for sulphonamide in animal food products (20–100 ng/g).

The procedure involves a rapid and efficient pretreatment by SampliQ QuEChERS kits. The homogenized liver sample was initially extracted in a buffered aqueous, 5% formic acid acetonitrile system. An extraction and partitioning step was performed after the addition of salts. Clean-up was done using dispersive solid-phase extraction (dispersive-SPE). The final extracts allowed determination of all compounds in a single run using LC–ESI-MS–MS operating in positive ion multiple reaction monitoring (MRM) mode. Norfloxacin was selected as the internal standard. The accuracy of the method, expressed as recovery, was between 62 and 113%. The precision, expressed as RSD, was between 2.2 and 13.4%. The established limit of quantification (LOQ) was 5 ng/g and is significantly lower than the respective maximum residue limit (MRL) for quinolones in food producing animals.

In the past decade, supercritical fluid chromatography (SFC) has experienced a steady growth in acceptance, particularly in pharmaceutical and chemical laboratories. In SFC, "supercritical" CO2 combined with one or more polar organic solvents, most commonly alcohols, are used as mobile phase. Preparative SFC is deemed by many to hold the greatest promise to attain mainstream acceptance. The reduction in solvent consumption and collection in relatively small volumes of volatile organic solvents, hence, a much less stringent post-purification endeavour, lead to significant savings on operation costs. For example, Ripka et al. calculated that 20000 samples purified by SFC instead of reverse phase liquid chromatography (RPLC) would realize a 48 times reduction in solvent consumption.1

The Application Notebook

The determination of BTEX in gasoline is usually performed in accordance with the standard test method ASTM D3606 using gas chromatography. However, particularly in the presence of ethanol, co-elutions are observed in one-dimensional chromatography. In this application note a method is described applying multidimensional GC to overcome the separation problem.

Medical/Biological
The Application Notebook

March 02, 2010

Viruses are packets of infectious nucleic acid (either DNA or RNA) surrounded by a protective coat consisting of a large number of protein subunits. Because viruses can cause various diseases - some life-threatening - characterizing virus particles thoroughly in terms of their size distribution, aggregation and absolute counts-per-unit volume is of extreme importance.

The Agilent 1290 Infinity LC has significant capabilities for a wide range of HPLC and UHPLC applications. It exhibits a broader power range (that is, the combination of pressure and flow capabilities) than any other commercially available system and has the flexibility to operate with a wide range of column dimensions and particle sizes. Additionally, advanced optical design in the diode array detector allows a wide dynamic range and high sensitivity, both of which are critical in the monitoring of small impurities in fine chemicals.

The characterization of pharmaceutical salts is critical to the drug development process. Active pharmaceutical ingredient (API) salts influence the solubility, stability and hygroscopicity of pharmaceuticals and affect the final drug formulation. Traditionally, the analysis of the API and its counterion salt requires separate applications. Nanopolymer silica hybrid (NSH) technology in the Acclaim Trinity P1 column along with the Corona ultra charged aerosol detector (CAD) enables simultaneous analysis of the API and counterion over four orders of magnitude.

The Application Notebook
Medical/Biological

March 02, 2010

Streamlined sample prep, LC and MS method development.

The stevia plant and its extracts have long been used as sweeteners in Asia and Latin America. Two steviol glycosides present in plant tissue, stevioside and rebaudioside A, are largely responsible for the sweet flavour.1 In December 2008, the US FDA placed rebaudioside A (also known as rebiana) on the Generally Recognized as Safe (GRAS) list of sugar substitutes to be used in foods, thereby allowing the use of rebiana as a commercial sweetener.2 The determination of steviol glycosides in these sweeteners is challenging due to their weak UV absorbance. Other detection methods, such as evaporative light scattering (ELS), can improve steviol glycoside quantification. In this proposed method, steviol glycosides were determined by UV and ELS detections in consumer sweeteners following separation on the Acclaim Mixed-Mode WAX-1 column.3

The Application Notebook

Polyols are alcohols with multiple hydroxyl groups. They are used for very different applications, such as, for example, in Feed and Food as sugar substitutes or in polymer chemistry as reactants to produce high quality products.

The combination of the small particle size and narrow particle size distribution coupled with the significantly shorter diffusion path provided by the Kinetex core-shell particle results in a material that yields significantly increased column efficiencies and chromatographic resolution. This is a significant benefit for separation scientists looking to increase efficiency and chromatographic resolution, especially for complex separations containing many compounds and very closely eluting compounds so that accurate identification and quantification can be achieved.

The method developed runs on a fully automated platform integrating solid-phase extraction (SPE) and LC (Symbiosis Pico) without any manual pretreatment step(s). Automatic pretreatment results in high throughput, low CVs and high precision. Sample preparation has been simplified to just one operator step before the system takes over the automated analysis of the samples.