There is no dearth of options—both in terms of variety of columns and vendors—for analyzing samples using column chromatography. The challenge is to be able to pick the right analytical column to analyze the right sample correctly. The decision is based on several factors: column specifications, dimensions, media particle and pore sizes, and chemistry of the bonded phase, all of which can affect separation efficiency, inertness, durability, pH range, batch-to-batch reproducibility, resolution, solvent usage, and more. There is also the complexity and quantity of the sample available and the desired cost and accuracy of analysis to be considered.
“What is really important to the consumer is lot-to-lot and column-to-column reproducibility,” says Dafydd Milton, product manager, LC and LC/MS columns, at Thermo Fisher Scientific Inc. “They have to have the confidence that the column will elute the analyte peaks at the same time, every time.” Along with elution times, the ability to get good peak shapes—sharp, narrow, symmetrical peaks— is also important, especially for applications such as method development.
Conventional liquid chromatography uses plastic or glass columns that can range in size from a few centimeters to several meters in length. Commonly used lengths vary from 10 to 100 cm, with longer columns being used for preparative scale separations. High-performance liquid chromatography (HPLC) columns are made of stainless steel and are typically shorter, approximately 10 to 30 cm in length. Short, highly efficient HPLC columns allow shorter analysis times, better peak shapes, and better quality data while also reducing cost per analysis. Narrower columns also offer better mass sensitivity and significantly reduce solvent use.
Milton mentions that in recent months there has been a shortage of acetonitrile, a solvent routinely used for HPLC analysis. Acetonitrile is a by-product of the automobile industry and, since there are no dedicated plants to manufacture acetonitrile, the recent slowdown in the production of cars has caused scarcity of the solvent. “We find many customers moving to smaller columns, packed with smaller particles (sub2-micron) because they use less solvent,” says Milton. Even before the solvent shortage occurred, the trend had been toward increasing the use of columns packed with smaller particles because of advantages associated with costs and efficiency, although slower, longer columns that offer better resolution are sometimes preferred to separate sample components in extremely complex samples. “It’s been a couple of years since the sub2- or 2.5-micron columns were introduced into the marketplace, and the smaller particle-size columns are now proving to be very important,” says Maureen Joseph, product manager in the Columns and Supplies division at Agilent Technologies Inc. These columns have proved very efficient in terms of cost and performance, and they cover a wide range of applications in industries that span food, environmental, pharmaceuticals, biofuels, and others.
There is also an increased demand for the analysis of polar analytes for applications in both drug discovery and development, such as the identification of metabolites. Hence, many companies have now introduced hydrophilic interaction chromatography (HILIC) columns for analysis of such polar analytes. “More and more drugs that are being developed seem to have a polar component, which the traditional C18-type columns don’t seem to retain very well,” says Milton. The HILIC columns use hydrophilic interactions to facilitate the transfer of polar analytes to the stationary phase for increased retention and better sensitivity.
There are a lot of changes also taking place in the area of biomolecule analysis. “Along with preparative and process analysis, there are also analytical columns being introduced for the fast and accurate detection of biomolecules,” says Taegen Clary, who is also a product manager in the Columns and Supplies division at Agilent but is involved more with biomolecule analysis. People involved in antibody sizing, analysis of protein isoforms, and clinical samples are becoming increasingly concerned about time and cost of analysis. Reducing the time needed to analyze samples, increasing efficiency, and improving data quality can result in significant savings for laboratories that run hundreds of samples per day. Such laboratories are also continually evaluating alternative methodologies that can overcome some of the limitations associated with column chromatography. Microfluidics, for instance, is beginning to play a role in analyzing samples that are rare and available in small quantities, such as for proteomics. However, it has yet to play a role in mainstream analytical applications. While there is definitely a trend toward miniaturization, microfluidics is unlikely to completely replace column technology. “There will always be a place for traditional column technology,” says Joseph.
Tanuja Koppal, PhD, is a freelance science writer and consultant based in Randolph, N.J.
SampliQ SPE is a solid phase extraction (SPE) product line for fast and cost-effective purification of extracts to improve the quality of analytical results. Researchers in food safety, pharmaceutical, environmental, and forensic industries use solid phase extractions to clean up samples prior to chromatography and mass spectrometry. SampliQ SPE is used widely throughout Asia and other regions to streamline detection and measurement of melamine in dairy products, for example. Formats range from the most popular cartridge sizes to 96-well microtiter plates. All SampliQ SPE products are manufactured under stringent quality-control standards to ensure consistent chemical composition, controlled particle size and lot-to-lot sorbent uniformity for consistent flow-through and performance.
PHENOMENEX Six new offerings in its Onyx™ monolithic HPLC column line include a 150 X 0.05 mm ID choice for proteomics research. This column is optimized for the low flow-rate systems commonly used for LC/MS proteomics analysis and delivers high sensitivity for biomarker discovery. Other Onyx column sizes include a 0.2 mm C18 column for higher flow rates, a 150 X .2 mm trapping column, a 150 x .1 C8 column and two ultra-highresolution C18 columns at 100 and 200 micron IDs. The permeable Onyx media separates viscous biological samples that clog traditional columns. These columns contain solid rods of fused silica and eliminate packing issues associated with particulate media. Run times are as much as 50 percent faster than with traditional columns.
Supelco, A Division of Sigma-Aldrich
The Ascentis Express HPLC column provides the analysis speed and resolving power of sub 2 μm columns at half of the backpressures. The technology inside the columns is the Fused-Core™ particles. These high purity silica particles are 2.7 micron overall with a 0.5 micron thick porous shell. Due to the high efficiencies at low backpressures, the columns can benefit conventional HPLC users as well as UPLC™ or other ultra pressure system users. Ascentis Express C18 and C8 provide classic reversed-phase selectivity with performance benefits for fast HPLC and high resolution HPLC. Ascentis Express HILIC provides all of the benefits of Fused-Core particle technology to the emerging technique of HILIC mode chromatography. Ascentis Express RP-Amide provides alternate selectivity to reversed phase separations.
Thermo Fisher Scientific
Based on improved, highly pure silica, Hypersil GOLD columns are designed to give outstanding peak shape. The novel derivatization and endcapping manufacturing processes result in fewer surface silanols, which in turn leads to reduced peak tailing, thus resulting in improved resolution and sensitivity. The columns are available in six bonded phases to provide flexibility in method development, all of which are also available in 1.9 μm particle size for enhanced speed and resolution. In addition to standard analytical columns, Hypersil GOLD columns are available in a variety of column designs to meet all separation needs. Specialized formats include preparative columns; KAPPA ™ capillary columns, PicoFrit™ and IntegraFrit™ nanobore columns with diameters as narrow as 75 μm; Javelin™ HTS and DASH™ HTS columns, designed for high throughput LC/MS screening; and guard columns for column protection.
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