Science Together AZURA® Preparative HPLC Flexible purification solutions 2 AZURA® Preparative HPLC The dependencies between throughput, purity and yield always have to be considered in HPLC purifications. Whatever priority you decide for, with the AZURA preparative systems you can success-AZURA® preparative systems are the perfect solution for frequently chang-fully adapt. ing separation tasks – from mil igram to kilogram scale. Design your AZURA preparative system to your needs and combine flexibility and reliability. Saving time Short run time, low purity or yield Customize Easy scale up Advanced your system from compact to separation modes pilot systems , Ine r xpensive pr ecovery ecovery , full r time saving Recovery and yield oduct, Purity Maximizing product Reducing impurities Low purity low AZURA® Prep systems are tai- Due to the flexible design of our AZURA® Prep systems can be Time efficient, lor-made for you. Configure your devices, you can easily change used for special separation system from injection to detec-parts like pump heads or flow modes like peak recycling and tion and choose between differ-cells and integrate all compo- stacked injections. We help you ent materials, flow rates, valves nents of the compact into the to configure your system and Valuable product, Higher yield, high recovery, slow Long run time, and detectors. pilot-scale system. choose the best software for you. lower purity baseline-separated The general objective of preparative chromatography is to isolate, purify and collect your target compounds. Preparative applications are often initially performed on an analytical level and need to be The AZURA SMB systems are the solution for your continuous upscaled. Depending on the desired scale, the requirements for a preparative system differ in eluent purification task. Get higher productivity and purity than with com-supply, sample injection, column, and detection. We customize our systems to meet your chromatogra-parable batch systems. Save up to 90 % of the solvent and reduce phy scale-up and purification challenges. Benefit from our experience in preparative chromatography. the solid phase costs up to 80 %. For more information: www.knauer.net/prep For more information see page 32. Scheme of SMB principle 2 www.knauer.net/prep 3 AZURA® Preparative HPLC waste UV detector fraction collector pump pump A sample B pump 6x ELUENT SAMPLE COLUMN SELECTION & DETECTION DELIVERY INJECTION THERMOSTAT Assistant Customizable combination of valves, Sample selection and injection detectors and pumps in one housing e.g. Most flexible Manual or automated injection: use a valve or a valve for column switching (see page 10) system solutions with feed pump (see page 11 + 12) on the market Multi Column Base Mobile Control Securely position up to three preparative Optional touch display and columns, customize and organize your control tool for AZURA systems system with a wide range of accessories (see page 27) (see page 24) Detection High or low Various detector types (UV/VIS, pressure gradient DAD, RI, FL, MS) and a selection HPLC column 50 – 1000 ml/min of flow cells for a wide range of Preparative separation flow rates (see page 16) columns (see page 15) Fiber optics flow cell Fraction collection Measure close to the Fractionation valve or fraction column to minimize peak collector for various flow rates User friendly broadening with fiber and powerful (up to 1000 ml/min) optics (see page 17) software (see page 20) Eluent delivery A choice of pumps with pump heads available in different materials al ows maximum flow rates from 50 – 1000 ml/min. Isocratic, low and high pressure gradient forming are possible (see page 8) 4 www.knauer.net/prep 5 AZURA® Preparative HPLC The modular AZURA Preparative HPLC platform offers you the opportunity to build a purification system best suited to your needs. 400 Pilot 300 Lab ar) (b 200 Pilot ssure 150 Compact reP 100 Pilot 50 Pilot 50 100 250 500 1000 Flow rate (ml/min) AZURA® Compact Prep LC AZURA® Lab Prep LC AZURA® Pilot Prep LC Flow rate max. 50 ml/min* Flow rate max. 50 ml/min* Flow rate max. 1000 ml/min* Isocratic Isocratic/HPG Isocratic/LPG/HPG AZURA system Available pump heads Gradient options Max. flow rate in ml/min* LPG HPG 50 100 250 500 1000 low pressure high pressure The AZURA Pilot Prep LC is the ideal solution for your upscaling For more information: AZURA Compact Prep LC • tasks. The 100 ml pump head allows you to run your system under www.knauer.net/prep AZURA Lab Prep LC • • analytical conditions before adapting your method to preparative scale. AZURA Pilot Prep LC • • • • • • * Information on best working conditions on pages 8–9. 6 www.knauer.net/prep 7 AZURA® Preparative HPLC Precise and reliable pumps covering a wide flow range for various The AZURA semi-preparative pump P 6.1L with 50 ml pump head is available as an isocratic or gradient and solvent selection options. binary HPG pump. It is made for medium-size purification tasks and upscaling processes. Pressure sensor Piston seal wash Piston seal wash The preparative HPLC pump AZURA P 2.1L covers a wide flow rate and pressure range. It has been Pressure sensor Solvent selection valve designed for the purification of milligram to gram samples. The integrated automatic RFID pump head recognition allows a quick adaptation to various applications. • Flow rate up to 50 ml/min • Best working conditions: • Flow rate up to 1000 ml/min 0.1 – 40 ml/min • LPG and HPG gradient options • Binary gradient with • Supports constant pressure mode solvent selection valve Exchangable pump heads (2x2 solvents) (stainless steel or titanium) • Up to 300 bar Gradient options of Pump P 2.1L • Supports constant pres- sure mode A low pressure gradient (LPG) module dynami-Pump Max. Best working cally composes the eluent on the inlet-side or low head pressure conditions High efficiency AZURA mixer pressure side of the pump head, by quickly switching between the different solvent channels. 100 ml 400 bar 1 – 80 ml/min We offer binary or ternary LPG upgrade modules for the isocratic P 2.1L. 250 ml 200 bar 2.5 – 200 ml/min Pump P 2.1L 500 ml 100 bar 5 – 400 ml/min The eluent in a binary high pressure gradient (HPG) system is composed by combining the sol-1000 ml 50 bar 10 – 800 ml/min For automated solvent change, a solvent selection vent flows of two isocratic pumps. valve can be attached to the pump P 2.1L. Covered flow rate For semi-preparative purification tasks, the pump P 6.1L features a built-in 2x2 solvent selection HPG valve (high pressure gradient version). ype Binary LPG ient t 8 Port solvent selection valve (PEEK) rad (stainless steel coming soon) Ternary LPG G Binary LPG valve block for the pump head 10 220 800 1000 Flow rate (ml/min) 8 www.knauer.net/prep 9 AZURA® Preparative HPLC A flexible combination module Adapt the sample injection mode to your preparative task. The assistant ASM 2.1L is a compact combination Depending on the integrated modules, the as-module which can be equipped with up to three sistant fulfills many different tasks such as sample device modules. Available for selection are valves, and solvent selection, sample injection, column The simplest way to inject your sample into the pumps and an UV detector. An assistant can be switching, fraction collection, eluent delivery or system. Use a manual injection valve and choose the basis of a whole HPLC system including a UV detection. from a large range of different sample loops. pump, valve and a detector, just like the AZURA The concept of the flexible combination of device compact prep system (page 30). As part of a larg-modules combines the highest functionality with er system, the ASM 2.1L is extremely versatile. minimum space requirements. KNAUER offers several injection valves for 1/16‘ and 1/8” tubing. The wetted parts are made of stainless steel or PEEK to cover a broad range of Can be equipped with combinations: valves, pumps, and one detector applications. Injection can be done either manual y via hand lever or automated with a valve drive. Valves For eluent, sample and solvent selection, UV Detector UVD 2.1S sample injection, column selection, fraction Variable single wavelength collection, automation and multiple swichting (see page 16) tasks. The KNAUER VariLoops are the perfect solution for or partially. This allows you to work very flexible the injection of medium up to high sample volumes and easily switch between different sample sizes (up to 40 ml). The sample loop can be emptied while keeping constant and reproducible injection completely or partially as well as filled completely volumes for every sample size. sample sample eluent eluent sample column sample column Compact pumps P 4.1S Up to 10 ml/min or 50 ml/min with pressure sensor waste pump waste pump 10 www.knauer.net/prep 11 AZURA® Preparative HPLC Sample injection can be easily automated with an autosampler. The AS 6.1L can inject up to 10 ml per injection. Sample tray temperature control from 4 – 40°C is optional available. It can waste handle either 30 samples in 10 ml vials or up to 768 samples in well plates. 1 2 3 1 2 3 UV pump column detector 4 5 6 4 5 6 Injection valve When automated selection of eluents is required, up to 12 different eluents can be attached to the The AZURA sample injection assistant for prepar-Sample pump preparative system. Multiposition valves fulfill ative LC is based on the multifunctional AZURA many different tasks: solvent and sample selec-Multiposition valve element ASM 2.1L. It is designed to automate in-tion, fractionation and column switching. jection of larger sample volumes and features a sample selection valve, a sample pump, and an injection valve. bypass Simply attach your sample vessels via 1/8‘‘ tubing column 1 to the multiposition valve and automate injection Sample selection valve column 2 with the integrated sample pump and injection Switching valves are ideal for screening and scale-valve. up. They can be easily integrated into your system Manual injection valve column 3 UV at pressures up to 400 bar and maximum flow rates pump detector column 4 of 300 ml/min. column 5 Standalone or integrated in an assistant module Injection: 1 2 3 4 ASM 2.1L: The compact pump AZURA P4.1S is perfect for feed injection. With the stacked injection function, it is possible • 10 and 50 ml exchangeable pump head to perform different runs automatically one after )U • Flow rate range: the other. The injection of the next run takes place A (m 0.01 – 50 ml/min (50 ml pump head) during the current run, so that the time until the al 0.001 – 10 ml/min (10 ml pump head) elution of the first peak can be ful y exploited. This Sign • Pump heads available in stainless steel increases efficiency, saves time and eluent. Stacked or ceramics injection can be operated with the chromatograph- • Best working conditions: ic data systems (CDS) PurityChrom® and OpenLab. 1 – 40 ml/min (50 ml pump head) Volume (ml) 0.1 – 8 ml/min (10 ml pump head) 12 www.knauer.net/prep 13 AZURA® Preparative HPLC Find the perfect column from the large KNAUER portfolio This flow chart gives you a guideline how to select the right column for your application. Start at the top and follow the decision lines all the way down to find a column recommendation. Substance An example to analyze Molecular weight or purify Molecular weight A separation can be much more demanding after < 2000 Da > 2000 Da upscaling from analytical to preparative scale. In many cases a baseline separation is not possible 5 µm 20 µm anymore, so time and money consuming method particle particle Soluble in Soluble in size size development or hardware adjustments are neces-water organic solvent sary. The AZURA Prep LC system is well-suited to apply the peak recycling technique to solve de-Soluble in Soluble in manding resolution tasks. Additionally, solvent ACN/H2O, Hexane/ Ionic Non-ionic recycling can be applied to save eluent, if it can MeOH/H2O Heptane be considered clean. Wide pore Time Time Reversed Ion-exclusion HILIC Reversed Phase (RP) Normal Phase (NP) Phase (RP) Comparison of analytical and preparative chromatogram. Collecting mode Cycle: 1 2 3 4 H-form Silica Classical C18 Silica Wide pore detector 400 (USP L17) (USP L3) (USP L1) (USP L3) classical C18 350 UV Ca-form NH2 Hydrophilic/ NH2 (USP L1) detector 300 (USP L19) (USP L8) aqueous (USP L8) C18 (USP L1) Wide pore 250 UV Pb-form Zwitter-ionic Diol C18A (USP L34) HILIC Hydrophobic/ (USP L20) hydrophilic/ 200 (USP -) pH stable aqueous pump column Na-form C18 (USP L1) Cyano (USP L1) 150 detector (USP -) (USP L10) Valve switching Classical Wide pore 100 pump column C8 (USP L7) C8 UV 50 (USP L7) detector Recycling mode Hydrophilic/ 0 aqueous Wide pore UV C8 (USP L7) C4 0 5 10 15 20 25 30 35 40 45 50 Minutes (USP L26) pump column Hydrophobic/ Peak Fractionation Solvent pH stable Recycling Recycling C8 (USP L7) pump column Successful peak separation with recycling mode. C4 (USP L26) CN (USP L10) Phenyl (USP L11) Eurokat KNAUER KNAUER Eurospher Eurosil Eurospher II Bioselect 14 AZURA® Preparative HPLC KNAUER gives you the opportunity to analyze nearly every compound due to a large portfolio of HPLC detectors. For the achievement of your More flexibility analysis goals and for matching your separation Fiber optic cables offer the possibility to separate scale, our detectors are flexible in the setup, in-the flow cell from the detector. This enables de-cluding flow cells and fiber optics. Our product manding applications such as measuring direct-line of UV/VIS detectors ranges from single vari-ly after a heated LC column or in hazardous enable wavelength to 8-channel diode array detec-vironments, allowing safe operation of the tors with 3D scan capability. instrument while maintaining performance. Detector UVD 2.1S UVD 2.1L MWD 2.1L DAD 2.1L DAD 6.1L Safe operation When working at high flow rates, separation of Compact and Reliable UV/ Robust multi- Versatility High-end the flow cell and the detector is a safety feature. versatile VIS detector channel UV/ through a diode array UV detector for a wide VIS detector wide flow cell detector with In case of leakages, no damage to the detector spectrum of range outstanding occurs. Fiber optics are available in a customized applications performance length of up to 10 meters. Flow cell with Wavelength 190 – 500 nm 190 – 750 nm 190 – 700 nm 190 – 700 nm 190 – 1 000 nm 1/4” TRI-Clamp connection Channels 1 1 8 8 8 3D scan • • Fiber optics • • • • • available The AZURA RID 2.1L HighFlow is a sensitive and competitively priced differential refractometer. It is suitable for detecting compounds with little or no UV activity such as alcohols, sugars, lipids or polymers in high concentrations. This instrument is designed for use in semi-preparative and preparative HPLC for flow rates up to 100 ml/min. Optional are higher flow rates possible with a flow Select from an impressive range of easily exchangeable preparative and semi-preparative flow cells splitter. The intelligent temperature control guarantees fast baseline stabilization and stable oper-for UV/VIS and DAD detectors. With capillary connections ranging from 1/16” to 1/4’’ and TRI-Clamp ation. adaptions, optional fiber optics technology and a variety of flow cell wetted materials, a wide spectrum of applications can be covered. Max. flow Connectors Path length Volume Max. pressure Fiber optics rate available 50 ml/min 1/16’ 3 mm 2 μl 300 bar • 250 ml/min 1/16’ 0.5 mm 3 μl 200 bar • 1000 ml/min 1/8’ 0.5/1.25/2 mm 1.7/4.3/6.8 μl 200 bar • 10000 ml/min 1/4’ 0.5/1.25/2 mm 1.7/4.3/6.8 μl 200 bar • 16 www.knauer.net/prep 17 AZURA® Preparative HPLC Choice of specialized detection technology, fully integrated in The KNAUER 4000 MiD is a single quadrupole MS PurityChrom®. Suitable for preparative LC with the help of a flowsplitter. with a spraychip ionization source. It is able to perform both positive and negative electrospray ionization and features the scan modes full scan, SIM and interleaved. With its integrated oil-free pump, it achieves a mass accuracy of +/- 0.3 m/z when performing a full scan. With a mass resolution of 0.7 m/z (FWHM), it is the perfect choice for mass directed purification. As a universal detector, an ELSD detector offers numerous possibil-ities for detecting substances that have few or no chromophores. Since the eluents are evaporated, the use of non-UV-compatible solvents poses no problems and the ELSD is gradient compatible. Target analytes: Carbohydrates and similar compounds, detergents, All-in-one solution Target analytes Easy to use ionic and non-ionics, artificial sweeteners, antioxidants, amino With the integrated vacuum sys-With a mass range of 800 m/z With the KNAUER 4000 MiD and acids, lipids, peptides, polymers, pestizides, proteins, steroids. tem and integrated electronics the KNAUER 4000 MiD can be its simple ‚plug and play‘ consu-inside of one box the KNAUER used for a broad variety of appli-mables mass spectrometry gets 4000 MiD brings mass spectro- cations. In combination with the as easy as possible. metry to places where no other KNAUER MiDas it is the ideal spectrometer can be deployed. choice for preparative chromato- graphy and direct indroduction methods. The Conductivity Monitor CM 2.1S can monitor salt gradients with flow rates of up to 100 ml/min and a maximum pressure of 100 bar. It supports a wide measurement range of 0.01 mS/cm - 999 mS/cm. Flow cel s in PEEK for both analytical and preparative scale are available. pH option available Purification of a natural product by mass directed fractionation Black: UV trace at 280 nm Blue: MS trace (SIM) for target compound 18 www.knauer.net/prep 19 AZURA® Preparative HPLC Collect large quantities or large numbers of fractions KNAUER offers different valves for fraction collection and variations of trusted fraction collectors. Whether you are doing research and development or production, there is an appropriate solution The Foxy® R1 fraction collector can be adapted tles and many more. For essentially unlimited vol-that suits your application. to a broad spectrum of applications. Flow rates umes, funnel racks can direct fluids to any collec-of up to 125 ml/min for Foxy R1 and 1000 ml for tion vessel or downstream process. Both devices Fractionation modes: Foxy R2 are possible. Fractions can be collected can be operated stand-alone or in the chroma-Manually – collection by direct control into 96 well microplates, standard tube sizes, bot-tography software PurityChrom®. Time-based – collection at defined time points Peak-based – collection according to detector signal Threshold function – collection according to any signal Rack type 144 Vials 12 mm 144 Vials 13 mm 100 Vials 16 mm 36 Vials 25 mm The LABOCOL Vario 4000 fraction collectors are fining the number of fraction vessels and their characterized by their high robustness and optimal position. The wide application area makes the Vario 2 Microwell plates 96 ratio of dimensions/benefit. The user is not limited 4000 series ideal for use in research and develop-to given rack types. The rack layout can be de-ment as well as in production. The Vario 4000 mod-60 Tubes 1.5 ml signed according to individual needs. Free rack els differ in the base area and the flow rate range. design. Any rack type can be integrated by de-72 Centrifuge tubes 15 ml 36 Centrifuge tubes 50 ml Rack type 2 x 9 Bottles 480 ml* 80 Tubes 18 mm 36 Funnels with vinyl tubing 125 Tubes 10.5 mm 26 Funnels with vinyl tubing* 20 Tubes 36 mm 39 Tubes 26 mm 24 Centrifuge tubes 50 ml * Foxy R2 only 20 www.knauer.net/prep 21 AZURA® Preparative HPLC Increase performance. Minimize solvent viscosity. When performing preparative LC at temperatures above 40°C in air-conditioned laboratories, a uni-8 Port Multiposition valve 12 Port Multiposition valve 16 Port Multiposition valve form temperature distribution is essential. With for 1/8”, PEEK for 1/8”, SST for 1/16”, SST the Eluent Heater, solvent temperature can 7 fractions + waste 11 fractions + waste 15 fractions + waste be precisely controlled using the integrated (stainless steel coming soon) touchscreen. It supports flow rates of up to 500 solvent recycling ml/min and is cleanroom compatible. Fractionation valves max. flow rate (in ml / min) HPLC system peak recycling 1/16˝ 1/8˝ Our column heating sleeves are the perfect solu-1/4˝ tion for thermostating your preparative column hardware. Available for all preparative KNAUER 100 500 1 000 waste collecting column dimensions at temperatures up to 100 °C. Custom dimensions, clean room compatible and Device Max. flow rate Racks Different rack Max. fractions autoclavable materials are available on request. (ml/min) types 1/16‘‘; 1/8‘‘; 1/4‘‘ 1/16‘‘; 1/8‘‘; 1/4‘‘ Valve 100 / 500 / 1000 16 / 12 / 10 Foxy R1 25 / 125 / – 1 15 up to 144 This oven can heat up to 80 °C. It can accommodate up to 8 KNAUER columns with max. 250 x 50 mm Foxy R2 – / 125 / 1000 2 15 up to 288 inner dimensions. Labocol Vario 4000 100 / 500 / 1000 3 5** 72* Labocol Vario 4000 100 / 500 / 1000 5 5** 120* Plus * For 50ml tubes ** Device supports other racks via user-defined position setting. Electrical heating element for pump heads. Temperature can be controlled using the eluent heat-er or a single device control unit. 22 www.knauer.net/prep 23 AZURA® Preparative HPLC Improve system performance, organize your lab bench, and work more conveniently with the right accessories. Accessory Features Benefit Accessory Features Benefit Pump head inlet • Connect one 1/4’’ tube to the AZURA For high flow rates and Column Base • Holds up to three preparative columns Flexible operation with up viscous eluent to three columns Pump P2.1L • Adapters for other diameters available Mass flow • Unmatched accuracy at flow rates up to Precisely monitor the eluent Benchtop Rack • Install AZURA systems at space-limited sites, Space-saving solultion for flow AZURA system setup controller 833 ml/min especial y in cold rooms. • Compatible with PurityChrom® Air Sensor • Detect end of buffer or end of sample Protect column from air with PurityChrom® damage and support automation ( e. g. sample Dynamic Mixing • Effective homogenization of eluents Better performance • Up to four air sensors per system injection) Chamber • For transparent tubings with 1/16” or 1/8” or 1/4” outer diameter AZURA® Click • Attach air sensor, pressure control, Organize your system. AZURA Organizer or your interface box Adapt the sample volume to to the side panel of your AZURA L VariLoop • Variable injection volume and multiple your application device injections AZURA® Organizer • Attach columns from 5 mm to Organize accessories directly at the system and 26 mm diameter, falcon tubes, reduce dead volume Interface Box IFU • Highly precise analog data acquisition Add any detector with pH flow cell analog output to your 2.1 LAN • 4-channel input/output system • Sample rates of up to 50 Hz (one channel only) Flow Splitter • Adjustable valve for precise direct Collect fractions while using control over split ratios your preferred detection • Ultra low dead volume fluidic desig method 24 www.knauer.net/prep 25 AZURA® Preparative HPLC ClarityChrom® CDS Mobile Control (Chrom) ClarityChrom is an easy-to-use chromatography The hand-held Mobile Control (Chrom) allows a data system (CDS) for workstations. Besides support complete overview of all devices of the AZURA PurityChrom is a powerful software to control your clicking one button. Create methods with highest of all KNAUER devices, components and systems systems on one screen. Remotely check import-preparative system. Get familiar with PurityChrom flexibility to realize complex application without from more than 45 manufacturers are also sup-ant parameters or control and monitor devic-in shortest time and with no effort due to the in-losing easy handling. Offline licenses for creating ported. ClarityChrom® includes the drivers for es. The touch-optimized user interface facilitates tuitive and clearly structured user interface. methods and data evaluation are for free. The several fraction col ectors and supports peak rec-navigation using just your fingers. The display Choose a time–or volume based workflow by just software is 21 CFR part 11 compliant. ognition by level and/or slope. The manual fraction software Mobile Control provides full access to control and the option to use the KNAUER electric AZURA devices. Change device settings, set op-valves for fractionation give you even more flexi-erating parameters, automate device control or bility. check the system status and GLP data… Mobile Control features all functionalities of a device dis- • Fraction collecting via peak recognition (lev-play. el only, slope only, level AND / OR slope – incl. Do you want to acquire data without the overhead self-learning) or single event (unconditional, of a chromatographic data system? Mobile Con-timed event) trol Chrom features data acquisition from AZURA • Easy to collect: waste, collect to position / detectors in addition to full device control. For collect to next, solvent recycling simple applications Mobile Control Chrom might • Direct control during a run – manually switch be the appropriate software solution – basic, easy-to: collect, waste, solvent recycling to-use and cost-effective! • Consecutive runs: easily find your chromatogram by clicking on your fraction Chromeleon™ 7 Chromeleon is one of the most wide-spread chro-OpenLab matography data systems. It offers a broad range OpenLAB CDS EZChrom Edition provides support of third-party drivers and can be easily used with of devices from KNAUER and many other manu-existing HPLC systems. Chromeleon drivers for facturers. The KNAUER fraction collector control Intuitive Control System visualization many KNAUER devices are available. option includes the drivers of several fraction PurityChrom includes intuitive data evaluation The system visualization offers a graphical repre-collectors and supports fractionation by time, the with peak recognition and integration. Due to sentation and allows easy handling even of com-peak recognition by level and/or slope, also with its high flexibility, methods can be developed plex flow processes. Furthermore, each device spectral confirmation. Collect Slices allows for according to specific demands. You have the which is displayed in the fluidic scheme can be setting a desired volume for each fraction, within option to create a method based on volume, manual y control ed, giving the opportunity to op-the defined fraction vial volume. The manual frac-column volume, or time. There is also the possi-timize, change and adapt your conditions during tion control and the option to use the KNAUER bility to pause your method during a run. The the run. electric valves for fractionation gives you more hold function provides you with complete control flexibility. The combination of virtual detector and over your chromatography process. Solvent vi-virtual fraction collector allows for optimizing the sualization calculates the consumption of solvent fractionation settings from an existing chromato-for the current run and prevents your column gram of your separations without any physically from running dry. existing device and, therefore, without the loss of solvent or target substance. 26 www.knauer.net/prep 27 AZURA® Preparative HPLC The AZURA® Prep Compact system is the perfect start into preparative chromatography. With the complete, semi-preparative HPLC system you master your isocratic purification tasks. sample injection A B C waste sample injection UV pump column detector fraction collector UV sample pump column detector fractionation pump 12x valve x12 Compact Prep System Pilot Prep System One manual injection can purify several hundred the compact system and regulates the fraction After starting preparative chromatography with • Pilot Ternary gradient HPLC system milligrams at up to 50 ml/min. Detection takes collection via a 12-port fractionating valve. Thanks the space-saving prep system, the requirements • Injection valve incl. 500 μl sample loop place via a versatile UV/VIS detector. The intui-to its compact design, the AZURA Prep Compact for your purification tasks can quickly increase. • Sample pump with automatic sample tive preparative software PurityChrom controls system finds its place in every laboratory. The existing Compact System (50 ml/min) can be selection expanded to a Pilot System (220 ml/min) by in- • UV/VIS detector with one variable vesting in a fraction collector and a preparative wavelength pump. All components of the Compact System • Intuitive PurityChrom® software are fully integrated into the Pilot System. • Fraction collector • Complete semi-preparative isocratic HPLC system with low space requirements • Injection valve incl. 500 μl sample loop • UV/VIS detector with one variable wavelength • Intuitive PurityChrom® software • Compact and expandable 28 www.knauer.net/prep 29 AZURA® Preparative HPLC Chamomile plants are known for their medical properties, having among others anti–inflammatory, analgesic and sedative effects. These are due to the various phenolic compounds, one of them ma-tricine is converted during the distillation process to chamazulene. The characteristic blue color of chamomile essential oils as “chamomile blue” is due to chamazulene. It has anti-inflammatory and anti-oxidant activity. The present application tested preparative HPLC to purify chamazulene from commercialy available “chamomile blue” oil. The Lab Prep LC system is designed for your more demanding semi-preparative separations. You can customize a highly flexible LC system with the freely combinable components. With a maximum flow rate of Fractionation/Purification The separation of chamazulene was op- 6000 50 ml/min it is possible to separate up to several hundred mil igrams per run. timized in analytical scale and the two step gradient method transferred to 5000 preparative scale. Chamazulene purifi- A1 A2 cation was performed on C18 250x20 4000 B1 B2 mm column, 25 ml/min. Fractionation of U] sample chamazulen was conducted by threshold [mA 3000 injection UV 245 function of PurityChrom software. waste 2000 pump pump A B UV 1000 column detector fraction collector selection (6x) 0 0 2 4 6 8 10 12 14 16 18 20 Time [min] Fig. 1: Chromatogramm of preparative separation of chamazulene blue, collected fraction highlighted in red, 1 mL sample injection Fraction analysis • Lab Prep HPLC system with binary high pressure gradient • Column selection • Injection valve incl. 500 μl sample loop • UV/VIS detector with one variable Fig. 3: Spectral view fraction wavelength • Intuitive PurityChrom® software • Fraction collector Fig. 2: Chromatogram overlay The collected fraction was analysed by analytical HPLC and revealed nearly 100 % purity. Chromatogram overlay of the fraction, chamazulen Fig. 4: Spectral view chamazulen standard and the sample clearly showed the succesfull purification of standard chamazulen (Fig. 2). Further the comparison of the fraction spectra (Fig. 3) and chamazulen spectra (Fig. 4) revealed that the purified fraction was chamazulen. 30 www.knauer.net/prep 31 AZURA® Preparative HPLC Steviol glycosides are the main sweetening com-The steviol glycoside rebaudioside A is the main pounds in Stevia rebaudiana and are often used compound of interest as it is the sweetest and less as natural sugar substitutes. To enable a commer-bitter compound of the extract. Often Stevia prod-cial usage, the plant extracts need to be purified. ucts contain a mixture of rebaudioside A and ste-In this work preparative online SPE (solid phase vioside. The development of a purification meth-extraction) with the AZURA Pilot Prep LC was in-od with high yield of rebaudioside A, only few Choose the Pilot Prep LC system if you want to increase your productivity vestigated for improvement of overall purity due stevioside impurities, and high throughput in-to reduction of matrix contamination. creases the economic output of Stevia production. even more. As for the AZURA Lab Prep LC system you can freely build up your system. Flow rates up to 1000 ml/min and loads up to several grams are possible. Optional peak and solvent recycling can be set up to increase separation power and reduce separation costs significantly. sample injection waste pump pump UV A B Overload experiments on preparative column, 200 μL Preparative online SPE, 10 mL loading; 1) rebaudioside sample column detector (red), 500 μL (blue), 2000μL (green); 1) rebaudioside A, A, 2) stevioside, blue bars – matrix, 25°C, 22 mL/min pump 2) stevioside, blue bars – matrix, 25°C, 22 ml/min • Pilot Prep HPLC system with binary high pressure gradient • Sample pump • Injection valve • UV/VIS detector with one variable Fraction analysis of preparative online-SPE purification (Fig.2) wavelength of rebaudioside A (1) and stevioside (2); a) F3 (blue), F4 (red), • 12 Port fractionation valve F5 (green), F6 (light blue); b) F7 (red dashed), F10 (blue dashed), F12 (green sashed), F15 (light blue dashed); c) frac- • Intuitive PurityChrom® software tionation of target peak, 5 mL fractions Fig. 1 shows the batch LC without online SPE. The matrix peak (1-5min) negatively affect the separation abilities. In comparision Fig 2 shows that the automated SPE process significantly decreased the matrix. The fraction analysis revealed that only a small part of the overlapping peak contained nearly pure rebaudioside A; fractions 3-5 approx. 15 mL with >90 % rebaudioiside A and <10% stevioside (Fig. 3, B). The later fractions contained high amounts of stevioside but also still rebaudioside A (Fig. 3 C). The results showed that purification of highly pure rebaudioside A is possible by an additional online-SPE . For more information visit www.knauer.net (Application Note VFD0171) 32 www.knauer.net/prep 33 AZURA® Preparative HPLC What is the difference between batch LC and SMBC? Simulated moving bed chromatography (SMBC) is increasingly applied as a separation technique Batch chromatography (single-column) SMB chromatography (multi-column) in the pharmaceutical industry, production of fine Unlimited number of fractions Two fractions, no waste chemicals and in the field of bioengineering. SMB is a method in process chromatography that en-Recovery typically below 80 % Recovery up to 100 % ables substance mixtures to be continuously sep-EITHER high purity OR high yield High purity AND high yield arated and extracted in two fractions. By repeat-ed use of the SMB process each partial fraction Isocratic or gradient Isocratic can be separated into a further fraction – down to binary substance mixtures. High solvent consumption Can be as low as 10 % of batch consumption Typically, the SMB process is set up in advance Very diluted product Product concentration comparable with for a two component mixture. Following this, both input concentration (feed) substances can be immediately extracted in pure form. Lab & pilot Operation Intuitive scale modes software For more information about SMB: www.knauer.net/smb Meet your continous separation Besides the standard SMB con- Based on our preparative soft- task in lab and pilot scale with figuration, different zone con-ware you adapt easily to our a biocompatible or a stainless figurations and Open/-Closed-continous chromatography soft- steel version. Loop are possible to optimise ware. your process. 34 35 Based in Berlin, KNAUER is a medium-sized, ow-tems and components for analytical HPLC / UHPLC, ner-managed company that has been serving the preparative HPLC, fast protein liquid chromato-sciences since 1962. We develop and manufac-graphy (FPLC), multi-column chromatography / ture scientific instruments of superior quality for simulated moving bed (SMB), and osmometry. liquid chromatography. The range includes sys-CEO and owner Alexandra Knauer The founder Dr. Herbert Knauer and his wife and owner of the company since 2000. Several Roswitha are still active as advisers in the awards for outstanding products and innova-KNAUER Academy company to this day. The couple‘s daughter, tions as well as entrepreneurial excellence make Alexandra Knauer, has been managing director KNAUER a „leading employer“. Worldwide partner in science since 1962 Independent and family owned www.knauer.net 36 37 M A K E YOU R PRE SE LEC TION Stainless steel Titanium 6x 6x 5x APPLICATION SERVICES bypass HPLC method development, HPLC method A1 A2 5x waste B1 B2 transfer & optmization, Rent-an-expert detector I detector II fraction collector air sensor COMPLIANCE pump pump sample A B pump 6x Qualification documents, Material certifica-KNAUER tion, FAT/SAT, Capillary labeling Services SOLVENT SELECTION SAMPLE COLUMN SELECTION DETECTION FRACTION & DELIVERY INJECTION & THERMOSTAT COLLECTION 50 ml/min binary Injection valve Column selection UV/VIS Fractionation valve TECHNICAL SERVICE & SUPPORT gradient pump P 6.1L (two columns or single wavelength Installation & Instruction, Maintenance, Sample pump module one bypass) Foxy fraction Repair, Support ..... x 100 ml/min pump P 2.1L UV/VIS multiwave collector with fixed Sample selection Column selection length rack types ..... x 250 ml/min pump P 2.1L ......valve: ..... x inlets high flow (5 columns, one DAD 2.1L Labocol fraction ..... x 500 ml/min pump P 2.1L Autosampler AS 6.1L bypass) collector with individual Conductivity rack types ..... x 1 000 ml/min pump P 2.1L ACADEMY pH Rack for fraction Ternary gradient collector HPLC trainings, Specialized courses and work-module for pump P 2.1L Refractive index shops for beginners and experienced users Flow splitter Binary gradient module Light Scattering for pump P 2.1L 4000 MiD ..... x solvent selection valve A/D-converter (integration of further detectors) All standard user instructions, helpful video tutorials, and a structured section of frequently asked questions is freely accessible on our web page www.knauer.net. If you need further support, our friendly Support team is happy to help you via e-mail, phone or Team Viewer. They will work with you personally until all issues are resolved. ACCESSORIES Phone: +49 30 809727-111 (workdays 9–17h CET) ..... x Airsensor ..... x Airsensor Mass flow controller AZURA Click AZURA Organizer Email: support@knauer.net main pump feed pump ..... x Tubing 1/16” ..... x Tubing 1/8” ..... x Tubing 1/4” Workstation (Windows) SOFTWARE COMMON APPLICATIONS ClarityChrom® OpenLAB® PurityChrom® Reversed phase Normal phase Chromeleon™ Mobile Control other... System Qualification 38 www.knauer.net/prep Technical data or prices are subject to change without All trademarks are the property of their respective owners. notice. Prices may vary by country and do not include Our general terms and conditions apply: taxes, customs duties or delivery. www.knauer.net/terms Distributed by: S/0.75/05.19U 7820 019 V ER 2UAN © K KNAUER +49 30 809727-0 Wissenschaftliche Geräte GmbH +49 30 8015010 (Fax) Hegauer Weg 38 info@knauer.net 14163 Berlin, Germany www.knauer.net