Hamilton Company
Derek Jensen and Mark Carrier, Hamilton Company
The Hamilton PRP-C18 is a new column designed to provide high-efficiency reversed phase separations over an extended column life in nearly any mobile phase or pH. The rigid stationary phase has excellent mechanical and thermal stability (> 100 °C), does not experience shrinkage or swelling and is completely inert to most conditions commonly encountered in reversed phase chromatography. In this study the pH stability of the PRP-C18 is evaluated. Even after prolonged exposure to concentrated (1 molar) NaOH and H2SO4 there was no measurable deterioration in performance.
Octadecyl silane (ODS) is the prevailing stationary phase in reversed-phase HPLC. Despite a well-celebrated, widespread use in chromatography, traditional ODS columns are not without limitations. Acidic conditions promote hydrolytic stripping of octadecyl functionalization, while alkaline conditions (pH > 7) attack the silica bed, both of which are principle sources for anomalous peak shape and shifting retention times that progressively worsen over the life of the column.
The PRP-C18 reversed phase column has similar mechanical stability (up to 5000 psi) and separation efficiency to that of traditional ODS, but without many of the chemical restrictions. The PRP-C18 stationary phase does not experience stripping or dissolution under the most extreme conditions. This allows for an expanded mobile phase repertoire for use in methods development or aggressive regeneration procedures.
Figure 1: Separation of standard mix after 200 column volumes 1 M NaOH, 1 M H2SO4.
Experimental Conditions
Column: PRP-C18, 4.1 × 50 mm, 5 μm
Instrumentation: Agilent 1100 quaternary pump with UV detector
Standards: acetone, phenol, benzyl alcohol, benzene, toluene, ethylbenzene, propylbenzene, napthalene, pentylbenzene, hexylbenzene
Mobile phase A: 0.2% Phosphoric acid
Mobile phase B: A + 95% CAN
Gradient: 5 to 100% B in 20 min
Flow rate: 2 mL/min
Temperature: Ambient
Injection volume: 2 μL
Detection: UV at 205 nm
Conclusion
Mobile phase pH is a useful tool in methods development, particularly for separation of neutral forms of amines or other organic bases under alkaline conditions. Although a few more recent C18 columns boast stability in alkaline pH, column life is still considerably shorter than if used under more favorable conditions. On the other hand, the PRP-C18 has genuine pH and chemical stability. The stationary phase is devoid of free silanols, does not strip, bleed, or dissolve at any pH, and therefore can be expected to perform reliably and reproducibly throughout the extended life of the column, regardless of mobile phase conditions.
Hamilton Company
4970 Energy Way, Reno, NV 89502
tel. (800) 648-5950; fax (775) 858-3026
Website: www.hamiltoncompany.com
Using LC-MS/MS Analysis for PFAS Detection in Biological Samples and Serum
November 1st 2024Per- and polyfluoroalkyl substances (PFAS) are exceptionally versatile chemicals and have seemingly endless applications. Due to their persistent nature; prevalence in blood, food and environment; and emerging toxicity, there is a significant concern for their presence in the human body. Analysis is especially complicated due to background contamination in laboratory supplies and system. This paper will discuss sample preparation and LC-MS/MS analysis of an expanded panel of PFAS compounds in serum.
Robust Oligonucleotide Molecular Weight Confirmation via LC/MS
October 31st 2024Oligonucleotides—therapeutic agents for difficult-to-treat diseases—rely on precise molecular weight confirmation to ensure quality during development. Experts from Agilent Technologies explore the challenges of liquid chromatography-mass spectrometry (LC/MS) analysis in oligonucleotide synthesis, as well as strategies for accurate deconvolution, method development, and optimal equipment selection.
Development of a Fully Scalable High Efficiency 5um Solid-Core Particle to Support HPLC Workflows
October 30th 2024In this work the development of two key attributes of the 5um CORTECS Columns is examined. First column efficiency is compared across CORTECS and other solid-core 5 μm columns. Next scalability from sub-2 μm to 5 μm particles is examined between the CORTECS Column lines and competitive column lines. It was found that CORTECS columns have higher efficiency compared to other solid-core columns and that CORTECS particles are fully scalable.