Diane M. Diehl

Effects of extra-column band spreading, liquid chromatography system operating pressure, and column temperature on the performance of sub-2-μm porous particles

The effects of extra-column band spreading, LC system operating pressure, and separation temperature were investigated for sub-2-microm particle columns using both a conventional HPLC system as well as a UPLC system. The contributions of both volume- and time-based extra-column effects were analyzed in detail. In addition, the performance difference between columns containing 2.5 and 1.7-microm particles (same stationary phase) was studied. The performance of these columns was compared using a conventional HPLC system and a low dead volume UPLC system capable of routine operation up to 1000 bar. The system contribution to band spreading and the pressure limitations of the conventional HPLC system were found to be the main difficulties that prevented acceptable performance of the sub-2-microm particle columns. Finally, an increase in operating temperature needs to be accompanied by an increase in flow rate to prevent a loss of separation performance. Thus, at a fixed column length, an increase in temperature is not a substitute for the need for very high operating pressures.

The application of novel 1.7 μm ethylene bridged hybrid particles for hydrophilic interaction chromatography

An un-derivatized 1.7 microm ethylene bridged hybrid (BEH) particle was evaluated for its utility in retaining polar species in hydrophilic interaction chromatography (HILIC), and was compared to a 3 microm un-derivatized silica material. Retentivity as a function of mobile phase pH, polar modifier and ACN content was examined. Also, the efficiency of the two particle substrates was compared by plotting HETP vs. linear velocity. Improved chemical resistance of the un-derivatized BEH particle was compared to un-derivatized silica at pH 5, demonstrating no performance deterioration over the course of 2000 injections for the BEH particle, while the silica particle deteriorated rapidly after 800 injections. Lastly, ESI-MS sensitivity as a function of particle size and separation mode was demonstrated. A 2.2 to 4.7-times higher ESI-MS response was observed on the 1.7 microm particle compared to the 3 microm particle, whereas a 5.6 to 8.8-times higher ESI-MS response was observed using HILIC as when compared to traditional RP chromatography.

Influence of stationary phase chemistry and mobile-phase composition on retention, selectivity, and MS response in hydrophilic interaction chromatography.

A comprehensive retention and selectivity characterization of several hydrophilic interaction chromatography (HILIC) stationary phases was performed with 28 test probes in order to study the influence of particle type, surface chemistry, and mobile-phase pH on chromatographic retention, selectivity, and MS response. Selectivity differences were compared for columns operated at both low and high pH, while ESI-MS was used to study the effects of mobile-phase pH on signal response. Additionally, acetone was explored as a potential alternative to ACN as the weak HILIC solvent. Moderate differences in selectivity were observed on the same column operated at different pH, mostly due to acidic compounds. In addition, the MS response increased when a high pH mobile phase was used, particularly for analytes that were ionized with negative ESI-MS. Even larger selectivity differences were observed for different stationary phases evaluated with the same mobile phase. Acetone was not a suitable replacement for ACN in routine HILIC separations due to differences in selectivity and MS response. Finally, the data from this study were used to establish guidelines for rapid HILIC method development of polar compounds, which is demonstrated with a mixture of histidine dipeptides and organophosphonate nerve agent metabolites.

Simultaneous analysis of morphine‐related compounds in plasma using mixed‐mode solid phase extraction and UltraPerformance liquid chromatography–mass spectrometry

A bioanalytical method using mixed-mode solid phase extraction and UltraPerformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the analysis of morphine, morphine-3beta-glucuronide, morphine-6beta-glucuronide, 6-acetylmorphine, morphine N-oxide, and 10-hydroxymorphine in porcine plasma. All six compounds, along with four deuterated internal standards, were simultaneously extracted using mixed-mode strong cation exchange SPE in a 96-well microElution plate format. Due to analyte instability, a neutralizing solvent was used during the elution step to minimize degradation of 6-acetylmorphine. Separation was subsequently performed in 8 minutes on a 2.1 x 100 mm, 1.8 microm C(18 )column designed for retention of extremely polar compounds using a formic acid and methanol gradient. Analytes were detected by positive electrospray ionization in multiple reaction monitoring mode using a fast-scanning triple quadrupole mass spectrometer. Recovery was 73-123% depending on the analyte, and inter-day variability was less than 6%. Linearity was determined in porcine plasma by spiking the analytes prior to SPE. Correlation coefficients were >or= 0.998, and% deviation from the actual concentrations was less than 15%. The lower limit of quantitation (LLOQ) for all compounds was between 0.1 and 0.25 ng/mL.

4 – HPLC Columns for Pharmaceutical Analysis

This chapter deals with the properties of high-pressure liquid chromatography columns. It is divided into two sections: column physics and column chemistry. In the section on column physics, we discuss the properties that influence column performance, such as particle size, column length and column diameter, together with the effect of instrumentation on the quality of a separation. In the section on column chemistry, we examine in depth the surfaces of modern packings, as well as the newer developments such as zirconia-based packings, hybrid packings or monoliths. We have also included a short section on hydrophilic interaction chromatography, a technique for the analysis of polar compounds that is drawing interest again in the pharmaceutical industry. Finally, we review what is currently understood about the selectivity of reversed-phase columns.

Chapter 2 Sampling and sample preparation

Publisher Summary This chapter presents various methods for collecting and preparing samples for analysis, from classical to more modern techniques. It provides a general overview, outlining some of the theory and practice of each technique, and focuses mainly on the analysis of organic compounds of interest (analytes) in a variety of matrices, such as environment, food, and pharmaceuticals. Sample preparation is frequently necessary for most samples and remains one of the major time-consuming steps in most analyses. A variety of methods for the sample collection and preparation of gases, solids, and liquids are illustrated in this chapter. Each technique discussed involves separating the analytes of interest from the components of the matrix. Selection of the best sample preparation technique involves understanding the physical and chemical properties of both the analytes and the matrix. These organic analytes can be subclassified into volatile, semivolatile, or nonvolatile. The matrix can be gas (or volatile samples), solid, or liquid. Both the anticipated concentration of the analyte and the type of sample dictate the instrumentation that can be used, as well as the sample preparation technique required.

UPLC-Methoden entwickeln und optimieren

Die hier vorgestellte Methode liefert schnell einen Uberblick uber die Selektivitaten von stationaren und mobilen Phasen. Die besten Ansatze werden dann uber Gradientenverlauf, mobile Phase und Temperatur weiter optimiert. So dauert es weniger als acht Stunden, bis eine Ubersicht uber die Selektivitatsoptionen vorliegt.

22 – NEW Developments in HPLC

This chapter is intended to serve as a general overview of new and emerging HPLC technologies and is divided into four sections: simplifying sample preparation, new column technologies, improvements in detectors, and improvements in HPLC throughput.