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    • HPLC Method Development and Transfer Utilizing Fused-Core® Particle Technologies HPLC Method Development and Transfer Utilizing Fused-Core® Particle Technologies David S. Bell, Ph.D. Recorded: Dec 5 2012 4:00 pm UTC 31 mins
    • Superficially porous (Fused-Core, Core-shell) particle technologies have gained acceptance in general high-performance liquid chromatography (HPLC) and ultra-high pressure liquid chromatography (UHPLC) practice over the past several years due to improved efficiency relative to comparably sized fully porous particles. The Fused-Core option has also been shown to be a superior approach toward improving column efficiency as compared to smaller porous particle (sub-2 µm, UHPLC) technologies owing to the lessened backpressure penalties that are paid for the efficiency gains. Ascentis® Express HPLC columns were initially introduced that employed Fused-Core particles with an overall 2.7 µm diameter. These columns provide efficiencies equal to sub-2 µm particles at much lower backpressures as well as superior efficiencies when compared to fully porous 3 µm phases. There are still instances, however, where the backpressure generated by a 2.7 µm particle may preclude their use and there are also situations based on available equipment or regulatory guidance where larger particles are preferred. For these reasons, a new generation of 5 µm Fused-Core columns has been developed.
      In this seminar we will provide an introduction to the new line of Ascentis Express 5 µm HPLC columns and explore several scenarios practicing analytical chemists might encounter:

      • Desire to develop method on the more efficient 2.7 µm column, but need to be able to transfer to different location or lab
      1. Can I easily transfer methods from 2.7 µm to 5 µm?
      2. Can I easily transfer methods from 5 µm to 2.7 µm?

      • Wish to transfer methods based on fully porous column technologies to Fused-Core
      1. Can this be done easily?
      2. What gains should I expect? What can be done to optimize the gains?
      3. Can I transfer both 3 µm and 5 µm fully porous particle method to Fused-Core?

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    • New! Introducing Ascentis Express 5 um HPLC Columns with Fused-Core Technology New! Introducing Ascentis Express 5 um HPLC Columns with Fused-Core Technology R. A Henry Recorded: Sep 21 2012 3:00 pm UTC 53 mins
    • Core-type particles are competing strongly with small porous particles to improve the speed and resolution of HPLC and UHPLC experiments. The pioneering Ascentis Express column with Fused-Core® 2.7µm particles has exploded in popularity because it operates more ruggedly at much lower pressure than current sub-2µm porous particles, yet delivers the same ultra-high performance. This unique performance has been largely attributed to very narrow particle size distribution. Fused-Core® design advantages have also become popular for LC-MS because Ascentis Express columns surpass performance of columns with 3µm porous particles and operate ruggedly at higher velocities and similar pressures.

      With new, narrow-distribution 5µm Fused-Core® particles, the same design advantages can now be realized over traditional 5µm and 3µm porous particles that remain very popular for HPLC columns. An Ascentis Express 5µm column brings 3µm performance and extreme ruggedness at 5µm pressures to your laboratory. Like Ascentis Express 2.7µm particles, the 5µm particles show flatter van Deemter plots than same-size porous particles and allow separation speed to be maximized with minimal loss of resolution. Extremely high plates per pressure are observed. The core-type 5µm design should replace porous 5µm columns in routine HPLC applications with traditional instruments, and should also compete with porous 3µm columns in many LC-MS applications. Performance will be compared to 5µm and 3µm particle columns, and examples of method transfer will be shown. Ascentis Express 5µm will be available in the same phase modifications as the original Ascentis Express 2.7µm ultra-high performance column. Highly stable columns are available in various IDs and lengths up to 25cm.

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    • HPLC of Peptides:  Speed and Resolution with Fused-Core Columns HPLC of Peptides: Speed and Resolution with Fused-Core Columns Hillel Brandes, Ph.D. Recorded: Jan 25 2011 7:45 pm UTC 24 mins
    • HPLC columns featuring 2.7 µm Fused-Core (superficially porous) particles with 90 Å pores demonstrate very fast separations of small molecules because of high efficiency and a flat van Deemter plot. These particles rival the efficiency of sub-2 µm totally porous UHPLC particles, but show only about one-half the backpressure. Fused-Core 2.7 µm particles with wider (160 Å) pores have been optimized for the rapid separation of peptides and small proteins. The higher efficiency and lower pressure drop of Fused-Core particles allows preparation of longer columns with very large numbers of theoretical plates. This dramatically increases the peak capacity of the column system, which facilitates qualitative and quantitative HPLC and LC-MS analysis.

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    • Using Silica HPLC Columns in HILIC Mode:  A Practical Guide Using Silica HPLC Columns in HILIC Mode: A Practical Guide Merlin K. L. Bicking, Ph.D. Recorded: Apr 27 2011 3:00 pm UTC 44 mins
    • Silica is a popular option for use in “HILIC” mode, but developing new methods can be difficult because the retention patterns are so different from C18. This presentation will provide some practical guidelines for using silica in a HILIC mode – with buffer/acetonitrile mobile phases. Some compound classes are retained under these conditions, and some are not. We will discuss which compounds are good candidates for this system, and summarize how to get started with your method development experiments.

      You will learn:
      •What compounds can be separated on silica in HILIC mode, and what compounds cannot be separated.
      •Starting points for separations, and suggestions for how to adjust conditions for your separation problem.
      •Advantages and limitations of silica in this mode.

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    • A Practical 2D-LC System Developed for Complex Pharmaceuticals A Practical 2D-LC System Developed for Complex Pharmaceuticals Lianjia Ma, Ph.D.* and Anthony Alexander, Ph.D. Recorded: Mar 23 2011 3:00 pm UTC 31 mins
    • A comprehensive two-dimensional liquid chromatograph (LC × LC) was constructed from commercially available conventional HPLC equipments. This system utilizes two independently configurable 2nd dimension binary pumping systems to deliver independent flow rates, gradient profiles and mobile phase compositions to dual Fused-Core secondary columns. Very fast gradient separations (30 seconds total cycle time) were achieved at ambient temperature without excessive backpressure and without compromising optimal 1st dimension sampling rates by using superficially porous stationary phases. A practical approach to optimize the various inter-related instrumental parameters will also be presented.

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    • Ultra-Fast LC/MS/MS in Bioanalysis Using Fused-Core Columns Ultra-Fast LC/MS/MS in Bioanalysis Using Fused-Core Columns Ethan R. Badman, Ph.D. Recorded: Feb 16 2011 4:00 pm UTC 39 mins
    • With recent advances in HPLC columns and LC/MS hardware, it is possible to increase the throughput of bioanalytical assays without sacrificing quality. By using fused-core columns, it is possible to decrease the run time from ~4 min to 1 min or less, without the use of UHPLC hardware. High quality methods at high flow rates (1-3 mL/min) using non-ballistic gradients as short as 20 seconds were developed that provide comparable or better performance for accuracy, precision, sensitivity, and specificity than traditional slower LC methods. Data will be presented that show that these assays meet regulatory requirements for bioanalytical work. Limitations in the ultimate speed possible for these assays will also be discussed.

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    • Rapid LC/MS/MS-Based Methods For Opiates & Benzodiazepines Rapid LC/MS/MS-Based Methods For Opiates & Benzodiazepines Dwight R. Stoll, Ph.D., Assistant Professor - Gustavus Adolphus College Recorded: May 18 2011 3:00 pm UTC 26 mins
    • Increasing case loads and budget and staffing cuts in forensic laboratories continue to motivate the development of higher throughput methods, particularly for confirmatory analysis of regulated intoxicants. In this work, we have focused on the development of rapid LC/MS/MS methods for the determination of nine opiates including two glucuronide metabolites, and 16 benzodiazepines, including two amino- metabolites. Here we aim to analyze both the parent compounds and important polar metabolites in a single analysis. To this end we have compared the retention of the target compounds on two different reversed-phase HPLC stationary phases: a conventional C18 type phase, and a perfluorinated phenyl (PFP or F5) phase built upon the increasingly popular Fused-Core particle morphology. We see that the F5 phase not only generally exhibits higher retention than the C18 type phase, but also exhibits very different selectivity such that the nine opiates can be nearly completely resolved in under four minutes. We find that the mixture of 16 benzodiazepines cannot be completely resolved in a reasonable (i.e., less than 20 min.) time, however we have developed a separation with no more than three overlapping peaks in an analysis time of five minutes.

      Full Title
      DEVELOPMENT OF RAPID LC/MS/MS-BASED METHODS FOR CONFIRMATORY ANALYSIS OF OPIATES AND BENZODIAZEPINES

      Authors
      SPENCER BONNERUP, D. CHRISTOPHER HARMES, TOMAS LISKUTIN, JONNA BERRY, AND DWIGHT R. STOLL

      Department of Chemistry
      Gustavus Adolphus College
      800 West College Avenue
      St. Peter, MN 56082

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    • Retention Mechanisms in HILIC Chromatography:  Robust Method Development Retention Mechanisms in HILIC Chromatography: Robust Method Development David S. Bell, Ph.D. Recorded: Jun 28 2012 6:00 pm UTC 55 mins
    • Hydrophilic interaction liquid chromatography (HILIC), especially in conjunction with mass spectrometry (MS), has become a powerful tool for the analysis of a wide variety of challenging analytes. Applications of the technique have increased dramatically over the past decade, especially for the analysis of polar analytes where reversed-phase chromatography suffers. HILIC conditions employ a high percentage of acetonitrile which enables facilitated solvent evaporation in LC/MS sources and thus often an increase in analyte response when compared to more aqueous based systems. The increased retention of polar analytes afforded by HILIC provides improved selectivity and decreases the impact of endogenous species, often leading to improved qualitative and quantitative analyses.

      Although HILIC has proven useful, it has also been thwarted with complications including difficulties in method development and method robustness.

      In this presentation, studies investigating the underlying retention mechanisms dominant in HILIC chromatography are presented and discussed. Along with reversed-partitioning HILIC is well known to exhibit, ion-exchange and the interplay of the dominant mechanisms are unveiled and used to develop a model of overall retention and selectivity. Interactions that operate using different stationary phase chemistries and conditions are presented. The impact of analyte polarity and charge as well as the variations caused by high percentages of organic on these physiochemical parameters are highlighted. Throughout the discussion, examples of use and misuse of HILIC are employed to illustrate these important concepts to build a solid fundamental foundation for efficient and effective use of this powerful technique.

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    • Increasing throughput and sensitivity of LC-MS assays Increasing throughput and sensitivity of LC-MS assays AB Sciex: Jason Causon, Senior Applications Specialist & Remco van Soest, Product Manager Recorded: Jul 28 2015 2:00 pm UTC 63 mins
    • Presentation 1: Jason Causon, Senior Applications Specialist, Sciex

      Development of bioanalytical LC-MS methods for the detection of therapeutic peptides and the proteolytic fragments of larger proteins and antibodies is growing along with the increasing number of peptide and protein drugs entering clinical research and development. Development of these methods present challenges that are different to small molecule method development because of the multiple charge states possible for a given peptide, the abundance of product ion possibilities and the large number of proteolytic fragments possible from a protein digest. In addition to optimizing for sensitivity, on-column HPLC method development is time consuming for the same reasons. In this webinar we present an automated tuning and optimization workflow with on-column validation using DiscoveryQuantTM 3.0 software with the new ChromaTune feature to overcome some of these challenges and make the optimization of peptide MRM methods easier and less time consuming.

      Presentation 2: Remco van Soest, Product Manager, Sciex

      Electrospray Ionization efficiency increases at lower flow rates. In this Webinar we will discuss how microLC can help you improve sensitivity in sample limited applications in bio-analysis by LC-MS. In order to analyse the same volume of sample as is typically injected in a conventional LC-MS system, up to 50 µL, we have developed a microLC system that allows for fast on-line pre-concentration on a short trap column. Because of the higher flow rates that can be used for loading the sample on the trap-column, sample throughput is not affected by using microLC flow rates for the LC-MS analysis.

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    • Colorectal Cancer and Immunohistochemistry Colorectal Cancer and Immunohistochemistry Jeff Gordon, Cell Marque- Rocklin, CA Upcoming: Mar 30 2016 3:00 pm UTC 75 mins
    • Colorectal cancer is the third most commonly diagnosed cancer in men and women, and is the second leading over all cause of cancer deaths in the United States. Each year, the CDC reports that approximately 135,000 people are diagnosed with colorectal cancer, and over 50,000 succumb each year to the illness. The National Cancer Institute cites a decline in the mortality rate of colorectal cancer due to advanced screening and early diagnosis. Immunohistochemistry has served as the catalyst for these advancements in colorectal cancer diagnosis. This presentation covers many of the basic science, facts, and statistics of colorectal cancer, as well as the utility of immunohistochemical testing with markers such as cadherin¬17, CDX¬2, SATB2, and the mismatch repair proteins in the accurate diagnosis and survival rates of colorectal cancer.

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    • The Analysis Workflow of the New EPA Method 325 for Fenceline Monitoring The Analysis Workflow of the New EPA Method 325 for Fenceline Monitoring Jamie Brown - Supelco R&D Scientist , Lee Marotta - PerkinElmer Senior Field Application Scientist Upcoming: Feb 17 2016 6:00 pm UTC 75 mins
    • The new EPA METHOD 325 for sampling Volatile Organic Compounds from Fugitive and Area Sources was promulgated in September of 2015. The petroleum refineries have 2 years to comply with the new ruling. Benzene emissions are the focus of the new ruling, but other VOC’s can be monitored using the same sampling tube. The air samples are collected using thermal desorption tubes that are deployed along fenceline of the property and remain deployed for 14 days. The samples are collected passively, without the using of an air sampling pump. After sampling the diffusive endcap is replaced with a metal storage cap and sent to a laboratory where they’re analyzed by thermal desorption gas chromatography. This is the first of a two series presentation that focuses on the analysis side of the workflow. It provides users the steps required to comply with the new EPA fence line regulations and what is needed to optimize the method for high sample throughput. Refineries and testing laboratories will benefit by gaining valuable information on the optimum techniques to perform this method and what it takes to comply. An introduction to the theory and operation of thermal desorption will also be included.

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