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Influence of viscosity on chromatographic injections using different (U)HPLC systems

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Influence of viscosity on chromatographic injections using different (U)HPLC systems

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Samenvatting

During the performance of dissolution analysis, drug product is dissolved in a non viscous medium. Samples are withdrawn on defined time points in order to create a release profile. Certain drug products however, contain high molecular weight polymers which causes the medium to become more viscous over time, resulting that the samples which are withdrawn become also more viscous.
To obtain a better insight, this study is conducted to investigate if, and how, the difference in viscosity between samples has an influence on their chromatographic results.
Since different (U)HPLC systems are available within the ARG laboratory, the investigation is applied on all systems, namely HPLC, UPLC and UPLC H-Class. These systems use different injection techniques and may therefore produce different results when dealing with increasing sample viscosities. HPLC and UPLC H-Class use a flow through needle injector, UPLC uses a loop injection technique (which is usable in three different injection modes: Full loop (FL), Partial loop uses pressure assist (PLPA) and Partial loop with needle overfill (PLNO)).
Five different solvents are prepared within the viscosity range of 1 to 100 mPa.s. In order to study the influence of injection draw speed, the samples are injected using three different syringe draw speeds (the recommended draw speed of water is used as the highest speed). Furthermore, to study the influence of injection volumes, the injection volume of each sample concentration is adjusted to obtain comparable peak heights for each injection, i.e. more diluted samples required a higher injection volume. The key parameter which is studied is peak area since dissolution profiles are composed from the sample assay values which are calculated directly from peak area. Furthermore, peak width, peak tailing and retention times are studied parameters.

As for peak area, HPLC shows by far the most satisfying results and is therefore the recommended system when dealing with variation in viscosity over multiple samples. Of course, this technique is not always favoured, especially when a large amount of samples is available and shorter analysis times with faster UHPLC are preferred. When UPLC or UPLC H-Class systems are then used, it is recommended to set the injection draw speed as low as possible in order to obtain areas which are not affected due to viscosity increase; however, it must be kept in mind that run times will be longer. Therefore, to obtain a benefit over HPLC, a good balance between draw speed and run time must be found. It is recommend not to use the UPLC PLPA injection mode when dealing with variable sample viscosity as even at a low viscosity changes, the obtained results showed a significant decrease in peak area, also at slower injection draw speeds.

The further a compound is eluted from the solvent peak, the less it is influenced by its viscosity. It is therefore recommended to obtain retention times for compounds which have a capacity factor which is not too low, i.e. larger than 2. Of course, peak width and run times will be larger when peaks elute at a later time point, so also for these factors a good balance has to be found.

The study shows that solvent mismatch between sample and mobile phase increases as injection volumes are higher. For this reason, it is recommended to keep sample volumes as low as possible. On the contrary, when dealing with loop injections, it is recommended to use a large loop volume since migration is faster due to larger loop diameters. Since slow migration directly affects peak area, which is the most critical parameter, the latter recommendation is more important than the first.

Toon meer
OrganisatieAvans Hogeschool
AfdelingATGM Academie voor de technologie van Gezondheid en Milieu
PartnersSynthon B.V. Nijmegen; Analytical Research Group (ARG)
TypeBachelor
TaalEngels

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