LCGC Europe-07-01-2016

The use of antibodies in “bottom-up” LC–MS workflows to determine low abundant biological active proteins in complex human samples has increased in recent years: immuno-capture analysis combines the workflow of conventional immunological assays with LC–MS analysis. This paper describes typical challenges, such as cross reactivity and the mass spectrometer’s dynamic range, as well as the advantages of isoform differentiation and multiplexing. Additionally, some experimental formats of immuno-capture bottom-up LC–MS analysis of biological active proteins in complex human samples will be discussed.

Ultrahigh-pressure liquid chromatography (UHPLC) instruments from different manufacturers and instruments with different configurations can produce significant variations in chromatographic separation. The variety in instrument configuration increases the complexity of the method development process, which now requires a more thorough evaluation of the effect of instrument variations on the method. The studies presented here determined the typical interinstrument variations in dwell volume, extracolumn dispersion, and mixing efficiency as measured by mobile-phase compositional accuracy. Additionally, the dwell volume and extracolumn dispersion were independently and systematically varied to evaluate the resulting impact on resolution for a small-molecule test mixture during gradient elution. To account for these interinstrument variations, dwell volume and wash-out volume method translation and adjustment techniques were evaluated.

A reliable autosampler is one key requirement for unattended operation of a liquid chromatograph.

While gas chromatographers may take their septa for granted, in fact these small and seemingly unremarkable polymer disks keep air out of the carrier-gas stream when used in an inlet and keep samples intact and uncontaminated when used in sample vials. Choosing the wrong septa can compromise method accuracy and repeatability as well as reduce column life in extreme cases. This instalment addresses septa for inlets and sample vials.

In recent years industry has been moving to columns with smaller and smaller inner diameters - moving from 4.6 mm and 3.0 mm i.d. columns to 2.1 mm, 1.0 mm, and even smaller. While small inner diameter columns have some clear advantages, they also bring challenges. Reduction of extracolumn volumes must be given greater consideration by both customers and manufacturers. This article focuses on the sources of band broadening within high performance liquid chromatography (HPLC) columns with an emphasis on eddy dispersion. The physical mechanisms of dispersion are discussed and a review of the current literature as it pertains to small inner diameter columns is presented.

Glycosylation of monoclonal antibody (mAb) therapeutics is widely recognized by the regulators and the industry as a critical quality attribute (CQA). Hence, it is necessary that glycosylation is measured and adequately controlled during production. This instalment reviews the various process parameters and raw material attributes that affect glycosylation, as well as the different analytical tools that are used for characterization, with greater emphasis on the chromatographic methods of analysis. Key recent advancements that have occurred in the past five years are also discussed briefly. While significant progress has been made in the monitoring of glycosylation, its real time control has yet to be demonstrated.

Selecting a gas chromatography (GC) column can be a daunting task. It may seem like there are a never-ending number of phase chemistries, or an inordinate number of column geometry options. However, when choosing a column for a new application (or to improve an existing one) there are some simple rules that can be followed.

The 10th annual International Conference on Packed Column SFC (pcSFC) will be held 5–7 October 2016 at the Melia’ Vienna hotel in Vienna, Austria.

Click the title above to open the LCGC Europe July 2016 regular issue, Vol 29, No 7, in an interactive PDF format.