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?
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.
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.Read more >
The Fused-Core particle technology behind Ascentis Express allows for twice the speed of traditional columns at half of the backpressure of sub 2 micron columns. Ascentis Express columns help you to turn any HPLC into a high powered workhorse like an UHPLC instrumentRead more >
10 minute introduction to the Ascentis Express family with an emphasis on the benefits of the new ES-Cyano phase. The presentation will then feature a 20 minute advanced level presentation on the molecular interactions contributing to alternative retention and selectivity using Cyano stationary phasesRead more >
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.
Kromasil Eternity is an innovative platform that allows users to work over an extended pH range. This presentation will highlight some of the benefits of developing methods with no pH limitations.Read more >
"With a BSc in Biochemistry and Molecular Biology from the University of Western Australia, I started my postgraduate career in the world of cell signaling and transgenic research at the Laboratories of Cancer Medicine at Royal Perth Hospital. After a number of years I relocated to the UK and started working in clinical diagnostic laboratories where I had a change of direction in my career. Moving into the development of HPLC and LC-MS/MS methods for clinical diagnostic analysis, I plied my trade at Guys Hospital and King’s College Hospital’s in London for 6 years before taking my current post at Addenbrooke’s hospital in Cambridge in late 2009. My work interests include new born screening, biogenic amines, steroids and vitamin analysis in an array of human sample types."Read more >
Workup and purification can be a complicated and time consuming process. The webinar will offer some available options for streamlining this part of your workflow, and provide some guidance and strategic advice depending on budget, equipment and experience. Workup of aqueous fractions will also be discussed.
With the need for results as quickly as possible we will discuss how to perform reaction monitoring as quickly as possible using standard HPLC systems. We will show, with an example, how you may be able to obtain your results in 1/3 of your current time if you are running isocratically, or with generic gradients, with 5um or 3um C18 HPLC columns.
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.Read more >
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.Read more >
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.
DEVELOPMENT OF RAPID LC/MS/MS-BASED METHODS FOR CONFIRMATORY ANALYSIS OF OPIATES AND BENZODIAZEPINES
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
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.
CRISPR Cas9 nucleases have revolutionized the field of gene editing and high-throughput lentiviral screens continue to hold ever-increasing promise for both basic research and development of future therapies to benefit human health. Even with such powerful technologies at hand, researchers new to the field may find screening of multiple targets to be challenging and time-consuming. This webinar discusses the Evotec partnership with Merck KGaA,Darmstadt,Germany and the screening services for drug discovery.Read more >
H&E is the most frequently used stain in histology and is the basis for diagnostics and further selected methods. Thus it is important to have brilliant staining. We will show which minor factors can give a negative effect or spoil the result completely. Tips will be given on improving the sensitivity of the stain. For PAS, we will give advice on how to avoid common errors and always get colourful results.
We will discuss:
•How to prevent "critical situations"
Webinar recording for sample preparation on Food Analysis.Read more >
The CRISPR/Cas genome editing system has revolutionized almost every aspect of the life science industry. Until recently, the most used formats for this technology have been plasmids, mRNA, or lentivirus. Each reagent has been successful in its own right, however, each approach has limitations. SygRNATM, the two-part synthetic crRNA and tracrRNA, increases the pace of research, decreases costs, and can be used with Cas9 protein, Cas9 mRNA and Cas9 expressing cells/models.
This webinar discusses the development of the SygRNATM system, protocol optimization, and proposes workflows that enable scientists to quickly incorporate CRISPR technologies into their research.
Part 2 of a 3-part presentation series about food analysis focused on sample preparation and including information about Reference MaterialRead more >
Part 3 of a 3-part presentation series about food analysis focused on sample preparation and including information about Reference MaterialRead more >
Childhood cancer and pediatric cancer are general terms used to describe a wide range of neoplasms found in children and teenagers. Occurring in approximately 1 in 300 people under the age of 20, compared to 1 in 6 adults, pediatric cancers are more rare than adult cancers. Because less is known about pediatric cancers, diagnosis can be quite challenging for pathologists.
This presentation covers the basic science, as well as facts and statistics about pediatric cancer. We will discuss how the utility of immunohistochemical testing along with the application of novel antibodies can contribute to accurate diagnosis and survival rates of pediatric cancer patients.
The ImageStream® and FlowSight® are multispectral imaging flow cytometers that generates high resolution images of cells at a rate of 1000’s of cells per second. This allows for the rapid acquisition of tens of thousands of images per sample. Using the IDEAS® image analysis software, the system calculates features based not only on fluorescence intensity but the morphology of that fluorescence as well. This novel approach is able to seamlessly combine the quantitative power of flow cytometry with the high content information associated with microscopy. The system can collect data on a wide range of applications including nuclear localization during a signal transduction cascade, measuring colocalization of two probes, or quantify features on the phagocytosed particles in macrophages.Read more >
In research areas such as academic, biofuel, food and pharmaceutical industries the determination of algal viability, lipid content, and cell concentration is important in the selection, monitoring, and maintenance of algal cultures. Flow cytometry has been shown to be an ideal method to assess health and lipid content of cultures but has been challenging to adopt due to high complexity and cost of traditional technologies. In this webinar, we present novel simplified methods for algal characterization using microcapillary cytometry on either a simple touch screen based cytometer, the Muse® Cell Analyzer, or a higher throughput cytometric platform, the compact Guava® easyCyte platforms. The MilliporeSigma algae kits utilize simple mix and read protocols, dedicated software modules, and provide quick results for the of count and viability measurements or relative lipid content on algae strains. The optimized and dedicated algae kits allow for high precision and comparable results to predicate methods. Data from applications to multiple common algal strains such as Chlorella vulgaris and Chlamydomonas reinhardti under different culture conditions will be presented. Availability of dedicated kits for algae research on simple and easy to use cytometric platforms will empower and enable algae researchers to rapidly select optimal culture conditions and strains for downstream experiments.Read more >
Analytical R&D teams are thought of as a core facility within a company. On top of their regular R&D duties, they are often asked to solve production problems, ranging from identifying a contaminant to investigating a competitor’s new product. Learning how to approach these challenges and considering the use of resources beyond your laboratory can save both time and money.
Three case studies will be presented. The case studies involve method development for sub-ppm detection, identifying an off-odor in food, and the detection of a low level pigment. A focus on the specific analytical technology will be presented in the case studies.
A scientific overview of the portfolio of cell lines University of Cambridge has deposited at ECACC with a focus on the KARPAS 299 and KARPAS 422 cell lines; the HeLa Mitotrap cell lines; and CHO cell lines. We will also provide an overview of the process of partnering with ECACC and Sigma-Aldrich for the storage and distribution of cell lines for research purposes. Topics include: The scientific applications of the cell lines; The types of companies and institutions we license to; Advantages of partnering with culture experts and specialised distributors; Our experience of working with ECACC and Sigma-Aldrich.Read more >
Skin cancer is by far the most prevalent cancer. Each year, approximately 3.4 million people in the US alone are diagnosed with some form of skin cancer. Skin cancer can be highly treatable if it is detected and classified early, and this detection and classification is often aided by immunohistochemistry. This presentation covers many of the basic science, facts, and statistics of skin cancer, as well as the utility of immunohistochemical testing with markers such as S-100, SOX-10, Ber-Ep4, and HHV-8 in the accurate diagnosis and survival rates of skin cancer. Continuing education credits for attending this webinar will be offered through the National Society of HistotechnologyRead more >