In this webinar you will learn:
- Learn how to optimize chromatographic systems, taking into consideration variables such as wavelength, polarity, and pH.
- Explore how different components of modern HPLC systems fit into analytical lab methodologies and gain a deeper understanding of its process and function.
- Learn about the relationship between UV-VIS detection in HPLC and spectroscopy, using a new line of internal standards, specially created for measuring very low sample concentrations in dynamic assays.
Chromatography is a simple concept wrapped up in a complex set of processes, chemistries, and instrumentation, which work together to produce accurate results for both detection and separation objectives. The choice of each component, chemical, and column affect the outcome and efficiency of analysis. By breaking down the function of each key area and component, we can gain a deeper understanding of the process, making method development and validation easier to achieve.
Chromatography is a critical tool that analytical scientists use every day, often without much consideration for how all the parts work together. If you work in an analytical lab, you may routinely run the same methods, without regard for process improvement, equipment choices or small adjustments to variables that may greatly enhance accuracy and efficiency.
Chemical selection for the solid and mobile phases is also critical for any successful separation; but sometimes such method development poses a challenge leading to a costly trial and error approach. With a deeper understanding of the nature and chemistries of the different columns and solvents, scientists can approach method development and validation in an organized fashion.
Over time, the portfolio of available liquid chromatography detectors has increased exponentially. New techniques continue to enter the market and inevitably become integrated into the laboratory protocols. With a strong knowledge of the fundamental principles, lab personnel can identify the technologies that will lead to increased productivity, with more confidence.
Whether you are involved in daily sample processing activities, such as sample preparation or the building and running of columns, in charge of purchasing equipment, or tasked with stocking reagents and consumables, a deeper understanding of chromatography fundamentals will enrich your performance within your designated role.
In this webinar, we will take a deeper look into the history, operation, and functionality of liquid chromatography to show analysts how the different components fit together to perform their function. From separation to solvent chemistry, we will break down the processes needed to make liquid chromatography work for your lab.
Meet the presenters.
Patricia Atkins — Senior Applications Scientist at SPEX® CertiPrep
Patricia graduated from Rutgers University in NJ and started her career as a laboratory supervisor for Ciba Specialty Chemicals. In 2008, Patricia joined SPEX CertiPrep within their Certified Reference Material’s division. As a senior application scientist, she spends much of her time researching industry trends and developing new reference materials. She is an experienced speaker and has presented at numerous conferences, including NACRW, NEMC, Pittcon and AOAC. She is also a published author and her work has appeared in various journals and trade publications, including Spectroscopy, LCGC and Cannabis Science and Technology, where she is a columnist for analytical issues in botanicals testing.
Alan Katz, PhD — Director, Global Product Manager - Chemicals
Alan holds a BA in Chemistry from the University at Buffalo and a PhD from Princeton University. His specialties include organic, computational, heterocyclic, and synthetic chemistry, drug discovery, biological assay design, structure-based molecular modeling, and lab automation. He has enjoyed combining his expertise with his interest in computer programming to develop a system to assist in drug discovery. At SPEX CertiPrep, he oversees the manufacturing and quality control of certified chemical reference materials, including inorganic and organic chemical standards, NPD and R&D.
Questions & Answers from the live event
There are now solvents designated as LCMS solvents not just HPLC solvents. Is there really a difference?
A: Sometimes there can be a difference between the HPLC and LCMS grades. These grades are not dictated by any regulation but are more marketing elements for different manufacturers. For most work with LCMS, HPLC solvents are often the most economical solvent. In some cases, scientists need ultra-low baselines such as in ppb work. In those cases, it is best to try the LCMS solvent on the system to see if it provides some better baseline resolution. Should my lab prefilter my mobile phases prior to use or are the filters in the system enough?
A: Most HPLC solvents have been filtered and are safe to add directly to the system. If you need to add buffers; particularly salts or solids, then you should filter the mobile phase after dissolving the solids. How long will a mobile phase last sitting in the reservoir? Are there any significant changes?
A: Mobile phases are exposed to the lab environment, therefore can change significantly in a short period of time from accumulating dust and growth to increasing contamination which can affect baselines. Important analysis should always be conducted with fresh solvent. No solvent should sit more than a few days before being replaced. Is there are mix of solvents which is best for flushing out an HPLC system?
A: Check with the manufacturer recommendations for the column you are using if you are planning on leaving the HPLC in place during a flush. The best mix of solvents is a blend of different polarities and functionalities. Typically, solvents like IPA, MeOH, ACN, cyclohexane, and acetone make a good mix for an RP system. Other solvents such as toluene, hexane, and dichloromethane can be used as a NP flush. Can we somehow get copies of the reference charts and tables in the presentation?
A: The presentation will be available for download on request and the charts and tables will be published on the SPEX website. Registrants will receive more information shortly regarding accessing the reference tables. Is the VapLock® system available for the different HPLC manufacturers or just the major ones?
A: Yes, the VapLock system can be fitted to accommodate all the different HPLC manufacturers. Please contact one of our representatives to help get you started. What are the best ways to transfer a method that has been found online? Can the method be used as written?
A: Methods found online can be a good place to begin method development. Try to find a method with the same type (phase) of column which you are using, and then amend the run time and other run conditions to adapt to your setup. Many instrument manufacturers provide tools to convert HPLC settings between columns. What is the best method for cleaning the column?
A: Columns should be flushed after methods testing for sticky analytes such as sugars, bulky organics, etc. During cleaning, you can flush with a range of high aqueous to high-organic phases for a low flow overnight to clean out a column. Further aggressive cleaning can use a mixture of cleaning solvents like was described in the clean flush questions. Please check with column manufacturers on the limitations or restrictions for your column before use. What is the advantage of SPEX standards over Sigma or Merck standards?
A: SPEX standards have almost seventy years of expertise behind their creation with a team of experts ready to assist the chemist with their analysis. Many of our in-stock standards can ship within 24 hours of order. Scientists can contact our sales and technical team and gain access to assistance with all their analytical needs. Is it safe to use an LCMS grade solvent for an HPLC or UHPLC?
A: Most HPLC and LCMS solvents are good for UHPLC unless you dissolve additives or salts into them, and then they will have to be filtered first. Any tips on how to appropriately choose buffer strength?
A: Start at the lowest concentration of your selected usually 1 to 10 mM or 0.01% and work up until you get the best effect and ionization. Which method is suitable for HPLC purification? Isocratic or gradient? How to select?
A: It depends on what is being separated and purified, and the method used. If separation of many compounds is needed, then gradients can be used. If there is easy separation between impurities, then isocratic may be used and can make it easier to remove excess solvent. Normal chromatography: what is the polar phase and what is the normal phase?
A: Polar stationary phases are used in normal phase chromatography and are molecules such as amino and cyano. Silica columns and other NP columns are used with nonpolar solvents such as alkanes, cycloalkanes, etc. Reverse-phase chromatography uses nonpolar columns like C18, but then use polar solvents such as ACN or MeOH.
Learn more about Chromatography: What is Chromatography
