Reporter Volume 26.4 High Resolution Separation of Human Serum Albumin Tryptic Digest using Fused-Core HPLC Columns s Liquid Chromatography s Sample Handling s Gas Chromatography Ascentis Express Fused-Core Particle and s Standards Results of Gradient Elution of Human Serum Albumin Tryptic Digest on Ascentis Express s Accessories C18 Columns 2 Reporter High Quality Innovative Solutions Volume 26.4 Visit us on the web at sigma-aldrich.com/thereporter Shyam Verma Market Segment Manager NEW Feature! Dear Colleague, Contributed Article Today’s analytical chemist needs not only sophisticated instruments or separation devices but also high quality reagents and chemicals to achieve the desired results with minimal instrument downtime due to “Contributed Articles” are submitted impurities. by our customers based upon their work with Sigma-Aldrich products. Fluka/Sigma-Aldrich offers a wide variety of superior quality, high purity solvents, reagents and chemicals We encourage you to submit articles that are used in sophisticated analysis as well as for general analytical purpose. Every year, many innovative describing your work for consideration reagents are included in our offering to fulfill the requirements of new analytical techniques and standard in future publications. methods for a variety of applications. Before highlighting a few specific high-quality offerings in the reagents and chemicals, I want to mention about the change in labeling of some of our products. The Riedel-de Haën logo is now replaced by the Table of Contents Fluka logo. This is only a brand name change as manufacturing, packaging, performance and quality of the impacted laboratory reagents and chemicals will remain the same. This change is made with a goal of Liquid Chromatography providing a logical structure to our Analytical product offering. Separation of Human Serum Albumin with Fused-Core HPLC Columns .........3 The Fluka (formerly Riedel-de Haën) superior quality LC-MS solvents, additives and blends for your HPLC undergo distinct tests to ensure quality for sensitive and specific LC-MS analysis. These reagents are Proper Injection and Wash Solvent for designed to optimize LC-MS performance with lower impurities and better baselines. HILIC Mode Chromatography ............6 Our portfolio also contains over 400 derivatization reagents, providing the broadest range to assist in a Ion Pairing for Analysis of faster and accurate chromatography analysis. Earlier this March a new guide for derivatization was Phosphonate Compounds ................22 produced that included a comprehensive list of our products and potential applications by industry. This guide can be ordered from the web: sigma-aldrich.com/derivatization. You will find this guide quite Sample Handling helpful in your day-to-day laboratory work. Comparison of Ion-pairing reagents are a widely used product group in reversed-phase HPLC requiring high purity SupelMIP SPE – Beta-agonists and Mixed-mode SPE .......................13 products. Fluka’s quality is unmatched in ion-pairing reagents. This focus on high quality leads to improved accuracy, reproducibility and reliability in the separation of complex mixtures of polar and ionic molecules. SPME for Bioanalysis ........................16 There are numerous types of buffers, indicators, and other reagents and chemicals that are used in Gas Chromatography chromatography analysis and separation techniques that Fluka/Sigma-Aldrich is proud to offer through our catalogs and on the web. In addition to these products, Sigma-Aldrich offers other innovative solutions to GC Analyses of Free Fatty Acids .........8 chromatographic analysis such as Solid Phase Microextraction, and a comprehensive selection of high GC Analyses of FAMEs by Boiling quality standards. Point Elution ......................................10 Fluka/Sigma-Aldrich works hard to assist you in today’s analytical needs and tomorrow’s new ventures Standards with our high quality and tested products, and related literature. We thank you for your confidence in Quality Reference Standards Sigma-Aldrich. for Monitoring Nitrosamine Regards, Contaminants ..................................18 AOCS Reference Mixes ....................19 Accessories Threaded TFE Funnels .......................20 Shyam Verma Hamilton Syringe Calibration Services ...........................20 Market Segment Manager [email protected] Reporter is published 5 times a year by Supelco Marketing, 595 North Harrison Road, Bellefonte, PA 16823-0048. sigma-aldrich.com/analytical AcceleratingVolume 26.4 Customers’ Success through Leadership in Life Science, High Technology and Service 3 High Resolution Separation of Human Serum Albumin Tryptic Digest using Fused-Core HPLC Columns above allowable limits. Thus, to fully exploit the higher Contributed Article efficiencies offered by the sub-2 μm particles, instrumenta- The following was generated by an outside source using tion beyond conventional HPLC is often required. The Sigma-Aldrich products. Technical content provided by: performance advantages that Ascentis Express columns Prof. Luigi Mondello packed with Fused-Core particles offer include efficiencies University of Messina, Italy equal to sub-2 μm columns at half the backpressure due to [email protected] their narrow particle size distribution, and a rugged design capable of high pressure operation and longer column Introduction lifetime in routine use. The last decades have witnessed the use of different analytical techniques to tackle such complex tasks as the Results and Discussion Liquid Chromatography analysis of biochemical systems like peptides and proteins. Initially, a conventional 5 μm C18 column was compared Liquid chromatography plays a central role in present-day to the 2.7 μm Ascentis Express for the separation of a proteome research because of its versatility as well as human serum albumin (HSA) tryptic digest (0.01 M in advances in LC column selectivity and resolution. Human ammonium formate buffer), under gradient conditions, Serum Albumin is a single-chain unglycosylated protein of at a temperature of 35 °C. The higher efficiency of the 585 amino acids that act as a depot and transport site for 2.7 μm column versus the 5 μm column is clear in the / 814-359-3041 fatty acids, drugs, bilirubin, heme, and hormones. It can (continued on page 4) undergo various modifications that significantly affect protein folding, stability, conformation, and function; Figure 1. Analysis of Human Serum Albumin Tryptic characterization of the types and locations of such Digest on Ascentis Express (top) Compared to 5 μm modifications is therefore of utmost importance in Column (bottom) (US and Canada only) understanding HSA’s role in human pathologies. A typical column: Ascentis Express C18, 15 cm x 4.6 mm I.D., 2.7 μm particles and C18, 15 cm x 4.6 mm I.D., 5 μm particles procedure involves the preparation of a tryptic digest from mobile phase A: 100% water with 0.1% TFA mobile phase B: 50:50, acetonitrile:water, with 0.1% TFA the protein, then generation of reliable LC data as a flow rate: 1.0 mL/min preliminary separation step prior to subsequent character- temp: 35 °C det: UV at 215 nm ization using complementary techniques such as MS or injection: 20 μL gradient: 0 to 100 %B in 80 min (linear), hold for 5 min MS-MS and a database search. Ascentis Express In the case of highly complex samples with closely eluting $P: 265 bar components, the slow mass transfer of solute molecules inside the stationary phase particles can severely limit resolving power. The new Ascentis Express® columns, based on Fused-Core™ particle technology, ameliorate this limitation by providing a small (0.5 μm) path for diffusion of solutes into and out of the particles, thereby reducing the time that solute molecules spend inside the particles. As a 0 10 20 30 40 50 60 result, these columns deliver more than twice the separat- Min G004282 / 814-359-3441 technical service: 800-359-3041 ing power of columns packed with 5 μm totally porous Conventional 5 μm particles, which have been the workhorse of most HPLC $P: 91 bar laboratories for many years, and over 50% more separating (US only) power (theoretical plates) than columns of the same length packed with 3.5 μm particles, which often require higher pump pressures due to lower bed permeability. On the other hand, in cases when resolution is critical, smaller particle columns are not suitable for separating closely eluting peak pairs, as the use of a longer column to 0 10 20 30 40 50 60 Min enhance separation is hampered by excessive backpressure G004283 ordering: 800-247-6628 ordering: sigma-aldrich.com/express Volume 26.4 Liquid Chromatography 4 Volume 26.4 Volume sigma-aldrich.com/analyticalsisigmgma-a-alaldrdricich.h cocom/m/ananalytytiiccalal high pressure drop generated bysmallparticles. need change to thesystem the overcome to configuration obtain higherresolution and/or higher speed, with no adding more columnlength athighertemperature to shorter runtime without sacrificing resolution, orby column allows method for optimization intheform of higher resolution andsensitivity oftheAscentis Express 34,000phase (i.e., mL/min). theoretical plates at1.0 The column wereobtained with theFused-Core stationary almostchromatograms inFigure1; twice theplates/ umn set). Regarding retention times, two-fold and backpressure (pressure drop: 389 baronthethree-col- temperature allowed reduce to solvent viscosity and length thesystem upto limits, while increasingthe Efficiency wasincreased three-fold byadding column