Science Together AZURA® Analytical HPLC/UHPLC Stay flexible regarding flow rates and upgrade options AZURA® Analytical HPLC In HPLC, components of a mixture are carried migration rates among the sample components. through the stationary phase by the flow of a mobile Therefore, the nature of your analytes defines not HPLC mode Mobile phase Stationary phase Analytes phase and separation is based on differences in only the method but also the HPLC system. Separation of small molecules (up to 2000 Da) Reversed Phase Polar Nonpolar Mid-polar – mid-nonpolar RP (<200 Å) (e.g. mixtures (e.g. C18) soluble in polar and from water and aqueous solvents Main characteristics of the analytes: acetonitrile) Normal Phase Nonpolar Polar Nonpolar MOLECULAR WEIGHT defines the pore size of NP (e.g. heptan) (e.g. SiOH) soluble in nonpolar solvents, insoluble in the stationary phase. water SOLUBILITY defines the HPLC mode, chemistry Hydrophilic Interaction Polar Polar hydrophilic and highly Liquid Chromatography organic sol- (e.g. SiOH) polar of stationary phase and eluent. HILIC vents + water not retained by RP CONCENTRATION AND MATRIX defines the Water layer between stationary detection parameters and column dimensions. and mobile phase Separation of biomolecules (larger than 2000 Da) Wide Pore Polar Nonpolar Mid-polar – mid-nonpolar Reversed phase soluble in polar and RP (>200 Å) aqueous solvents The solubility of your analytes defines the HPLC mode. The elutropic series defines Polymer gel based stationary phases the solvent strength for the most often used Ion Exclusion & Water, acidic Organic resin with Sugars, organic acids, chromatography modes normal phase and Ligand exchange water charged groups alcohols reversed phase. 2 www.knauer.net/azuraanalytical 3 AZURA® Analytical HPLC thermostat A1 A2 column B1 B2 pump pump A B detector detector ll Efficient and adaptable to your needs autosampler The analytical HPLC and UHPLC systems of the tool to overcome your analytical challenges. KNAUER AZURA liquid chromatography instru-Choose between different gradient forming tech-ELUENT SAMPLE COLUMN SELECTION & DETECTION ments are designed to support and facilitate your nologies and maximum flow rates to find the best DELIVERY INJECTION THERMOSTAT work. Whether doing routine analysis or demanding configuration for your task. separation tasks, AZURA systems are the right Most flexible Supporting OpenLAB, Matching detectors system configurations ClarityChrom® and according to Chromeleon™ your analytes Eluent delivery Detection A choice of pumps Various detector types available in different (UV/VIS, DAD, RI) and flow rates and flow cells pressure ratings (see page 15) (see page 6) • Isocratic binary high pressure gradient (HPG) or quaternary low pressure gradient (LPG) pump • Pump heads allowing flow rates up to 10 ml/min Tempering at 700 bar or 1 000 bar up to 2 ml/min Column thermostats • Autosampler with 0.1 μl sample aspiration at max. 1 240 bar with Injection modules and column switching zero sample loss Automatic or solutions • Choice of highly sensitive UVD, MWD, DAD, or RID detectors with manual injection (see page 10) intelligent temperature control (see page 8) • Wide range of flow cells available, including remote cells • Finger-tight high pressure stainless steel capillary connections “K-Connect” • Extensive safety features such as leak management and sensors • Frontal access of detector lamp and pump head for easy mainte-nance 4 www.knauer.net/azuraanalytical 5 AZURA® Analytical HPLC Advantages and disadvantages of HPG and LPG Choose your HPLC pump according to your ap-or pump heads with a flow rate of 5 ml/min and plication’s needs. Gradient formation, mixer size 1000 bar backpressure. Special pump heads for and pulsation compensation will have an exten-normal phase applications will help to deliver HPG (Binary HPG pump) LPG (Quaternary LPG pump) sive influence. robustly even demanding eluents like heptane Choose between pump heads with a maximum or hexane. Pumps without a degasser offer a cost Small gradient delay volume Higher gradient delay volume flow rate of 10 ml/min and 700 bar back pressure, effective alternative. Fast methods For classical HPLC methods: no difference For UHPLC: slower gradients AZURA® pump filter Piston seal wash Only binary gradients possible Ternary and quaternary gradients possible protects the column from will clean the back piston contamination. space for longer seal life. 2 pumps that have to be maintained Only 1 pump, but shorter maintainance intervalls because of higher usage Solvent selection valve is integrated in every binary HPG eluent eluent eluent eluent pump. Each channel can be used A B A D with two different solvents. mixer eluent eluent B C P 6.1L pump pump A B HPG LPG valve Additional 2- or 4-channel online degasser column dwell volume eliminates gas bubbles and reduces in LPG pump baseline noise, resulting in a high- er sensitivity. column P 6.1L LPG The dwell volume The dwell volume is responsible for the time de-AZURA® mixer lay for a gradient. By definition it is the volume of Highly efficient microfluidic solvent a gradient HPLC system between the mixing Whether an LPG or an HPG pump is recom-mixer combines high mixing per- chamber and the column inlet. It is typically high-mended highly depends on the userś pref-formance with a low dead volume. er in LPG systems caused by the setup of the erences. The user-changeable mixer is pump. Therewith, gradients are faster with HPG Only if very fast UHPLC gradients are need-available in different volumes systems. ed, an HPG will be recommended. (50/100/200 µl) allowing best If a quaternary gradient is inevitable, an LPG possible adaption to any applica- pump has to be applied. tion. Multi-proportioning valve In any other case: The user decides! will precisely blend up to 4 eluents. 6 www.knauer.net/azuraanalytical 7 AZURA® Analytical HPLC Most important factors in injection are precision, accuracy and carry- over. They are effected by the injec- Principle of manual injection tion technique and equipment and in case of manual injection also by Manual injection valves are the most cost-effective Load position Inject position the user. option to introduce samples. Normal y, valves with sample sample 6 ports and 2 positions - for loading and injection sample sample injection injection - are used. In the load position a sample loop is filled injection injection The AZURA Autosampler AS 6.1L is a high preci-with sample while the system is equilibrating. waste waste waste waste sion device available for a maximum back pres-When turning to the inject position, the sample loop sure of 1 240 bar. This autosampler can inject from is switched to the high pressure part of the HPLC up to 768 positions when equipped with microtiter system. The flow delivered by the pump flows plates or from up to 108 standard 2 ml sample through the loop and feeds the sample onto the column column column vials. The AS 6.1L is equipped with an ILDTM valve, column. column consisting of a rotor-stator combination with a pump pump pump pump central port for depressurizing the sample loop before receiving the sample. This way, the sample is not diluted with solvent. The AS 6.1L is available KNAUER valves feature a wide back pressure range of up to in two analytical version for 700 and 1240 bar, 1200 bar with a 0.3 mm bore size. All valves can be equipped with respectively. In addition, also a biocompatible ver-an automatic valve drive. In addition, an integration into the assis-sion and one for preparative purposes is available. tant module housing AZURA ASM 2.1L is possible. For automated injection of numerous samples, we recommend to use the autosam- • Up to 1 240 bar (700 bar) pler AS 6.1L. The injection principle - based on a valve and a sample • Optional cool/heat function (4 – 40 °C) loop - is similar, but the AS 6.1L additionally features several injec- • 0.1 μl – 10 000 µl (depending on configuration) tion modes as described below. sample injection volume • Up to 768 samples (microtiter plates) or Injection modes of the autosampler AS 6.1L 108 standard vials • Intermediate Loop Decompression – ILD™ Device Key features Full loop • Sample loop is completely filled with the sample. • Maximum possible reproducibility but not the maximum accuracy is achieved as the specified sample loop volume may deviate by 10 %. • Maximum injection volume equals the loop volume. • Sample loss caused by the need of overfilling of the loop. Partial loop • Sample loop is filled with both sample and mobile solvent. • High precision of the sample volume with minimal loss of sample The Integrated Intermediate Loop • Maximum injection volume equals 50 % of the loop volume decompression valve reduces sample • Sample loss per injection equals the adjusted flush volume of the dilution and increases measurement needle tubing. reproducibility. Microliter pick-up • Sample loop is filled with a very small amount of sample and transport liquid or wash solution (mobile phase). • Very high precision • No loss of sample Rack for 108 standard 2 ml vials 8 www.knauer.net/azuraanalytical 9 AZURA® Analytical HPLC When running HPLC at temperatures above 40 °C, The Eluent pre-heating car-a significant temperature gradient within the col-tridge can easily be retro- fitted to the oven chamber umn is always present when mobile phase pre-of the CT 2.1 by any endus- heating is not applied in air-conditioned labora-er by just connecting 2 tories. screws. Wide space, This device will converge solvent temperature The AZURA CT 2.1 is a forced air column thermostat capable of easy handling with column temperature before entering the heating or cooling from 5 to 85 °C. The powerful fan and robust column and will therefore reduce temperature peltier element keep the column at a very stable temperature, thus gradients within the column. allowing reproducible analysis results. Up to 6 columns Be flexible with KNAUER valves with maximum Temperature gradient 300 mm length in column bed KNAUER valves are designed for a wide range of chromatograph-Eluent pre-heating ic applications. Flexibility is provided by choice of different mate-cartridge for CT 2.1 rials and sizes as well as drivers for various software packages. Columns up to 16 mm ID Available with 0.10 or Eluent pre-column 0.18 mm inner diame- Column thermostat tempering ter (5 µl/18 µl inner vol- Cooling and ume) heating from 5 – 85 °C Rule of thumb Uniform temperature For flow rates higher than 500 µl/min or distribution temperatures above 50°C, the use of a Simplified column switching with KNAUER valves pre-heating cartridge is recommended. Valves are driven either manually or automatically through a valve drive. Flexible capillaries and finger-tight connections provide easy handling while ensuring high pressure stability. Precision manufactured surfaces and edges significantly enhance fluid transfer. K-Connect 1/32” flexible capil- Fingertight up to Adjusts on any laries with 1/16” fitting sleeves 700 bar column port are available in different inner diameters: Column thermostats are an essential part of each HPLC system since most HPLC 0.10 mm ID separation parameters are tempera- ture-sensitive. Hence, the control of the 0.18 mm ID column temperature is one of the most UNF 10/32 0.45 mm ID important prerequisites for reproducible Optional: threads fit in any results in HPLC. wrench-tight up standard HPLC Fit on any capillary to 1200 bar port with 1/16“ OD 10 11 AZURA® Analytical HPLC Inner diameter of the Recommended analytical column safety solution Advantages S TAY F L E X I B L E B E S A F E Any commercially available Find the matching guard col- Minimized dead volume via sintermetal filter 2 mm UHPLC precolumn standard HPLC column can be umn solution for your analytical filter instead of a packed precolumn cartridge, easy exchange. used in an AZURA analytical column and extend its lifetime system. KNAUER HPLC columns even if more agressive eluents 3 – 4.6 mm, KNAUER Vertex I I Low dead volume because no capillary is are compatible with any LC sys-or samples are used. column hardware Integrated precol- used between analytical column and precolumn umn. Can easily be installed later on any tem. KNAUER Vertex III column without a precolumn. F I N D YO U R P E R F E C T M AT C H M A K E I T S I M P L E 3 – 4.6 mm, any column External precol- Precolumn can easily be flushed in reversed hardware umn cartridge and We offer a wide range of station- Easiest up- and downscaling holder flow for cleaning. ary phases for nearly every ap- with the same selectivity in High capacity for analytical and semi- pre-plication field. many different particle- and col- 8 mm External precol- umn parative purposes, easy cleaning by flushing umn dimensions as well as pres-in reversed flow. sure ranges. Which column dimensions are recommend? A guard column system is always recommended to protect the valuable analytical column, but especially when: While the stationary phase always depends on the application, the recommended column dimensions are mainly dependent on the • Agressive eluents are used. pump integrated in your AZURA analytical system: • Injected samples are dirty. • Not only standards but different samples are analyzed. Analysis AZURA pump Typical column dimensions Classical HPLC P 6.1L 700 bar 250 x 4 mm ID / 250 x 4.6 mm ID, 5 µm Smaller column dimensions An integrated precolumn HPLC Plus P 6.1L 700 bar 150 x 3 mm ID, 3 µm = can be exchanged very Shorter analysis time easily by the user. = UHPLC P 6.1L 1000 bar 100 x 2 mm ID, 2 µm Higher back pressure and performance 12 www.knauer.net/azuraanalytical 13 AZURA® Analytical HPLC Modification USP code Highest flexibility When do I use which detector? The detection technique depends C18 P L1 on the characteristics of the analyte. C18 H L1 13 different modifications are available for a wide range of C18 L1 small molecule applications (< 2000 Da) C18 A L1 Nonpolar Chromophor phases: • Reversed phase mode for samples, soluble in water and Phenyl L11 RP UV/VIS and DAD detectors water/organic solvent mixes C8 L7 • Normal phase mode for water insoluble samples Matching Fluorophor C8 A L7 • HILIC mode for highly polar and water soluble samples that detectors Fluorescence detectors C4 L26 are not retained by RP chromatography according to NH₂ L8 your analytes • Ion pair chromatography for acids and bases that are poor-Electrochemically active CN L10 Polar ly retained in RP mode ECD detectors HILIC - phases: NP & Diol L20 HILIC No special characteristics Universal detectors like RI or ELSD Si L3 Variable or multiple wavelength detectors (MWD or VWD) Classical UV detectors record 2D data as a chromatogram. These devices are cal ed VWD or MWD. Why do I need wide pores? Which modification should I choose? Nevertheless, 3D-like measurements can be done by scanning the wavelength range. VWD or MWD small molecule Biomolecules detectors cannot record 3D data over the whole 2 000 – 5 000 Da C18 analysis time but just in programmed time frames. Larger molecules Biomolecules 5 000 – 20 000 Da larger biomolecule Larger pores C8 Shorter Eurosil Bioselect is the first C chains Biomolecules X choice for the analysis of 20 000 – 100 000 Da 3D data means that full UV spectra are mea- • Proteins C4 • Peptides sured and these spectra are plotted over • Oligonucleotides Diode array detectors (DAD or PDA) the whole time. This is useful for unknown analytes or target compounds with different Devices called DAD or PDA detectors are able to UV characteristics. Additionally 2D chro-record 3D data in addition to the 2D data over matograms can be extracted from 3D-data the whole analysis time. at any wavelength. A complex separation consisting of ligand ex-fonated cross-linked styrenedivinylbenzene change, ion exclusion, ion exchange, size exclu-copolymer, 4 ionic forms are available for special sion and partition mechanisms. Based on a sul-applications: Eurokat is ideal for the organic H Sugars, organic acids and alcohols solvent free analysis of: PB, Ca Small carbohydrates (mono- and disasccharides) • Fruit beverages • Soft drinks Na Sugar oligomers (up to DP 8) • Wine • Beer • Fermentation broths 14 www.knauer.net/azuraanalytical 15 AZURA® Analytical HPLC Choose between single variable wavelength, multiple variable wavelength and 3D diode array detectors. Fluorescence detection These detectors allow to develop highly selective methods with sensitivities three to six orders of magnitude greater than UV detection. Single variable wavelength detectors Stackable AZURA L housing + Very sensitive method AZURA® UVD 2.1L + Easy handling Target analytes: Molecules carrying a chromo- - Only for fluorescent molecules phore, all analytes in a sample that absorb at the same wavelength Electrochemical detection Very sensitive, selective detection of oxidizable/reducable compounds. In amperometric electrochemical detection the electrical current is measured resulting from oxidation or reduction reactions. Wavelength range: Data rate up to 50 Hz + Very sensitive method 190 – 750 nm + Lowest LODs Modern detectors can record data rates up - Only for special applications to 100 Hz or even more. These high data - Handling is not for beginners rates are needed in very fast UHPLC. For classical HPLC, 20 or 50 Hz are typically suf-ficient. Refractive index detection (RI) Normal RI detection is a cost-effective solution for the analysis of sugars, Multiple variable wavelength detectors and diode array detectors polymers, surfactants and other compounds that do not contain 30˚ a chromophore. These detectors measure the ability of analyte AZURA® MWD 2.1L, DAD 2.1L and DAD 6.1L molecules to bend or refract light. Target analytes: Molecules carrying a chromo-45˚ phore, adsorption at different wavelengths or for + UV absorbing solvents usable - No gradient elution method development + Very price attractive - Comparably low sensitivity Wavelength range: + Easy to use MWD 2.1L & DAD 2.1L: 190 – 700 nm Data rate DAD 6.1L: 190 – 1 000 nm up to 100 Hz Evaporative light scattering detection (ELSD) Incident light As an universal detector, an ELSD offers numerous possibilities for detecting substances that have few or no chromophores. Since the eluents are evaporated, the use of non-UV-compatible solvents Easy lamp poses no problems and the ELSD is gradient compatible. exchange due to frontal Scattered light + Gradient elution possible - Comparably cost-intensive access + Comparably high sensitivity - Very clean solvents needed LightGuide flow cells and - Nitrogen source needed Polka-Dot technology - Not suitable for volatile compounds 16 www.knauer.net/azuraanalytical 17 AZURA® Analytical HPLC PressureProof Flow cells • Price attractive and robust • Possibility to couple a second detector or LC dimension caused by the pressure stability up to 300 bar Analysis Typical HPLC column dimension Main objective Recommended flow cell • Also suited for higher flow rates up to 20 ml/min Robust method Analytical PressureProof Classical HPLC 250 x 4 mm ID / LightGuide Flow cells 250 x 4.6 mm ID, 5 µm High sensitivity High sensitivity LightGuide • Total reflection technology for high sensitivity Robust method Analytical PressureProof • Minimized cell volume for high resolution High resolution and HPLC Plus 150 x 3 mm ID, 3 µm fast runs Standard LightGuide Comparison of flow cells with 10 mm path length: High sensitivity High sensitivity LightGuide PressureProof vs. LightGuide High resolution and UHPLC 100 x 2 mm ID, 2 µm fast runs Standard Lightguide Higher peaks with LightGuide caused High sensitivity High sensitivity LightGuide by total reflection technology Micro and 100 x 0.3 mm ID, 5 µm Sharper peaks with LightGuide caused Nano LC 50 x 0.075 mm ID, 3 µm Lowest volume Nano flow cell with fiber optics by lower cell volume The great advantage of LightGuide flow cel s and their KNAUER flow cells in cartridge design are low dispersion volume seen in UHPLC does not play very easy exchangeable by just clicking Blue: Analytical PressureProof a major role in classical HPLC as the complete system them out without any tools. Red: Standard LightGuide dispersion volume is much larger. Comparison of LightGuide flow cells: Standard vs. High Sensitivity Blue: Standard LightGuide with 10 mm path length Separate the flow cell spatially from the detector Red: High Sensitivity LightGuide with 50 mm path length via fiber optic cables in a distance up to 10 meters. Peaks get around 4 times higher with Lightguide High Sensitivity flow cell Fiber optics adapter cartridge Nano flow cell 18 www.knauer.net/azuraanalytical 19 AZURA® Analytical HPLC One of the smallest single variable wavelength detectors on the market Temperature Wide linear dynamic range: AZURA® RID 2.1L AZURA® UVD 2.1S control: 30 – 55 °C > 1000 µRIU The AZURA RID 2.1L is a highly competitive and The AZURA UVD 2.1S offers excellent technical Highly competitive sensitive refractive index detector, ideal for fast specifications for routine laboratory work. With UV detector and reliable routine analysis of non-UV absorbing its small footprint, it is one of the smallest detec-substances. The intelligent temperature control tors for HPLC on the market. Wavelength range: guarantees fast baseline stabilization and stable 190 – 500 nm operation. Flow rate: Data rate up to 100 Hz Target analytes: Alcohols, sugars, saccharides, up to 10 ml/min fatty acids and polymers Fluorescence Detector RF-20A Pressure resistant The fluorescence detector RF-20A provides world-class sensitivity, flow cell (5 bar) excellent maintainability and diverse validation / support functions. It supports a wide range of applications in the wavelength range Light Scattering Detector Sedex 85LT, Sedex 90LT and Sedex 100LT of 200 to 650 nm from conventional analysis to high-performance analysis. Sensitive universal detection with the possibility to run gradients Target analytes: Fluorecent molecules like polycyclic aromatic hy-As an universal detector, an ELSD offers numerous drocarbon, fluorescence tagged analytes like amino acids or proteins possibilities 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 gradi-Electrochemical Detector AZURA® ECD 2.1 ent compatible. With its measurement frequency up to 100 Hz, this electrochemical detector is specially designed for super-fast highly sensitive Target analytes: Carbohydrates and similar com-and selective measurement of oxidizable and reducible substanc-pounds, detergents, ionic and non-ionics, artificial es in (U)HPLC. The AZURA ECD 2.1 comprises of a thermostat-con-sweeteners, antioxidants, amino acids, lipids, trolled Faraday’s cage, accommodating column and flow cell. peptides, polymers, pestizides, proteins, steroids Target analytes: Vitamins, neurotransmitters, carbohydrates, antioxidants, antibiotics, phenols and polyphenols, bisphenol A, pesticides ELSD is a good replacement for a Charged Aerosol Detector (CAD). Just choose the Interface Box IFU 2.1 LAN right fitting nebulizer and get the best out The KNAUER interface box IFU 2.1 LAN allows highly precise ana-of the ELSD for your analyte detection. log data acquisition of third party modules over analog and relay outputs. Example: MALS-detectors for molecular weight determination. 20 www.knauer.net/azuraanalytical 21 AZURA® Analytical HPLC Product Features Page AZURA Analytical This system features a AZURA P 6.1L HPG pump, an autosampler 24 UHPLC system 6.1L, a column thermostat CT 2.1, a DAD 6.1L UV/VIS detector KNAUER’s monitoring and control software with 8-variable UV measuring channels from 190 - 1000 nm, as well as a Tablet PC with AZURA Mobile Control. It is the UHPLC for AZURA® devices and systems system with a backpressure range of 1000 bar. It perfectly fits the demanding needs of a method development system, as With the hand-held Mobile Control (Chrom) option you have your well as the robust fitness of a routine analysis machine. AZURA devices at your fingertips. Remotely control and monitor your system and enjoy the touchscreen-optimized user interface. AZURA Educational Complete isocratic analytical HPLC system, UV/VIS detector 25 Choose Mobile Control as an easy-to-use and cost-effective soft-system with one variable wavelength, pump unit with pressure sensor ware solution! for a low pulsation eluent supply, optional AZURA Mobile Control app allows direct control of all integrated modules. Mobile Control Mobile Control provides full access to AZURA devices. Change device settings, set operating parameters, automate device control or check the system status… Mobile Control features all function-alities of a display. Do you want more than a display without using AZURA Analytical HPLC up to 700 bar in LPG version for quaternary gradients, 26 an advanced chromatographic data system? system for analysis of 10 mL pump head, 8-channel DAD detector with 3D UV/Vis carbonyl emissions in air detection at 190-700 nm and up to 100 Hz, column thermostat, Mobile Control Chrom automated injection via autosampler, Mobile Control for Mobile Control Chrom features data acquisition from AZURA de-monitoring and control of the integrated modules, Clarity-tectors in addition to full device control. Chrom CDS for instrument control, data acquisition and data processing. OpenLAB® CDS EZChrom Edition Chromeleon™ 7.2 Drivers OpenLAB CDS EZChrom Edition is the next gen-Thermo Scientific™ Dionex™ Chromeleon™ is one eration of chromatography data systems. It proof the most wide-spread chromatography data AZURA GPC Cleanup The system features 15 sample loops controlled by two 16 port 27 vides chromatography data acquisition, process-systems. Its intuitive handling benefits laboratory system multiposition valves. With an additional multiposition valve 15 ing and control of GC and LC chromatographs workflow and the highly developed algorithms fractions and waste can be collected. All multiposition valves and is used in chromatography operations rang-simplify data processing. It offers a broad range and the manual injection valve are integrated into a compact ing from single user/single instrument to of third-party drivers and can be easily used with AZURA® Assistant ASM 2.1L. Elution of separated substances/ multi-user/multi-instrument laboratories. existing HPLC systems. KNAUER offers drivers for standard components is monitored by a variable single wave-a lot of its devices. length UV detector. A second assistant harbors the small detector, the system pump with a pressure sensor and a valve ClarityChrom® CDS to bypass the GPC column or select between two columns, ClarityChrom is an easy-to-use chromatography Mobile Control for system control. data system for workstations. The optional GPC-AZURA sugar system HPLC up to 700 bar in isocratic version, 10 mL pump head, re-28 and Knauer FRC control modules and extensions fractive index detector, column thermostat, manual injection via for PDA, SST, SEC/GPC and MS allow using the an injection valve, Mobile Control for monitoring and control of software for a wide range of applications. the integrated modules, ClarityChrom CDS for instrument control, data acquisition and data processing. 22 www.knauer.net/azuraanalytical 23 AZURA® Analytical HPLC Determination of 14 compounds in 1.5 minutes using the For tomorrow’s HPLC professionals AZURA® UHPLC system Your AZURA Educational System includes printed training material that explains several applications UHPLC for quality control of dark chocolate of HPLC. For example, the quantitative determi-12 • Polyphenols and alkaloids like caffeine or nation of caffeine and paracetamol in samples of theobromine your choice (e.g. coffee, soft drinks, medicine) is explained in detail. AUm 9 14 13 In simple steps, students are introduced to 11 basic concepts and terms used in HPLC such 4 as calibration curve and internal standard. 2 5 The easy to understand instructions guide 6 10 1 3 7 8 them through the complete experiment, including setup, run and analysis. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Min UHPLC system 1 Acesulfam K; 2 Theobromine; 3 Saccharin; 4 Theophylline; Analytical HPG configuration with DAD detection 5 Caffeine; 6 Chlorogenic acid; 7 Catechin; 8 Epicatechin; What is included? 9 4-Hydroxbenzoic acid; 10 Vanillin; 11 Guaiacol; 12 Sorbic acid; 13 Methylparaben; 14 Propylparaben Devices Key features HPLC system AZURA AZURA Compact HPLC with pump P 4.1S and detector UVD 2.1S with Educational System flow cell (10 mm path length, 1/16“ connectors) Devices Key features AZURA Pump P 6.1L HPG pump, 0.001–5 ml/min, Sample application Manual injection valve including 10 μl sample loop HPLC injection sst, max. 1000 bar syringe (straight needle), 100 μl volume injection valve mounting AZURA Detector Diode array detector, bracket DAD 2.1L D lamp, 190-700 nm, 2 Start-up kit AZURA Capillaries and fittings (PEEK, 1/16“) max. 100 Hz Accessories Eluent tray E 2.1L set of mobile phase bottles (2 x 1000 ml) tool kit for AZURA Column Forced air column thermo- HPLC system setup Thermostat CT 2.1 stat, 5 – 85 °C PC Laptop with pre-installed Windows AZURA Auto- Analytical autosampler, sampler AS 6.1L 0.1 µl – 10 ml injection volume, cool/heat option, 108 vial PC communication Router for LAN connection (8 x) positions, max. 1240 bar Software for controlling ClarityChrom software and licence for the AZURA Compact HPLC and recording system HPLC Column Eurospher II 100-5 C18 with integrated precolumn, 125 mm x 4 mm ID, magnetic clip to attach column 24 www.knauer.net/azuraanalytical 25 AZURA® Analytical HPLC In the monitoring of industrial air, the determination of Preparative system for general cleanup tasks relying on gel carbonyl (aldehyde and ketone) emissions is crucial to prevent permeation respiratory, pulmonological, autoimmune diseases or cancer. GPC Cleanup system by KNAUER HPLC solution by KNAUER • Saves time with automated sample prepara-According to the analytical method described in For method optimization DryLab® has been used tion DIN ISO 16000-3, the carbonyls must be converted and the detailed application note (VEV0078) is available • Improves the reproducibility and quality of to their corresponding hydrazones with 2,4-dinitro-on the KNAUER website: “Systematic HPLC method your analytes phenylhydrazine (DNPH) in order to be detected via development and robustness evaluation of 13 car- • Fully automatically processes up to 15 sam-UV detector and analyzed by reversed phase HPLC. bonyl DNPH derivatives using DryLab®”. ples while requiring minimal bench space • Can be customized to fulfil individual require-1 ments 1 2 3 2 AU 8 m 6 9 11 4 4 5 7 10 12 13 This gradient HPLC system perfectly meets The AZURA GPC cleanup system was 3 even sophisticated analysis demands. selected by the Analytical Scientist for „The Innovators 2017“ product. Min 1 Formaldehyde-DNPH; 2 Acetaldehyde-DNPH; 3 Acetone-DNPH; 1 3 4 Acroleine-DNPH; 5 Propionaldehyde-DNPH; 6 Crotonalde-2 hyde-DNPH; 7 2-Butanone-DNPH; 8 Methacroleine-DNPH; 9 n-By-taldehyde-DNPH; 10 Benzaldehyde-DNPH; 11 Valeralde-4 hyde-DNPH; 12 m-Tolualdehyde-DNPH; 13 m-Tolualdehyde-DNPH System (A46001) Four pesticide standards with US EPA method 3640A and HPLC Plus System with 8-channel DAD detector, LPG pump a chromatogram of spiked olive oil. (700 bar), autosampler and column thermostat, DNPH-column Devices Key features Optional hardware and software AZURA UVD 2.1S Chromatography workstation for OpenLab/Chromeleon, Assistant 6 port 2 pos valve, 1/16”, sst, 300 bar Mini PC, WIN 7, 64 bit, 24” Monitor ASM 2.1L P 4.1S, 10 ml, sst, 50 bar Chromatography workstation for ClarityChrom®, Mini PC, AZURA 3 valves: WIN 10, 64 bit, 24” Monitor Assistant 16 port 16 pos, 1/16”, sst, 50 bar ClarityChrom® single instrument license one time base ASM 2.1L OpenLAB® CDS EZChrom edition workstation for one sys-AZURA GPC 16 sample loops (1 ml each) tem with SMA and 4x system suitability tubing guide Chromeleon™ 7.2 driver Column BioBeads SX-3 Optional service: Installation 26 www.knauer.net/azuraanalytical 27 AZURA® Analytical HPLC Honey and its substitutes AZURA Eluent Tray E 2.1L • Differentiate between natural honey and pos-The eluent tray E 2.1L for AZURA devices with a sible substitutes of food industry capacity of 6 x 1000 ml bottles is stackable onto • Determination of sugar monomers like fruc-all AZURA devices. The inlay is removeable for tose (1), glucose (2), sucrose (3) and maltose (4) cleaning. The eluent tray possesses a drainage • Highest reproducibility in shortest time system and a removable front that facilitates access to a capillary guide. AZURA Inlet Tubing Kit with solvent filter The AZURA Tubing kit with solvent filter (stainless steel, 10 µm) is suitable for all analytical HPLC systems (isocratic, HPG, LPG). The filter can be used with all flow rates of the pump heads provided. HILIC amino phase for fast and high resolution separation of sugars. Safety caps Why should I use safety caps? • Stop the liquid flow line via stopcocks when-ever no flow is delivered. Be safe from eluent loss! • Filters and air-inlet valves prevent the eluent from evaporating and result in maximized Overlay of 12 replicates of the sugar standards and different stability of retention times due to stable elu-honey and honey substitute samples. ent composition. • Filters at the waste bottle keep the air around your AZURA system clean. Devices Key features AZURA Pump P 6.1L LPG pump, 0.001–10 ml/min, sst, max. 700 bar AZURA Detector Refractive index detector, RID 2.1L max. 5 bar, max. 100 Hz AZURA Column Forced air column thermo- Thermostat CT 2.1 stat, 5 – 85 °C, 2 °C/min AZURA Valve V 2.1S 6 port 2 pos injection valve, 1/16”, sst, 400 bar Vertex Plus column 150x4 mm, 100 Å, 3 µm Eurospher II NH2 28 www.knauer.net/azuraanalytical 29 AZURA® Analytical HPLC AZURA Autosampler AS 6.1L Method HPLC Plus UHPLC Autosampler Flow Path Analytical Analytical Low pressure Low pressure High pressure High Pressure Maximum back 700 bar 1240 bar AZURA Pump P 6.1L gradient HPLC Plus gradient UHPLC gradient HPLC Plus gradient UHPLC pressure Max. flow rate 10 ml/min 5 ml/min 10 ml/min 5 ml/min Vial/plate dimensions max. plate/vial height: 47 mm (incl. septa max. plate/vial height: 47 mm (incl. or capmat) septa or capmat) Flow rate range 0.001 – 10 ml/min 0.001 – 5 ml/min 0.001 – 10 ml/min 0.001 – 5 ml/min Injection volume range 0.1 µl - 10 ml depending on sample loop 0.1 µl - 10 ml depending on sample loop Flow rate increment 0.001 ml/min 0.001 ml/min 0.001 ml/min 0.001 ml/min Sample loop 100 µl 10 µl Maximum delivery 10 150 psi 14 500 psi 10 150 psi 14 500 psi pressure [psi] Dispenser syringe 250 µl 250 µl Maximum delivery 700 bar 1 000 bar 700 bar 1 000 bar Headspace pressure built-in compressore, only for sample vials built-in compressor, only for sample pressure [bar] with septum vials with septum Maximum delivery 70 MPa 100 MPa 70 MPa 100 MPa Switching time inj. valve < 100 ms < 100 ms pressure [MPa] Piercing needle precision ± 0.6 mm ± 0.6 mm Pump head materials Stainless steel Stainless steel Stainless steel Stainless steel Sample tray cooling none none Gradient Low pressure Low pressure High pressure High pressure Vial detection missing vial/well plate detection by sensor missing vial/well plate detection by gradient (quaternary) gradient (quaternary) gradient (binary) gradient (binary) sensor Leak management Yes Yes Yes Yes Needle wash programmable: wash between injections programmable: wash between injec-and wash between vials tions and wash between vials Best working 0.1 – 8.0 ml/min 0.02 – 5 ml/min 0.1 – 8.0 ml/min 0.02 – 5 ml/min conditions Wetted materials Tefzel® (ETFE), Glass, Teflon® (PTFE), Tefzel® (ETFE), Glass, Teflon® (PTFE), Kel-F® (PCTFE), stainless steel, PEEK Kel-F® (PCTFE), stainless steel, PEEK Continous working 0.1 – 4.0 ml/min 0.1 – 4.0 ml/min 0.1 – 4.0 ml/min 0.1 – 4.0 ml/min conditions Injection modes full loop filling, partial loop filling and mi-full loop filling, partial loop filling and croliter pickup; PASA™ (pressure-assisted microliter pickup; PASA™ (pressure-as-sample aspiration) sisted sample aspiration) Injection precision RSD (Relative Standard Deviation): full RSD (Relative Standard Deviation):full AZURA Column Theromostat CT 2.1 loop injection: <0.3%partial loop injection loop filling: <0.3%partial loop injection at injection volume>5 µl: <0.5%microliter at injection volumes>5 µl: <0.5%micro-Heating and cooling microprocessor controlled Peltier element for heating and cooling, fan supported pickup injection at an injection volume>5 liter pickup at injections>5 µl: <1.0% system 2-way air circulation µl: <1.0% Temperature range 5 – 85 °C Sample carryover < 0.05 % with needle washing < 0.05 % with needle washing Heating/cooling rate 2 °C/min Injections per vial max. 9 injections max. 9 injections Temperature accuracy ± 0.2 °C Injection cycle time minimum 7 s from the same vial, 14 s from minimum 7 s from the same vial, 14 s different vials;< 60 s for>100 µl sample from different vials;< 60 s for>100 µl Temperature stability ± 0.1 °C injection in all injection modes, incl. 300 sample injection in all injection modes, µl needle wash incl. 300 µl needle wash Dimensions, interior 90 x 390 x 47 mm (W x H x D) Analysis time max. 9 h, 59 min, 59 s max. 9 h, 59 min, 59 s Safety self-check and auto-calibration at power-on, selectable turn-off temperature Tray Tempering Optional (4-40°C) Optional (4-40°C) 30 www.knauer.net/azuraanalytical 31 AZURA® Analytical HPLC AZURA Detector MWD 2.1L UVD 2.1L UVD 2.1S Detector type Variable multiwave- Variable single wave- Variable single wave- length detector length UV detector length UV detector Detection channels 8 (Digital)/4 (Analog) 1 1 Light source Deuterium (D²) lamp Deuterium (D²) lamp with Deuterium (D²) lamp with with integrated GLP integrated GLP chip integrated GLP chip chip Wavelength range 190 - 700 nm 190 - 750 nm 190 - 500 nm AZURA Detector DAD 2.1L DAD 6.1L Spectral bandwidth < 10 nm at Ha line 11 nm at Ha line (FWHM) 13 nm at Ha line (FWHM) Detector type Diode array detector Diode array detector (FWHM) /Note: digital bandwidth 1 - 32 nm Number of diodes 256 1024 Wavelength accuracy ± 1 nm ± 2.5 nm ± 3 nm Pixel pitch 2 nm/diode 0.8 nm/diode Noise ± 5 µAU at 254 nm ± 1.5 x 10-5 AU at 254 ± 2.0 x 10-5AU at 254 nm Detection channels 8 (Digital)/4 (Analog) 8 (Digital)/4 (Analog) nm (ASTM E1657-98) (ASTM E1657-98) Drift 400 µAU/h at 254 nm 3.0 x 10-4 AU/h at 254 3.0 x 10-4AU/h at 254 nm Light source Deuterium (D²) lamp with integrated High brightness deuterium (D²) lamp nm (ASTM E1657-98) (ASTM E1657-98) GLP chip and halogen lamp with integrated GLP chip Linearity > 2.0 AU at 274 nm > 2.0 AU at 270 nm > 2.0 AU at 270 nm (ASTM E1657-98) (ASTM E1657-98) Wavelength range 190 - 700 nm 190 - 1000 nm Maximum data rate 100 Hz (LAN)/12.5 Hz 50 Hz (LAN)/20 Hz 50 Hz (LAN)/20 Hz Spectral bandwidth <10 nm at Ha line (FWHM) /Note: < 3.5 nm at Ha line (FWHM) /Note: (analog) (Analog) (Analog)/10 Hz (RS-232) digital bandwidth 1 - 32 nm digital bandwidth 1 - 32 nm Flow cell Not included Not included Not included Wavelength accuracy ± 1 nm ± 1 nm Time constants 0.00 / 0.01 / 0.02 / 0.05 0.0 / 0.1 / 0.2 / 0.5 / 1.0 / 0.00 / 0.02 / 0.05 / 0.1 / Noise ± 5 µAU at 254 nm ± 3.5 µAU at 254 nm / 0.1 / 0.2 / 0.5 / 1.0 / 2.0 / 5.0 / 10.0 s 0.2 / 0.5 / 1.0 / 2.0 s 2.0 / 5.0 / 10.0 s Drift 400 µAU/h at 254 nm 300 µAU/h at 254 nm AZURA Detector RID 2.1L Linearity > 2.0 AU at 274 nm > 2.5 AU at 274 nm Detector type Refractive index detector Maximum data rate 100 Hz (LAN)/12.5 Hz (analog) 100 Hz (LAN)/12.5 Hz (analog) Flow cell Not included (see Accessories / Not included (see Accessories / Spare Light source Long-life LED Spare parts) parts) Detection channels 1 Time constants 0.00 / 0.01 / 0.02 / 0.05 / 0.1 / 0.2 / 0.00 / 0.01 / 0.02 / 0.05 / 0.1 / 0.2 / 0.5 0.5 / 1.0 / 2.0 / 5.0 / 10.0 s / 1.0 / 2.0 / 5.0 / 10.0 s Refractive index range 1.00 - 1.75 RIU Integration time Automatic Automatic Noise ± 2.5 nRIU Drift 200 nRIU/h Linearity > 1000 µRIU Flow cell 5 bar back pressure resistance Flow cell included Maximum flow rate 10 ml/min (pure water) Flow cell volume 15 µl Wetted materials Stainless steel / fused silica / PTFE / PEEK Temperature control OFF, 30 - 55 °C (1 °C increment) Time constants 0.00 / 0.01 / 0.02 / 0.05 / 0.1 / 0.2 / 0.5 / 1.0 / 2.0 / 5.0 / 10.0 s Maximum data rate 100 Hz (LAN)/20 Hz (Analog) 32 www.knauer.net/azuraanalytical 33 AZURA® Analytical HPLC M A K E YOU R PRE SE LEC TION UHPLC HPLC Bio-Inert (SST, max. 1000 bar) (SST, max. 700 bar) (metal-free, max. 400 bar) column Worldwide partner in 2x/6x science since 1962 A1 A2 B1 B2 detector I detector II 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-pump pump A B 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-ELUENT SELECTION SAMPLE COLUMN SELECTION & DETECTION & DELIVERY INJECTION THERMOSTAT 5 ml/min binary Manual injection 2 columns UV/VIS DAD 2.1L gradient pump P 6.1L valve single wavelength (UHPLC) 6 columns DAD 6.1L Autosampler AS 6.1L UV/VIS multiple 5 ml/min quaternary Column thermostat wavelength Fluorescence gradient pump P 6.1L Autosampler AS 6.1L Independent and Detector RF-20 A (UHPLC) cool/heat Column kit HPLC Conductivity family owned Fluorescence 10 ml/min binary Column kit UHPLC pH Detector RF-20 Axs gradient pump P 6.1L Eluent preheater Refractive index ECD 2.1 10 ml/min quaternary 0.1 mm ID UHPLC pump P 6.1L Light Scattering The founder Dr. Herbert Knauer and his wife owner of the company since 2000. Several Eluent preheater ..... x solvent selection 0.18 mm ID HPLC A/D-converter Roswitha are still active as advisers in the com-awards for outstanding products and innova-valve (6 further inlets) (integration of pany to this day. The couple‘s daughter, Alexand-tions as well as entrepreneurial excellence make further detectors) ra Knauer, has been managing director and KNAUER a „leading employer“. ACCESSORIES 0.1 mm tubing 0.18 mm tubing PEEK tubing ..... x Back pressure Workstation regulator (Windows) We separate molecules FLOW CELLS FOR UV-DETECTOR 10 mm/10 µl 10 mm/2 µl 50 mm/6 µl 3 mm/2 µl (up to 100 ml/min) and unite people. Pressure proof LightGuide® LightGuide® Pressure proof SOFTWARE ClarityChrom® OpenLAB® Chromeleon™ Mobile Control www.knauer.net COMMON APPLICATIONS Reversed phase Normal phase other... System Qualification 34 www.knauer.net/azuraanalytical 35 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/0.5/09.19 7852U 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