In this section you'll find a selection of valuable learning materials relating to the use and application of HPLC and other liquid chromatography techniques, including technical articles, applications, webinars and microsites.
Developing, Testing and Troubleshooting Chromatography Method Transfer
Presenter: Tom Wheat (Waters Corp., USA)
Advances in HPLC of Proteins and Peptides
Presenters: Hillel Brandes (Principal R&D Chemist, Large Molecule Bioanalytical Group, MilliporeSigma, USA)
Accelerating Large Molecule LC-MS Method Development
Presenters: Ian Moore (Global Technical Marketing Manager, Pharma Applications, SCIEX)
Reversed Phase Method Development: Retention and Selectivity of Biphenyl Stationary Phase Chemistry
Presenter: Dan Shollenberger (Supelco, a division of Millipore Sigma)
In the previous instalment of HPLC Solutions (HPLC Solutions #134) we took a brief look at why we might decide to use an internal standard and how a calibration plot is constructed and used in HPLC analysis. This time, we’ll consider some properties that are necessary when choosing a compound to use as an internal standard. These are listed in the figure.
In the next few instalments of HPLC Solutions , we’ll look at several aspects of the use of internal standards (IS) in HPLC and LC-MS applications. The topic of this discussion is how an internal standard is used in quantitative analysis.
This is the fourth instalment of HPLC Solutions related to resources you should have at your fingertips when you are working in an HPLC laboratory (see also #130, 131,132). In the last discussion (#132) we looked at online resources from the International Conference on Harmonization (ICH) and the United States Food and Drug Administration (FDA) regarding validation of HPLC methods. This time I’d like to mention a few books that I find indispensable in my day-to-day work as a chromatographer. The first three form my trifecta of HPLC books that I refer to most often.
The separation power of HPLC combined with the very high sensitivity and selectivity of MS detection is widely used to develop analytical methods that are required to achieve ultra-low detection and quantification limits. Examples of such demanding HPLC-MS applications comprise clinical and preclinical pharmacokinetic studies, proteomic studies and analytical methods for food safety and environmental monitoring.
The sample solvent, also called dissolution solvent or injection solvent, is the solvent used to prepare the solution in which the compound to be analysed by HPLC is dissolved. The choice of the sample solvent is fundamental for a successful separation, since this determines the initial retention of the compound on the stationary phase. Sample solvents with stronger elution strength than the initial conditions of the mobile phase cause a less effective retention with undesirable consequences, such as peak distortion, peak broadening and earlier elution.
It is matter of fact that the samples most chromatographers have to face are rather complex for sample number and compound variety. In these situations, gradient elution is largely preferred for the separation of analytes with different retention factors. In this way it is possible to narrow the peak width of the later eluted compounds and shorten the run time. In this article, the importance of column equilibration is considered for optimum efficiency.
Mostly in HILIC conditions, by increasing column temperature a decrease in the retention time is observed.
Presented is a rapid, three step SPE procedure for the identification and quantification of fentanyl and its major urinary metabolite norfentanyl, in addition to four “designer” compounds: U-47700, W-18, W-15 and furanyl fentanyl. Due to the rapid use and abuse of fentanyl in medical and recreational settings, respectively, it is important to develop a method that would accurately extract this Schedule II drug from any other novel compounds that may be present.
Efficient and fast separation for low molar mass polymers and proteins can be achieved using a combination of micro columns packed with smaller particles and optimized detectors. Although traditional analytical RI detectors, such as the SECcurity RI, profit from the higher resolution of micro columns, less peak broadening and higher resolution are the benefits that dedicated micro cell detection units offer.
Analysis of Glufosinate, Glyphosate, and AMPA in Drinking Water Using a Triple Quadrupole LC/MS/MS System
Glufosinate is a popular amino acid-based herbicide and glyphosate a popular foliage treatment herbicide. Glyphosate metabolizes in soil or water to form aminomethylphosphonic acid (AMPA). In this example, Appendix Method 22 is used to analyze glufosinate, glyphosate, and AMPA. In addition, by using the LCMS-8050, samples can be analyzed directly without the pretreatment process of concentrating samples by solid phase extraction.
Separation Science, in collaboration with Phenomenex, has developed a series of HPLC Tutorials covering the following topics:
- Understanding HPLC Particle Technology
- A Survey of Reversed Phase-HPLC Stationary Phases - from Selectivity to Applicability
- Basic HPLC Troubleshooting and Column Care
Presenters Jeff Layne and Scott Krepich provide expert advice on these key issues for LC users.