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Efficiency within Quality Control, UHPLC conquering PHARMA

An introduction on UHPLC technology

Like its little sister High Performance Liquid Chromatography (HPLC), Ultra High Performance Liquid Chromatography (UHPLC) is an analytical technique to separate, identify and quantify components in mixtures. Each chromatography system consists of a mobile and a stationary phase. HPLC and UHPLC use liquids as mobile phase and small solid particles as stationary phase.  Compounds are dissolved in the mobile phase and passed through columns, packed with the stationary phase. Separation is achieved by interactions of compounds, dissolved in the mobile phase, with particles of the stationary phase. Due to its robustness, ease of use and changeable selectivity, HPLC is a widely used analysis method. However, compared to gas chromatography (GC), HPLC shows more limitations in efficiency.

The easiest way to increase the efficiency of liquid chromatography, is to decrease the particle size of the packing material. Using smaller particles enables more efficient chromatographic separation at higher flow rates. The practical disadvantage of using smaller sized packing material is the immense increase of backpressure. To overcome this problem, UHPLC technology using ultrahigh pressure pumps was introduced by Waters Corporation® in 2004. While HPLC systems use packing materials with particles of 3 to 10 µm diameter and are limited to back-pressures of 400 bar, UHPLC systems are characterized by the small particle size of less than 2 µm diameter and are able to handle back-pressures of up to 1000 bar.

Theoretical Background

Taking a close look at the Van Deemter equation will help to understand, why particle size is so important. Efficiency of a column is measured in theoretical plates. The measurement of theoretical plates, takes into account the dispersion of a peak, also known as band broadening. It can be estimated by the retention time of each component and its standard deviation. A high plate count accounts for narrow and high peaks, which is the desired shape.

Plate count can be normalized with the length of the column to give the height equivalent theoretical plate. The empirical van Deemter equation describes efficiency as function of linear velocity.

Efficiency is best at the lowest height equivalent of a theoretical plate.

A-term (eddy diffusion) describes the diffusion occurring because of different flow channels in the packing material. It relates to the particle size, quality and uniformity of the packing material. Reducing the particle size decreases the A-term in a linear manner. B-term (longitudinal diffusion) is a dispersion occurring because of longitudinal diffusion of the molecules traveling through the column. The B-term is lowered by increasing the linear velocity.

C-term (mass transfer) describes the interactions of the molecules with the internal surface of the stationary phase and their distance of diffusion into and out of the pores of the packing material. It is related to the linear velocity and the square of the particle size. Reducing the particle size decreases the C-term in an exponential manner.

Photo by sindlera/iStock / Getty Images
Photo by sindlera/iStock / Getty Images

Benefits of using UHPLC technology

UHPLC technology provides high analysis speed, better chromatographic resolution, performs more sensitive analysis, consumes less time, reduces solvent consumption. Although these facts are proven, the implementation if this technology shows only slow progress within the quality control of pharmaceutical.

We have developed a strategy how methods can be transformed from HPLC to UHPLC. Benefits including cost savings can be seen in short time.

We provide Business case, Regulatory Strategy and Registration Documents, Method Validation Documents according to ICH, Testing Procedure and Project Plan.

The entire process takes approximately 2 Month. If you are interested, please don’t hesitate to contact us.

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