Application Notes: General

The introduction of UHPLC revolutionized bio/pharma analytical laboratories and advances in technology promise more for the future. In this ebook, experts explain recent advances in UHPLC, and the basics of glycan analysis. Sample preparation technologies that can increase sample throughput and improve data quality for peptide quantitation are explored. In addition, the characterization of intact antibodies using reversed-phase chromatography is detailed. Review UHPLC workflows for: • Glycan analysis • Peptides • Monoclonal antibodies

As instrumentation and analytical methods are becoming increasingly sensitive, the ability to perform trace and ultra trace analyses relies on the high quality and purity of the reagents used.

Chromatographers take great care in the selection of salts and organic solvents used in mobile phase preparation, but selecting the best type of water is sometimes an arduous task.

Ultrapure water is highly prone to contamination, e.g. it easily leaches contaminants out of container surfaces and absorbs contamination from the laboratory environment. As ultrapure water is the most frequently used solvent in any LC-MS laboratory, its purity plays a critical role in analyses. There are a number of high purity water handling pitfalls that result in degradation of its quality. To help analysts critically evaluate the potential risks involved in poor ultrapure water handling, we discuss here (I) the effect of laboratory environment and long high purity water storage, (II) the effect of the container material used to collect ultrapure water, (III) the effect of laboratory ware and equipment cleaning, and (IV) the effect of poor practices of water purification system usage.

As the sensitivity of analytical instrumentation is constantly improving, and ultra-traces of compounds are being analyzed, the purity of reagents is becoming of paramount importance. Trace impurities in the water used to prepare LC-MS mobile phases, standards or blanks may lead to erroneous results or difficulties in analyzing data.

‘Old’ HPLC methods with long run times are being altered or surpassed by newer UHPLC or core-shell methods in order to save time and cost. In this application note we show how with the use of 3 simple equations transfer of older methods can be easily achieved onto newer core-shell particles. We show the example of a pharmaceutical drug and its impurities being reduced from a 30minute run time down to less than 10minutes. Using the calculations correctly means that no loss of resolution is seen even with the decrease in retention time.

‘Old’ HPLC methods with long run times are being altered or surpassed by newer UHPLC or core-shell methods in order to save time and cost. In this application note we show how with the use of 3 simple equations transfer of older methods can be easily achieved onto newer core-shell particles. We show the example of a pharmaceutical drug and its impurities being reduced from a 30minute run time down to less than 10minutes. Using the calculations correctly means that no loss of resolution is seen even with the decrease in retention time.

Starch is used for a variety of industrial and nu¬tritional purposes. Its functional properties are influenced by the ratio and molar masses of its mac¬romolecular constituents, which vary with source, crop year, and climate. Starch contains large homopolymers of amylose (AMY) and amylopectin (AMP).

In the example shown here we seek to provide an alternative to HILIC methods by using a novel multi-mode column called the Scherzo SS-C18. This stationary phase has both reversed phase (C18) and polar components with the addition of a high density of strong anion and cation exchange ligands. This strategy for increasing polar retention is much more predictable run to run and does not require lengthy column pre-treatments.

Multi-angle light scattering (MALS) is primarily used to determine molecular weight, size-and shape-related parameters of macromolecules or nanoparticles as they elute from a separation device. In batch (unfractionated) mode, MALS provides the weight-average molecular weight wand the z-average mean square radius z. The DAWNHELEOSIIis particularly well suited for these tasks owing to its flow cell design, holding a mere ~67µl and readily connecting toa fluid line through HPLC-type fittings.

The dioxin family consists of 210 compounds, of which 17 contain the 2,3,7,8 pattern of chlorination. These 2,3,7,8 containing compounds are of extreme human health concern due to their high level of toxicity. Approximately, 12 of the 209 polychlorinated biphenyls have also been identified as human toxins. For this reason, the US FDA and EU have established strict regulations for the monitoring of food products for human consumption, in particular edible oils. Manual extractions of oils can be a time consuming procedure often delaying lab turnaround times. By automating the process with the EconoPrep®, food oil samples can be reliably processed with routine 24 hr turnaround times.

The dioxin family consists of 210 compounds, of which 17 contain the 2,3,7,8 pattern of chlorination. These 2,3,7,8 containing compounds are of extreme human health concern due to their high level of toxicity. Approximately, 12 of the 209 polychlorinated biphenyls have also been identified as human toxins. For this reason, the US FDA and EU have established strict regulations for the monitoring of food products for human consumption, in particular edible oils. Manual extractions of oils can be a time consuming procedure often delaying lab turnaround times. By automating the process with the EconoPrep®, food oil samples can be reliably processed with routine 24 hr turnaround times.

This note describes the use of TSKgel UP-SW3000, 2µm SEC columns for the analysis of proteins, with data demonstrating the operation of these columns using a simple and well established method for use in both HPLC and UHPLC systems. TSKgel UP-SW3000 columns have superior resolution for proteins and the shorter column dimension, 4.6 mm ID × 15 cm, allows runs to be completed 2 times faster than its longer column dimension counterpart without compromising resolution and reproducibility.

Centrifugal Partition Chromatography (CPC) also known as Counter Current Chromatography (CCC) is a preparative, pilot and industrial liquid purification technique that does not require traditional solid supports. CPC was used to purify few mg of gingerol from crude extract.

TSKgel UP-SW3000 columns are 2 µm SEC columns designed for the analysis of monoclonal antibodies and other biopharma products. Higher resolution can be achieved for the separation of antibody monomers, dimers, and higher order aggregates with a TSKgel UP-SW3000 column compared to a competitor UHPLC column. The TSKgel UP-SW3000 column provided excellent reproducibility for the peak parameters of retention time, asymmetry, and column efficiency. As demonstrated by the %RSD values, injection-to-injection reproducibility was superior to the competitor column.

Scientists employ fused silica capillary tubing routinely in a wide range of analytical applications, encompassing GC, CE, capillary LC, and CEC. It has proven as an ideal substrate for interfacing in hyphenated techniques.

Due to the highly polar nature of saccharides, the analysis of sugars is typically achieved using hydrophilic interaction chromatography (HILIC).

The challenging retention of polar organic acids in reversed phase conditions is accomplished using a unique multi-mode stationary phase.

Reflectron lenses are used in time-of-flight (TOF) mass spectrometers to create an electrostatic field to alter ion flow, providing for a longer flight path and therefore greater resolution.

Direct exposure probe (DEP) coupled to the Pegasus HT, time-of-flight mass spectrometer, was used for the identification of synthetic cannabinoids in MTN-series compounds.

Photonis has combined two of our superior detection and analysis technologies into a new digital ion beam profiling unit. The new Ion Beam Profiler combines a large microchannel plate (up to 120 mm) in a complete assembly with the Photonis Nocturn, a high-resolution digital CMOS low-light camera which is immune to sudden light damage.

Silica-based cyano stationary phases have historically exhibited poor stability and short column lifetime at pH extremes. This application note details an investigation of the stability of YMC's Cyano-HG (high strength) 10 ?m silica-based stationary phase, which was performed at a customer's request.

In water samples, OCPs are monitored by the U.S. Environmental Protection Agency by various methods including methods EPA 508, EPA 608, and EPA 8081. The use of solid phase extraction (SPE) can rapidly increase both extraction efficiency and reduce lab solvent usage. By implementing the FMS TurboTrace® ABN system with its dual cartridge functionality, an in-line Florisil cartridge can be added to perform automated extract clean-up during the elution step.

The unique surface chemistry of the Flare diamond core-shell column combines ionic and hydrophobic separation mechanisms to effectively retain a variety of chemical species in a single run.

Natural and synthetic phenolic compounds have been used in a wide range of applications, from antiseptics and fungicides to food additives.

In mass spectrometry instrumentation, the sensitivity and resolution of the instrument is often limited by the detector.

The unique surface chemistry of the Flare diamond core-shell column combines ionic and hydrophobic separation mechanisms to effectively retain a variety of chemical species in a single run.

As the cost of helium increases and its availability decreases, an increasing number of Gas Chromatography (GC) professionals are considering switching to hydrogen gas for their carrier gas.