AZURA® Prep LC  What do you want to purify today? 2 Select the right purification process for your sample A preparative HPLC system should be as versatile as possible, since purification tasks can change frequently. AZURA Prep LC was designed for flexi bility and to comfortably handle large sample volumes. Easy to operate and maintain the AZURA Prep LC systems are perfectly suited for the purification of your products such as synthesis stages or active ingredients. The systems can be optimally adapted to the scale you need. The KNAUER team will support you in designing the system of your choice. About KNAUER KNAUER is an owner-managed middle-sized company situated in Berlin, Germany. Since 1962, we have been developing and manufacturing laboratory instruments for customers around the world. We are the oldest German manufacturer of instruments for HPLC, FPLC, simulated moving bed (SMB) chromatography and osmometry. 2 What’s your priority? The dependencies between throughput, purity and yield always have to be considered in LC purifications. Whatever priority you decide for, with the AZURA Prep LC systems you can successfully adapt. Throughput Saving time Short run time, low purity or yield Cheap product, low recover y, time saving Low purity Time efficient, full recover y, Valuable product, high recovery, slow Recovery and yield Purity Maximizing product Reducing impurities Long run time, baseline- Higher yield, lower purity separated www.knauer.net/azuraprep 3 Flexibility and performance! Mobile Control Optional display and control tool for KNAUER AZURA systems (page 22) HPLC column Preparative separation columns including axial compression (page 16) Fiber optics flow cell Flow cell for remote operation and other flow cells (page 13) Column stand (page 20) www.knauer.net/azuraprep 4 Assistant Customizable combination modules integrate up to three units in one device e.g. for column switching (page 8) Injection Manual or automated injection: using a valve, a valve with feed pump, or an autosampler (page 9) Detection Various detector types (UV/VIS, DAD, refractive index, fluorescence) and a selection of flow cells for a wide range of flow rates (page 12) Fraction collection Fractionation valve or fraction collector for various max. flow rates (up to 1000 ml/min) (page 14) Control software Eluent delivery Several solutions for A choice of pumps with pump heads available in different materials purifications available allows maximum flow rates from 50 to 1000 ml/min. Isocratic, (page 21) low and high pressure gradient forming are possible (page 10) 5 AZURA® Prep LC Purification solutions What is your scale? The modular AZURA Prep LC platform offers you the opportunity to build a purification system best suited to your needs. n AZURA® Compact Prep LC Flow rate max. 50 ml/min Isocratic AZURA system features Available pump heads Gradient options and options Max. flow rate in ml/min 50 100 250 500 1000 LPG HPG AZURA Compact Prep LC  AZURA Lab Prep LC   AZURA Pilot Prep LC       6 n AZURA® Lab Prep LC Flow rate max. 50 ml/min n AZURA® Pilot Prep LC Isocratic/HPG Flow rate max. 1000 ml/min Isocratic/LPG/HPG Peak Recycling options Injection volume Fractionation options All parking LC detectors Feed pump usable Peak Solvent Manual valve Autosampler option Valve Foxy R1/R2    up to 40 ml up to 10 ml        up to 40 ml up to 10 ml        up to 40 ml up to 10 ml     www.knauer.net/azuraprep 7 How often have you wished for an assistant? AZURA® ASM 2.1L is your LC assistant A unique multifunctional device that can be equipped with combinations of up to three integrated modules. Configuration examples • Injection (feed pump and two valves) • Fractionation (valve/s, cascadable) • Column switching and optional back flush (two to three valves) • AZURA Compact Prep LC (pump, valve and UV detector) Can be equipped with combinations of: valve(s), pump(s), or detector n Column Switching Assistant Switching valves are ideal for screening, up-scaling or just for being flexible. These valves can be easily integrated into your system and can be used at pressures up to 400 bar and maximum flow rates of 300 ml/min (depending on the valve type). Column Column Column Column Column 8 Injection Available sample injection methods range from manual to automated using injection with feed pump or autosampler. n AZURA® Pump P 4.1S Integrated into an assistant or as a standalone module: The compact pump AZURA P 4.1S is perfect for feed injection. • Flow rate range: 0.01 – 50 ml/min • 10 and 50 ml exchangeable pump head • Pump heads available in stainless steel or ceramics n Sample Injection Assistant ASM 2.1L The AZURA sample injection assistant for pre parative LC is based on the multifunctional AZURA element ASM 2.1L. This assistant includes a sample pump and two 6-port-3-channel valves. It is possible to inject small sample volumes via a sample loop and injection syringe or large sample volumes via the integrated sample pump. n Autosampler 3950 Do you want to process many different samples fully auto - mated? The Autosampler 3950 can inject up to 10 ml per injection and is well-suited for an automated purification system. For an overview of available sample loops see ... www.knauer.net/sample-loops www.knauer.net/azuraprep 9 Eluent delivery Precise and reliable pumps covering a wide flow rate range, gradient and solvent selection options. n AZURA® Pump P 2.1L The preparative HPLC pump AZURA P 2.1L covers a wide flow rate and pressure range. It has been designed for the purification of milli gram to gram samples. The integrated automatic RFID pump head recognition allows for quick adaption to various appli cations. • Flow rate up to 1000 ml/min • LPG and HPG gradient options • Supports constant pressure mode Exchangeable pump heads (stainless steel or titanium) High or low pressure gradient? A low pressure gradient (LPG) module dynami - cally composes the eluent on the inlet-side or LPG HPG low pressure side of the pump head, by quickly switching between the different solvent chan nels. Low investment costs Less wear We offer binary or ternary LPG upgrade modules for the isocratic P 2.1L. Limited flow rate range No flow rate limitation The eluent in a binary high pressure gradient (HPG) system is composed by combining the Channel usable for feeding or solvent selection High accuracy solvent flows of two isocratic pumps. Covered flow rate HPG Binary LPG Ternary LPG 10 220 800 1000 Binary LPG valve block ml/min ml/min ml/min ml/min for the pump head 10 Pressure sensor Automatic piston seal wash Solvent selection valve High efficiency AZURA mixer n AZURA® Pump P 6.1L The AZURA semi-preparative Pump P 6.1 with 50 ml pump head can be used as an isocratic or binary HPG pump. It is made for medium-size purification tasks and upscaling processes. It also supports constant pressure mode. • Flow rate up to 50 ml/min • Flow rate increment: 0.01 ml/min • Binary gradient with solvent selection valve (2 x 2 solvents) • Up to 300 bar Solvent selection Do you often have to change your solvent? The Pump P 6.1L features a built-in 2 x 2 solvent selection valve, but any AZURA system can be extended with a valve for solvent selection, useful e.g. in method development. Choose your valve with up to 12 ports. www.knauer.net/azuraprep 11 Detection Which detector do you need for your target molecule? We provide a choice of UV/VIS detectors, ranging from single variable wavelength to 8-channel diode array detector with 3D scan capability. Wavelength range 190 –1000 nm 190 –700 nm 190 –700 nm 190 –700 nm 190 – 500 nm AZURA detector UVD 2.1S UVD 2.1L MWD 2.1L DAD 2.1L DAD 6.1L Compact and Reliable UV/VIS detector Robust multi- Versatility High-end diode array versatile for a wide spectrum of channel UV/VIS through a wide detector with outstanding UV detector applications detector flow cell range performance Channels 1 1 8 8 8 3D data acquisition   Data acquisition rate 50 Hz 50 Hz 100 Hz 100 Hz 100 Hz Fiber optics available      n AZURA® RID 2.1L Refractive index detector • Active temperature control • RI range: 1.00 –1.75 RIU For cost-effective, fast and reliable analysis of non-UV absorbent compounds such as carbohydrates and sugars, etc. n AZURA® CM 2.1S Conductivity and pH Monitor • Flow rates up to 100 ml/min • 1 μS/cm – 999 mS/cm Basic and versatile monitor • pH range 2 –12 for biopurification applications. 12 Flow cells for UV/VIS and DAD detectors Select from an impressive range of easily exchangeable preparative and semi-preparative flow cells for UV/VIS and DAD detectors. With capillary connections ranging from 1/16” to 3/4”, optional fiber optics technology and a variety of flow cell wetted materials, a wide spectrum of applications can be covered. Max. flow rate Connectors Path length Volume Max. pressure Fiber optics version 100 ml/min 1/16’’ 3 mm 2 μl 300 bar  100 ml/min 1/16’’ 0.5 mm 3 μl 200 bar  500 ml/min 1/8’’ 0.5/1.25/2 mm 1.7/4.3/6.8 μl 200 bar  1000 ml/min 1/4’’ 0.5/1.25/2 mm 1.7/4.3/6.8 μl 200 bar  Process-scale flow cells also available with TRI-Clamp connection up to 3/4’’ Fiber optics technology for more flexibility Fiber optic cables offer the possibility to separate the flow cell from the detector. This enables demanding applications such as measuring directly after a heated Fiber optics flow cell with TRI-Clamp LC column or in hazardous environments, allowing safe connection for high flow applications operation of the instrument while maintaining performance. Fiber optics also allow working in hazardous environments www.knauer.net/detectors 13 Fraction collection How do you typically collect your sample? Manually – valve switching triggered by user via instrument or chromatography software Time-based – collection in time intervals Peak-based – collection according to detector signal … KNAUER supports all of these methods Fraction collecting with KNAUER valves The simplest, yet very effective option for fraction Solvent recycling collecting are our multi-position valves. Regardless if you HPLC system just want to collect your product or want to improve Peak recycling your purification by solvent and peak recycling – our valves offer you all the options – available as a single device or integrated into an Assistant ASM 2.1L. Fractionation valves max. flow rate 1/16’’ Waste Collecting 1/8’’ 1/4’’ 100 500 1000 ml/min ml/min ml/min 14 Which collection device fits your method? Device Max Racks Different Max. fractions Stand alone Cooling/ RFID rack flow rate rack types (1/16’’; 1/8’’; heating recognition (ml/min) 1/4’’) option Valve 1000 16/12/10  Foxy R1 25/125 1 15 up to 144   Foxy R2 125/1000 2 15 up to 288   n Fraction collectors Foxy R1 or R2 The fraction collectors Foxy R1 and Foxy R2 can collect samples in the working range of up to 1000 ml/min (R2), depending on rack type used. The Foxy R1 can be cooled. The two-rack fraction collector Foxy R2 uses radio frequency identification to automatically recognize collection racks for easy setup. • Choice of racks: capacities from two 96 well microtiter plates up to nine 480 ml bottles, or virtually unlimited fraction volumes with funnel racks • Your sample is kept stable either via cooling option or coldroom usage • User-friendly touch screen • In stand-alone mode, fractions can be collected by time, drop count or pumped volume www.knauer.net/fractionation 15 KNAUER preparative columns and AZURA Prep LC systems – a perfect match! Available column dimensions High performance • Excellent performance due to optimum packed bed density and stability in a universal design • Reproducible column efficiency Application flexibility with ... n Eurospher II • Silica material is available in 13 different modifications • Particle sizes of 5 μm, 10 μm, 15 µm, 20–45 µm, ideal for prep. LC • Can replace e.g. Kromasil, Nucleosil, Inertsil, Betasil … n Eurokat Pre-columns High performance polymer phases for the separation of complex mixtures of organic acids, carbohydrates, and alcohols. 16 n Vertex Plus AX Column hardware with axial compression. Axial compression for extended life time Extend the life time of your column with column bed regeneration by axial compression. Regenerated column After 700 injections New column 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Minutes Principle of axial compression 1. After extended use: 2. Regeneration by bed compression: fixed column part adjustable column part Demanding application conditions can deteriorate separation performance through small bed fissures. Regain performance by compressing the bed with the adjustable column part of the Vertex Plus AX column. Refill service Cost-effective Only for Vertex Plus AX: Save up to 20 % of the Taking the features and advantages of the KNAUER original column price.* Vertex Plus AX preparative HPLC columns into account, *no refill service for precolumns we offer a highly competitive product. www.knauer.net/prepcolumns 17 Eurospher II, a true application allrounder Eurospher II is a high performance column material for analytical, semi-preparative and process-scale applications. • Ultra pure spherical silica gel • Narrow particle and pore size distributions • Outstanding mechanical stability • Particle sizes: 5, 10, 15, 20 – 45 µm • Pore size: 100 Å With the wide range of different surface modifications available, all application fields in reversed phase and normal phase modes are covered. Normal phase mode Phase/USP code Key application CN Allrounder for applications Pharmaceuticals USP L10 in normal as well as reversed phase mode Diol Alternative to the silica Phospholipids in lecithine USP L20 packing with shorter equilibration time and comparable selectivity Si Wide range of different Fat soluble vitamins USP L3 applications, also for normal phase HPLC; good choice for analytical and preparative purposes to separate polar compounds Eurospher II is also available in the following modifications: Phenyl, C8, C8 A, C4, NH2 , HILIC 18 C18 HR C18H HR 10.49 11.80 IEX pH 2.7 HS IEX pH 2.7 HS 1.55 1.56 0.07 0.07 0.12 0.12 1.40 1.41 0.43 IEX pH 7.6 0.46 SS IEX pH 7.6 SS HBC HBC C18A HR C18P HR 15.20 IEX pH 2.7 HS 4.29 IEX pH 2.7 HS 1.45 1.64 Selectivity plots for phases are based 0.07 0.06 on Tanaka tests: 0.32 0.12 HR: hydrophobic retention 1.45 1.48 HS: hydrophobic selectivity 0.38 IEX pH 7.6 SS 0.71 IEX pH 7.6 SS SS: steric selectivity HBC: hydrogen bonding capacity IEX pH 7.6: ion exchange capacity at pH 7.6 HBC IEX pH 2.7: ion exchange capacity at pH 2.7 HBC Reversed phase mode Phase/USP code Key application C18 Classical C18 phase for Steroids USP L1 acidic, basic and neutral analytes C18A Polar endcapped C18 phase Water soluble vitamins USP L1 for alternative selectivity and 100 % aqueous eluents C18H For acidic, basic and neutral Acidic and basic analytes USP L1 analytes with extended pH range from pH 1 to 12 C18P High 20 % carbon load, Enantiomers like hop USP L1 fully endcapped; excellent constituents shape selectivity (steric cis-trans separations) and pH stability www.knauer.net/eurospherii 19 Accessories Improve system performance, organize your lab bench, and work more conveniently with the right accessories. n Eluent and column heater Minimize solvent viscosity. Increase performance. Column Heating Sleeve* With the KNAUER electrical eluent heating element and Up to 100 °C* the column heating sleeve combination you can easily adjust the temperature of your preparative HPLC system, while minmizing unwanted inner column temperature gradients. *Temperature range and dimensions can be customized to your needs. Eluent Heater* n Multi Column Base n Benchtop Racks A robust column stand for up to three preparative Available in different heights to easily stack LC columns. The included stand rod allows for flexible components with different footprint like attachment of other accessories. fraction collectors or autosamplers. n Prism Column Holder Made to reliably hold a single column (max. ID: 50 mm). www.knauer.net/accessories 20 Software solutions n ClarityChrom® More supported software packages: ClarityChrom is an easy-to-use chromatography data n OpenLAB® CDS EZChrom system for workstations. The optional extensions for GC control, PDA, GPC and MS make the software Edition adaptable to a wide spectrum of applications. Practi - ti cally all KNAUER HPLC devices can be controlled. The OpenLAB CDS EZChrom Edition offers advanced functionality with a user-interface known from the Additionally, devices and systems from more than 45 ChromGate CDS. The direct fraction control and the manufacturers are also supported. ClarityChrom® option to use the KNAUER electric valves for fractiona-includes the drivers for several fraction collectors and tion give you additional flexibility. supports peak recognition by level and/or slope. The manual fraction control and the option to use the KNAUER electric valves for fractionation give you even n PurityChrom® more flexibility. The software PurityChrom is designed for liquid • Fraction collecting via peak recognition (level only, chromatography purification tasks. Although pre-set slope only, level AND / OR slope – incl. self-learning) for volume-based operation, you have the freedom or single event (unconditional, timed event) of creating methods also based on volume or time. Advanced fractionation options include logical com- • Easy to collect: waste, collect to position / collect to binations of detection criteria and can be carried out next, solvent recycling using a fractionation valve as well as a fraction collector. • Direct control during a run – manually switch to: collect, waste, solvent recycling • Consecutive runs: easily find your chromato gram n Chromeleon™ by clicking on your fraction Chromeleon is one of the most wide-spread chromatography data systems. Its intuitive handling benefits laboratory workflow and the highly developed algo-rithms simplify data processing. It offers a broad range of third-party drivers and can be easily used with existing HPLC systems. KNAUER offers drivers for a lot of its devices. www.knauer.net/software 21 Mobile Control AZURA® devices at your fingertips n AZURA® Mobile Control With the Mobile Control app you have your AZURA devices at your fingertips: Easily check the status or set parameters of several devices or several systems simul-taneously, even outside the laboratory. With the possibility of data acquisition, the Mobile Control is the perfect addition to the AZURA Compact Prep LC system. This combination represents a truly space-saving HPLC solution. No additional computer or software is needed. Control every AZURA system in your laboratory with one tablet. Mobile Control is safe: it ensures that device parameters can only be changed by authorized operators. The Mobile Control app can be run from a tablet or PC with Windows 8 or higher. Depending on the hardware control is possible via WLAN or Ethernet. • Control your devices or systems from your desktop • Easily switch between your AZURA systems and monitor all of them • Create different user accounts to securely manage your systems • Work effortless with the intuitive user-interface • Optionally record data and control fraction collection www.knauer.net/mobilectrl 22 Benchtop purification n AZURA® Compact Prep LC This system fits on any lab bench and allows purifi cation tasks up to 50 ml/min. Additional devices can be easily added any time due to the modular design of AZURA. Multi-position valve for fractionation Manual Recycling option available injection valve Detector UVD 2.1S Pump P 4.1S 190–500 nm with 50 ml pump head, Variety of flow cells made of stainless steel Fiber optics option for remote flow or ceramic cell applications 23 How flexible do you need to be? n AZURA® Lab Prep LC 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 50 ml/min it is possible to separate up to several hundred milligrams per run. Assistant Combine up to three additional devices (valves, additional pumps or UV detector). Fractions can be collected via multi-position valve or – if you desire even more flexibility – with the fraction collector Foxy R1/R2. Detector Choose between detection types: UV (variable wavelength, multiple wavelength, diode array), RI, ELSD, conductivity, pH, or fluorescence. Pump Regardless if an isocratic or HPG system is needed, the P 6.1L is the perfect pump for your semi-preparative separations up to 50 ml/min and 300 bar. 24 Peak and solvent recycling Reduced cost and increased purity Vitamin E An example A separation can be much more demanding after upscaling from analytical to preparative 5 µm 20 µm scale. In many cases a baseline separation is particle particle size size not possible anymore, so time and money consuming method development or hardware adjustments have to be done. The AZURA Lab Prep LC system is well-suited to apply peak recycling technique to solve demanding resolution tasks. Additionally, solvent recycling can be applied Time Time to save eluent, if it can be considered clean. Comparison of analytical and preparative chromatogram. Collecting mode Cycle: 1 2 3 4 400 350 300 250 200 150 Valve switching 100 50 Recycling mode 0 0 5 10 15 20 25 30 35 40 45 50 Minutes Peak Fractio- Solvent Recycling nation Recycling Successful peak separation with recycling mode. www.knauer.net/azuraprep 25 The upscaling expert! n AZURA® Pilot Prep LC Choose the Pilot Prep LC system if you want to increase your productivity 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 Assistant Feed assistant for manual injection or sample injection via pump. Detector Variable wavelength UV detector. Other detectors supporting up to 1000 ml/min can also be combined with the AZURA Pilot Prep LC. Fraction collection Fraction collection is possible via multi-position valve for up to 16 fractions. Alternatively the Foxy R2 can be used if more fractions need to be collected (flow rate up to 1000 ml/min). Pump The pump AZURA P 2.1L with 100 ml pump head can be used for flow rates from the analytical range to 100 ml/min. The pump performs very well in the whole flow rate range. This makes the system very suitable for up-scaling tasks. The LPG module connected to the pump head adds binary gradient functionality to system (ternary also available). Pump heads can be easily exchanged. The max. available flow rate is 1000 ml/min. 26 Upscaling in one system Absorbance Absorbance 0 10 Minutes 20 0 10 Minutes 20 Analytical Preparative The AZURA Pilot Prep LC is the ideal solution for your upscaling tasks. The 100 ml pump head allows you to run your system under analytical conditions before adapting your method to preparative scale. Scaling up of a separation of apple flavors from extract 1 1 trans-2-Hexen-1-al 2 trans-2-Hexen-1-ol Blue Eurospher II 100-10 C18, 150 x 4 mm 3 Methyl 2-methylbutyrate, Ethyl isobutyrate, Green Eurospher II 100-10 C18, 150 x 20 mm Ethyl butyrate, n-Butyl acetate 4 Ethyl 2-methylbutyrate, Ethyl valerate, 3-Methylbutyl acetate, 2-Methylbutyl acetate 5 5 other compounds (elution with higher Ethanol 2 concentration) 3 4 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Minutes Get started! With the KNAUER ScaleUp Converter, you can easily calculate the method parameters to start from, e.g. how to move from a 4 mm ID column to a 30 mm ID column. www.knauer.net/azuraprep 27 Preparative HPLC columns KNAUER ScaleUp Converter HPLC columns for lab- Method transfer from scale purification tasks, analytical to preparative featuring axial compres- HPLC becomes easy with sion of the column bed. this free software tool. Significantly longer- Enter analytical parame- lasting performance ters and get the required than standard columns. parameters for your scaled-up LC method. www.knauer.net/precolumns www.knauer.net/scaleupconv How do you control your Preparative HPLC intro HPLC system? This presentation gives a brief introduction to the With the AZURA Mobile topic of LC purification. Control App you can control selected AZURA devices from your tablet. As addition to your CDS or for stand-alone use. www.knauer.net/mobilectrl www.knauer.net/prepintro Distributed by: Application support Benefit from our expe- rience in preparative chromatography and our extensive laboratory equipment. Our HPLC experts support you to find the best solution for your task. Technical data are subject to change without notice. www.knauer.net/appsupp All trademarks and registered trademarks are the property of their respective owners. www.knauer.net HPLC · SMB · Osmometry KNAUER Phone: +49 30 809727-0 Wissenschaftliche Geräte GmbH Telefax: +49 30 8015010 Hegauer Weg 38 E-Mail: info@knauer.net 14163 Berlin, Germany Internet: www.knauer.net © KNAUER 2015 V7820US/3.0/11.15/studio.w Document Outline Introduction Table of contents Overview of AZURA features and options Features in detail Injection Eluent delivery Detection Fraction collection Preparative columns Accessories Control solutions System examples Further reading