Thermo Scientific Accucore XL HPLC Columns. Technical Manual

Thermo Scientific Accucore XL HPLC Columns Technical Manual

Thermo Scientific Accucore XL HPLC Columns Based on Core Enhanced Technology using µm solid core particles, Accucore XL HPLC columns allow users of conventional HPLC methods to enjoy performance far beyond that of columns packed with µm, µm or even µm fully porous particles. Very high separation efficiencies using standard HPLC instruments and conditions provide increased peak resolution and lower limits of detection. An ultra-stable packed bed results in exceptionally robust columns that demonstrate excellent retention and response reproducibility. Accucore XL HPLC columns Ultimate core performance for conventional HPLC methods μm solid core particles for all users Same system, same method, better results Robust, fast and easy to use

The Key Components of Core Enhanced Technology Solid Core Particles µm diameter particles with a solid core generate very high efficiencies with conventional HPLC methods Tight Control of Particle Diameter Enhanced selection process keeps particle size distribution to a minimum and produces high efficiency columns Automated Packing Process Enhanced automated procedures ensure that all columns are packed with the highest quality Advanced Bonding Technology Optimized phase bonding creates a series of high coverage, robust phases Features and Benefits of Accucore XL HPLC Columns Compatible with conventional HPLC methods High resolution Sharp, tall peak shape Reproducible chromatography Long column lifetime No need to change methods or invest in new equipment Separate difficult to resolve peaks Lower limits of detection detect trace levels of analytes Confidence in your results Thermo Scientific Accucore XL HPLC Columns Technical Guide

Ultimate Core Performance for Conventional HPLC Methods Solid Core Advantage The solid core particle design, tight control of particle diameter and automated packing process used for Accucore XL HPLC columns result in minimized eddy diffusion. Accucore XL µm solid core particle Particle Size Distribution Accucore XL versus typical fully porous material Porous layer depth =. µm Number (%) 9 8 Fully porous μm Accucore XL μm 8 9 Particle Diameter (µm) Material Accucore XL µm Particle Size Distribution (D9/). Minimized eddy diffusion results in higher efficiency sharper taller peaks Thermo Scientific Accucore XL HPLC Columns Technical Guide

Adjusting Conventional HPLC Methods For users of conventional HPLC methods working in regulated environments there may be regulatory issues to consider when changing columns in order to realise the improvements offered by newer technologies. For example USP (United States Pharmacopeia) General Chapter <> Chromatography-System Suitability describes the maximum adjustments that can be made to an analysis so that a method still fulfils the requirements of the system suitability test. Column Parameter Allowed Change Column length ± % Column internal diameter ± % Particle size Reduction of up to %; no increase Method Parameter Flow rate ± % Injection volume Column temperature ± % Mobile phase ph ±. UV wavelength Concentration of salts in buffer ± % Composition of mobile phase Allowed Change System suitability testing (SST) criteria must be met No changes outside manufacturer specifications Minor component adjustment ± % or ± % absolute, whichever is smaller Transferring a method from a column packed with a µm fully porous material to an Accucore XL µm HPLC column requires no changes to method parameters and involves only a % reduction in particle size thus meeting the above requirements. Thermo Scientific Accucore XL HPLC Columns Technical Guide

Accucore XL HPLC Columns Two different phases are available in the Accucore XL HPLC column range, C8 and C8. Accucore XL phases are characterized using three tests based on the Tanaka testing protocols. This detailed phase characterization allows the retentivity, selectivity and secondary interactions demonstrated by HPLC packing materials under specified conditions to be objectively compared. T: Hydrophobic Interactions Parameter Term HR Hydrophobic Retention Retention of compounds based on their hydrophobicity k' HS Hydrophobic Selectivity Separation of compounds that have similar structure, but differ slightly in hydrophobicity α SS Steric Selectivity Separation of compounds that have similar structure, but differ in shape α HBC Hydrogen Bonding Capacity Separation related to degree of end capping α T: Secondary Interactions Under Neutral ph Parameter Term BA Base Activity Peak shape for basic analytes resulting from total silanol activity (all dissociated at ph.) t f C Chelation Peak shapes for chelating analytes resulting from silica metal content t f IEX(.) Ion Exchange Capacity (ph.) Separation between basic and neutral compounds resulting from total silanol activity (all dissociated at ph.) α T: Secondary Interactions Under Acidic ph Parameter Term AI Acid Interaction Interactions resulting in poor peak shape for acidic analytes t f IEX(.) Ion Exchange Capacity (ph.) Separation between basic and neutral compounds resulting from acidic silanol activity α The results of the phase characterizations are shown in the radar plots on the next page. Thermo Scientific Accucore XL HPLC Columns Technical Guide

Accucore XL C8 HR/ Hydrophobicity AI HS Low ph Range High IEX (.) SS Pore Size to 8 Å Carbon Load (% ) C HBC Particle Size µm USP L BA IEX (.) Optimum retention of non-polar compounds Hydrophobic interaction mechanism Separates a broad range of analytes Accucore XL C8 The carbon loading of Accucore XL C8 provides high retention of non-polar analytes via a predominantly hydrophobic interaction mechanism. The highly retentive nature of the phase means that it can be used to separate a broad range of analytes. HR/ Hydrophobicity AI HS Low ph Range Pore Size to 9 High IEX (.) SS 8 Å Carbon Load (%) C HBC Particle Size µm USP L BA IEX (.) Similar selectivity to C8 with lower retention Recommended for analytes with moderate hydrophobicity Accucore XL C8 offers lower hydrophobic retention than columns packed with longer alkyl chain length material, such as C8. It is then therefore recommended for analytes with moderate hydrophobicity, or when a less hydrophobic phase provides optimum retention. Thermo Scientific Accucore XL HPLC Columns Technical Guide

Column Formats Accucore XL HPLC columns are offered in analytical and micro formats. Optimum conditions and ratings are shown in the table below. Column ID Optimum Flow Rate Optimum Injection Volume Backpressure Rating. mm. ml/min μl bar C. mm. ml/min μl bar C. mm. ml/min μl bar C Temperature Rating Analytical and Micro Columns Accucore HPLC columns are packed into our high pressure hardware. These stainless steel columns are engineered to the highest quality and have a pressure rating of bar. Guard Cartridges Guard cartridges are designed to protect your column from particulates introduced from the matrix or instrument and from any strongly retained components in the injected sample. Thermo Scientific Accucore XL HPLC Columns Technical Guide

µm Solid Core Particles for all Users The µm solid core particles used in Accucore XL HPLC columns have been specifically designed to get the optimum chromatographic performance from conventional HPLC instruments. Very high efficiencies Little decrease in efficiency as flow rate is increased Moderate backpressures Efficiency Accucore XL HPLC columns generate higher efficiencies than columns packed with µm and µm fully porous material as shown in the van Deemter curve below. Plate Height (H, µm) 8..... 8.... Increasing Efficiency......... Linear velocity (u, mm/s) Fully porous C8, μm Fully porous C8, μm Accucore XL C8, μm Identical instrument and method conditions for all columns Column Dimensions: x. mm Mobile Phase: % water : % acetonitrile Temperature: C Injection Volume: µl UV at nm (. s rise time, Hz) Sample: o-xylene % higher efficiency than µm fully porous % higher efficiency than µm fully porous Backpressure Accucore XL HPLC columns generate reasonable backpressures, moderately higher than fully porous µm and lower than fully porous µm, that are compatible with conventional HPLC instruments. Pressure (bar). bar conventional HPLC pressure..... Flow rate (ml/min) Fully porous C8, μm Fully porous C8, μm Accucore XL C8, μm Identical instrument and method conditions for all columns Column Dimensions: x. mm Mobile Phase: % water : % acetonitrile Temperature: C Backpressures between those generated by µm and µm fully porous Within conventional HPLC instrumentation pressure limit - even at high flow rates Thermo Scientific Accucore XL HPLC Columns Technical Guide

Impedance Impedance (E) combines retention time, efficiency and backpressure in a single term. Lower impedance values indicate fast and higher efficiency separations performed at lower backpressures. Impedance (E).E+.E+.E+. Decreasing Impedance P t E = N η P Backpressure t Retention time N Efficiency h Solvent viscosity Increasing Linear Velocity.. Linear Velocity (u, mm/s) Fully porous C8, μm Fully porous C8, μm Accucore XL C8, μm Identical instrument and method conditions for all columns Column Dimensions: x. mm Mobile Phase: % water : % acetonitrile Temperature: C Injection Volume: µl UV at nm (. s rise time, Hz) Sample: o-xylene 8% lower impedance than µm fully porous % lower impedance than µm fully porous Resolution The high chromatographic efficiencies offered by Accucore XL HPLC columns represent tall, narrow peaks. This provides significant advantages in terms of better peak separations (resolution) and lower limits of detection. Rs =. Fully porous C8, µm Rs =.8 Fully porous C8, µm Rs =. Accucore XL C8, µm 8 9 Minutes % higher resolution than µm fully porous % higher resolution than µm fully porous 8 Thermo Scientific Accucore XL HPLC Columns Technical Guide Identical instrument and method conditions for all columns Column Dimensions: Mobile Phase A: Mobile Phase B: Gradient: Flow Rate: x. mm water Temperature: C Injection Volume: µl Sample: acetonitrile to % B in min. ml/min UV at nm (. s rise time, Hz). uracil (t). tebuthiuron. metoxuron. monuron. chlorotoluron. diuron. linuron

Sensitivity S/N =.8 Fully porous C8, µm S/N =. Fully porous C8, µm S/N =. Accucore XL C8, µm 8 Minutes Column Amount Average Limit of Detection on Column S/N (based on S/N = ) Fully porous C8, μm ng.8. ng Fully porous C8, μm ng.. ng Accucore XL C8, μm ng..8 ng Identical instrument and method conditions for all columns Column Dimensions: Mobile Phase A: Mobile Phase B: Gradient: Flow Rate: x. mm water Temperature: C Injection Volume: µl Sample: acetonitrile to % B in. min. ml/min UV at nm (. s rise time, Hz). uracil (t). tebuthiuron. metoxuron. monuron. chlorotoluron. diuron. linuron (each at ng/µl) % more sensitive than µm fully porous % more sensitive than µm fully porous Thermo Scientific Accucore XL HPLC Columns Technical Guide 9

Same System, Same Method, Better Results The following applications show the improvements in performance that Accucore XL HPLC columns offer without any changes in instrument configuration or method conditions. Ibuprofen and Valerophenone (USP) P = 9 bar Fully porous C8, µm mau N = 8 plates S/N = -........... Time (min). P = bar Accucore XL C8, µm N = 8 plates S/N = mau -............ Time (min). Analytes. Valerophenone. Ibuprofen Columns: Accucore XL C8 µm, x. mm Fully porous C8 µm, x. mm Mobile phase:.:. (v/v) water with phosphoric acid, ph.:methanol Flow rate: ml/min Column temperature: C UV at nm Injection volume: µl % higher efficiency % higher sensitivity Thermo Scientific Accucore XL HPLC Columns Technical Guide

Fat Soluble Vitamins. ml/min P = bar Fully porous C8, µm mau Rs =.9 N = plates - 8 9 Time (min). ml/min P = bar Accucore XL C8, µm Rs =. N = 9 plates mau - 8 9 Time (min). ml/min P = 9 bar Accucore XL C8, µm mau Rs =. - 8 Time (min) Analytes. Vitamin K. Vitamin D. Vitamin D. Vitamin E. Vitamin E acetate. Vitamin K Columns: Mobile phase: Flow rates: Accucore XL C8 µm, x. mm Fully porous C8 µm, x. mm 8: (v/v) acetonitrile:methanol ml/min Column temperature: C Injection volume: µl. ml/min UV at 8 nm % higher efficiency 8% better resolution of critical pair Reduced run time with good resolution at increased flow rate Thermo Scientific Accucore XL HPLC Columns Technical Guide

Endocrine Disruptors mau - P = bar S/N = PW. =.9 min Fully porous C8, µm 8 9 Time (min) Rs =. P = bar Accucore XL C8, µm mau S/N = 9 PW. =. min Rs =. - 8 9 Time (min) Analytes. Desethyl Atrazine. Simazine. Atrazine. Diuron. Bisphenol A Columns: Mobile phase A: Mobile phase B: Accucore XL C8 µm, x. mm Fully porous C8 µm, x. mm water acetonitrile Gradient: Time (min) % B Flow rate: Column temperature: C Injection volume: µl..... ml/min UV at nm % better resolution of critical pair % narrower peaks % higher sensitivity Thermo Scientific Accucore XL HPLC Columns Technical Guide

Triazines P = bar Fully porous C8, µm mau S/N = PW. =. min Rs =.9-8 9 Time (min) P = bar Accucore XL C8, µm S/N = 8 PW. =. min mau Rs =.9-8 9 Time (min) Analytes. Simazine. Simetryn. Atrazine. Prometon. Ametryn. Propazine. Prometryn Columns: Mobile phase A: Mobile phase B: Accucore XL C8 µm, x. mm Fully porous C8 µm, x. mm water acetonitrile Gradient: Time (min) % B Flow rate: Column temperature: C Injection volume: µl..... ml/min UV at nm % better resolution of critical pair 9% narrower peaks % higher sensitivity Thermo Scientific Accucore XL HPLC Columns Technical Guide

Catechins. ml/min P = 8 bar PW. =.9 min Rs =. Fully porous C8, µm. ml/min P = 8 bar Rs =.8 Accucore XL C8, µm mau PW. =. min. ml/min P = bar Rs =. Accucore XL C8, µm PW. =. min 8 9 Minutes Analytes. Epigallocatechin. Catechin. Epigallocatechin gallate. Epicatechin. Gallocatechin gallate. Epicatechin gallate. Catechin gallate Columns: Mobile phase A: Mobile phase B: Accucore XL C8 µm, x. mm Fully porous C8 µm, x. mm water +.% formic acid methanol +.% formic acid Gradient: Time (min) % B Flow rate:.... ml/min Column temperature: C Injection volume: µl. ml/min UV at 8 nm 9% better resolution of critical pair % narrower peaks Reduced run time with good resolution at increased flow rate Thermo Scientific Accucore XL HPLC Columns Technical Guide

Robust, Fast and Easy to Use Robustness Accucore XL HPLC columns are extremely robust offering excellent performance over extended use. Stability Retention Capacity Factor (k') 9 8 Ibuprofen Fenoprofen Naproxen Number of Injections Stability Efficiency Normalized Effciency % % % % % % 9% 8% % % % Ibuprofen Fenoprofen Naproxen Number of Injections Column: Mobile Phase: Flow Rate: Temperature: C Injection Volume: µl Sample: Accucore XL C8 µm, x. mm : acetonitrile: mm ammonium formate ph. ml/min UV at nm Non-Steroidal Anti Inflammatory Drugs (NSAIDs) ibuprofen, fenoprofen, naproxen Stable retention and efficiency over thousands of injections Thermo Scientific Accucore XL HPLC Columns Technical Guide

Productivity In addition to using established conventional methods, the high efficiencies offered by Accucore XL HPLC columns, across a wide range of flow rates, allow methods to be optimized to reduce run times and increase productivity. Rs =. Fully porous C8, µm Standard method t = 8. Rs =. t =. Accucore XL C8, µm Standard method Rs =.99 t =. Accucore XL C8, µm Optimized method 8 9 Minutes Column Method t R of last peak Reduction in time Fully porous C8, μm Standard 8. min % Accucore XL C8, μm Standard. min % Accucore XL C8, μm Optimized. min % Column Dimensions: Mobile Phase A: Mobile Phase B: x. mm ID water acetonitrile Standard Method Optimized Method Gradient: to % B in min to % B in min Flow Rate:. ml/min. ml/min Temperature: C Injection Volume: µl Sample: UV at nm (. s rise time, Hz). uracil (t). tebuthiuron. metoxuron. monuron. chlorotoluron. diuron. linuron Run time reduced by over a third with an improvement in performance Thermo Scientific Accucore XL HPLC Columns Technical Guide

Ordering Information Accucore XL HPLC Columns Description Particle Size Length (mm). mm ID. mm ID. mm ID Accucore XL C8 µm Accucore XL C8 µm - - - - - - - - - - - - - - - - - - - - - - - - Accucore XL Guard Cartridges (/pk) Description Particle Size Length (mm). mm ID. mm ID. mm ID Accucore XL C8 µm - - - Accucore XL C8 µm - - - UNIGUARD Direct-Connection Guard Cartridge Holders Description. mm ID. mm ID. mm ID UNIGUARD Drop-In Guard Cartridge Holder 8-8- 8- Standard Replacement Tip 8-RT 8-RT 8-RT Thermo Scientific Accucore XL HPLC Columns Technical Guide

Resources for Chromatographers Thermo Scientific Chromatography Columns and Consumables Catalog This extensive catalog offers pages of proven chromatography tools and product selection guides. Available online, with a robust search tool and optimized for your ipad. Visit www.thermoscientific.com/catalog Chromatography Resource Center Our web-based resource center provides technical support, applications, technical tips and literature to help move your separations forward. Visit www.thermoscientific.com/crc For more information visit: www.thermoscientific.com/accucorexl Thermo Fisher Scientific Inc. All rights reserved. ipad is a registered trademark of Apple Inc., registered in the US and other countries. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. USA and Canada + 8 France + () 9 8 Germany +9 () 9 or United Kingdom + ()98 Japan +8 8 PS8_E /S China +8 888 +8 8988 +8 899 8 8 8 India +9 99 +9 Australia 9 (free call domestic) New Zealand 8 9 9 (free call domestic) All Other Enquiries + () 98 Technical Support North America + 8 Outside North America + () 98