PURIFICATION. Preparative Chromatography Modular Solutions from Manual Systems to Full Automation. Scale Up. Reverse. Phase. Impurity Isolation

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1 PURIFICATION Preparative Chromatography Modular Solutions from Manual Systems to Full Automation PREPARATIVE CHROMATOGRAPHY SOLUTIONS Lab scale Scale Up Reverse Phase Medicinal Chemistry Natural Products Peptides Chiral Oligonucleotides Impurity Isolation Normal Phase Small molecules

2 PLC 2020 Purification System LC out of the Box! Fully-functioning purification system, the PLC 2020 Personal Purification System features: Compact footprint Easy-to-use The PLC 2020 Personal Purification System is ideally suited for low-throughput, high pressure purification needs and is intended to support an individual or a small group of chemists. Wide Range of Purification Modes Multigrams range High Pressure Reverse Phase Normal Phase Chiral Low Pressure Flash Solid Injection Capability Save time High Pressure Purification brings more resolution with no need to purify multiple times to achieve required purities. Running the System Simple as 1 Tap Run on the main menu 2 Select Method & tap Run 3 Load & inject sample 2 A robust system true to its name to last through the years

3 Easy-to-Use Software Intuitive interface. Test tubes 13 x 100 mm (240) 15 x 45 mm (144) 16 x 100 mm (132) 16 x 150 mm (132) 18 x 150 mm (132) Fraction Capacity (3 Racks) Scintillation vials 20 ml (42) 40 ml (42) Custom racks available Storage for up to four 4L solvent bottles. NEW! PLC 2020 Purification System Real-time, graphical gradient modifications. Real-time sample tracking during run. Conditional logic fraction collection based on two detector channels using any combination of Slope and/or Level. Auto-Print and Auto-Export of reports at the end of a run. Integrated touch screen control. Column holders for FLASH & Reverse Phase columns. Accommodates any FLASH style cartridge as well as dry load configurations. Monitor the progress of the system, run lights illuminate the racks during a run and flash if an error has occurred. Automatic repetitive injection with external pump to increase purification throughput. 3 rack positions dedicated to fractions. Interchangeable pump head design to accommodate wide flow rate and pressure range. 3 USB ports for easy download/upload methods and results. Easily connect your printer, keyboard and mouse. Selection of up to 4 solvents. The purification throughput is highly related to compounds solubility, matrix complexity and column resolution. PLC2020 Overview Low Pressure High Pressure Column Type # NP Flash Cartridge C18 Flash Cartridge HPLC Column Size g g mm ID mm ID mm ID Flow Rate ml/min ml/min ml/min ml/min ml/min. Loading Capacity g g Up to 0.5 g Up to 2 g Up to 6 g Injection Volume > 10 ml and solid injection 2-10 ml ml ml >10 ml Pressure < 100 psi psi Calculation based on Methanol/Water solvents - 5 µm column 150 to 250 mm length 3

4 Analytical to Preparative HPLC System Gilson High Performance Liquid Chromatography (HPLC) systems provide complete solutions for all purification needs. Front-end liquid handlers and autosamplers are available in a range of bed capacities and optimized bench space footprints. GX Series Platforms The GX series platforms feature a selection of bed capacity adapted to the users needs. Allow injection and Fraction Collection on the same instrument bed. The revolutionary GX Solvent system accommodates sample volumes ranging from microliters to hundreds of milliliters. The GX series Liquid Handler offer numerous injection possibilities ranging from Analytical to Preparative. When size matters GX-281 & GX-271 Bed Capacity Tube Diameter GX-281 Sample Quantity GX-271 Sample Quantity 10 mm mm mm mm mm mm mm mm The GX Series instruments features: GX series collection options Low Mount Fraction Collection Separate Injection and collection pathways. High Mount Fraction Collection Collection in closed vials and micro-plates. GX Z-Injection Module For fast injection with low carryover by aspirating the sample directly into the injection valve. Decreases cycle times. GX series Prep Solvent System Syringeless technology with access to five off-bed solvents. Injection volumes ranging from µl to 10s of ml. Multiple dilution and rinsing solvents. GX Direct Injection Module Innovative injection port design to reduce void volumes and minimize carryover associated. Possibility to combine two modules for analytical and preparative injection. 4 Perfect blend of performance, capacity and size...

5 Seamless Automation Unique Solutions Built On Specific Users Needs Because all our users have different needs and constraints, we do not have a one fits all solution but we build specific solutions that answer current and future needs of our users. Some of the solutions that can be offered: Columns Selection and Column Back Flush Columns selection for universal systems: Analytical method development and Scale Up to Preparative. Multiple purification modes (RP, NP, Chiral,...) Purification followed by direct analysis of fractions in analytical. Column Backflush. HPLC Purification System Fully Automated Samples Pre-Treatment and Injection Liquid handling and injection capabilities on the same instrument allowing multiple injections from the same sample, sample dilution, internal standard addition, sample cleanup by SPE,... GX-281 Preparative HPLC System GX-271 Semi-preparative HPLC System To Answer Solubility Issues Sandwich injection to avoid sample precipitation during the injection process: Unlimited injection volumes through solvent system and 1/8 injection loop. Direct Fractions Re-Injection The GX series platforms feature dual injection path allowing for analytical and preparative injections without compromises. The Trilution LC software can automatically inject the collected fractions for purity confirmation or for secondary purifications.... for mg to gram scale purification 5

6 HPLC Pumps Analytical, Semi-Prep, to Preparative Pumping Solutions Gilson offers unsurpassed flexibility in HPLC pumping systems with an extensive product range for all type of pumping applications. Capable of fast separations with high-efficiency columns in normal and reversed-phase modes, Gilson Pumps are the premier solvent delivery solutions for the analytical to preparative chromatography. Gilson dual-piston pumps for semi preparative and preparative chromatography feature high pressure mixing to guarantee gradient precision and reproducibility. Programmed Composition (% E) Gradient Linearity and Accuracy Time (minutes) Detected Composition (mau) Variable mixer and high pressure mixing solvents for optimized gradient performance. Solvent selection capable of up to 4 solvents per pump for a total of 8 solvents in a binary pumping system. mvolts Retention Time Repeatability Time (minutes) % Mobile Phase Series of Ten Injections Methyl PB Ethyl PB Propyl PB Average Retention Time Standard Deviation CV 0.72% 0.34% 0.29% Pump Overview 32x-H2 Analytical to Semi Preparative 331/332 Analytical to Semi Preparative 333/334 Analytical to Preparative Flow Rate ml/min ml/min ml/min. Flow Rate Accuracy 2% 2% 1% Pressure 4300 psi / 300 bars 8700 psi / 600 bars 3040 psi / 210 bars Column Types ID: 4.6 to 22 mm from 5 µm particles ID: 4.6 to 30 mm from 2.5 µm particles ID: 4.6 to 50 mm from 5 or 10 µm particles Typical Application Method development and purification up to 2 grams Method development, purification up to 6 grams and purity check on fast analytical column Scale up an purification up to 15 grams 6

7 HPLC Detectors From Analytical to Preparative Detection Solutions UV/VIS Detection 15X Series UV/VIS Detectors Rugged detectors operate in single wavelength or dual wavelengths mode. 171/172 Diode Array Detectors Accurate detectors with an optional remote flow cell design. Ideal for compounds identification (spectral library) and peak purity calculation. Specifications 15X detector Wavelength nm nm nm Diodes HPLC Purification System Flow Cell Selection Chart for Optimizing Sensitivity on 15X Series Detectors Selecting the correct flow cell will optimize sensitivity and peak shape. Analytical Flow Cell Semi-preparative Flow Cell Preparative Flow Cell Product # Internal Volume 12 µl 0.7 µl 0.16 µl Path length 5 mm 0.2 mm 0.05 mm Column Size 4 mm - 10 mm 20 mm > 40 mm Flow Rate 1 15 ml/min ml/min ml/min. Purify µg - 5 mg mg 300 mg - 15 g PrepELS II Detector The prepels II Detector is ideal for obtaining the purest fractions with the ability to detect virtually any analytes with or without a chromophore, including compounds that have poor UV absorption. Specifications Light Source 670 nm laser diode, < 5 mw Gas Requirement 65 ± 5 psi Nitrogen or Argon Evaporative Zone Temperature Ambient 120 C Nebulization Temperature 0 70 C Others Detectors Preparative Refractive Index Detector, Fluorimeter, Radioactivity, The Gilson 506 interface allows the collection of analogic signals (up to 4) to trigger fraction collection and for reporting. Mass Spec Single Quad Settings 7

8 LC/MS Purification System It s Your Chemistry... Simplified! Your time is valuable. The quality of your data is crucial. That s why Gilson has developed an LC/MS Purification System that is not only extremely easy-to-use and easy-to-maintain, but has one of the largest mass ranges on the market today. Your Specifications Modular design means each component can be configured to your specifications. Your Own Probe Just walk up and snap in to start, without worry of cross contamination in an open access, multi-user environment. Your Choice Choose to collect fractions using conditional logic based on any combination of UV, DAD, ELSD and up to seven MS Channels. Mass Based Purification Simple as: Select scan range Enter target masses and adducts Select conditional logic collection to trigger off both UV and MS RUN 8 Meeting the growing demand for...

9 NEW! LC/MS Purification System MS Detector Rugged & Simple Quick-connect Rugged and simple to maintain source allows for cleaning and maintenance without breaking vacuum limiting instrument down time and increasing productivity. Quick-connect feature allows each user to have their own probe, preventing contamination between users in open access environments. Multiple Mode Operation Automation & Calibration Ability to monitor in Full Scan, Extracted Ion and SIM (Selected Ion Monitoring) simultaneously. Integrated calibration vials for Auto-Tune allows for automated and effortless mass calibration. Range & Speed Source Features Mass Range 50 3,000 amu The source features XYZ adjustments of probe positioning to optimize spray in relation to the capillary entrance for higher flows and/or sample concentrations. Transparent window to easily view the spray. Scans Speeds up to 10,000 amu/ sec. Selective Collection of Compounds with Weak UV Response Immediately identify fraction of interest Sample tracking. Scan of the fraction collected. Real time access to MS spectra for each fraction collected. Gilson TRILUTION LC Software Conditional Logic Fraction Collection Purification Based on UV and MS Signals Fraction Tracking Function Split Ratio is Independent of Changes in The Hplc Flow HPLC HPLC 1 Stream Stream 1 Mass to be Mass to be Transferred Transferred HPLC HPLC Stream Stream 1 1 Mass Spec Spec Spec 3 Mass 3 Mass Stream Stream Mass Spec 3 Stream 3 Stream Aliquot Fill Allows for large, setable split ratio to allow the system to operate with a large flow range and column conditions. Mass Mass Active Splitter 4 4Transferred Transferred 2 2 Aliquot Transfer...simplified LC/MS based purification solutions 9

10 Trilution LC Software Gilson has redefined purification with the HPLC system control software, Trilution LC. This liquid chromatography software, specifically designed for purification, allows for complete control to achieve the challenging results that applications require. Configuration & Virtual Lab Screen Fraction Collection Simulator Full Hardware Control All aspects of the equipment are set in the Virtual Lab Screen. Graphical Method Creation Gradient profile. Hardware tasks. Fraction collection parameters. Fraction Collection Settings Advanced Fraction Collection Capabilities Collect by time, volume, level and slope. Individual parameter for front and back slope. Co-eluting peak. Collect non peak. Divide peaks by time or volume. Collect on different tubes for each injection or collect always in the same set of tubes in case of repetitive injections. Fraction Collection Optimization Change fraction collection parameters. Call a previous chromatogram. Check the fraction collection. Conditional Fractions Collection Set the collect on conditions from up to 8 channels. Select condition on AND, OR, AND/OR. Conditional Fraction Collection Screen 10

11 Control Software Sample Re-Injection Set Up Screen Application Run Screen Automatic Fractions Reprocessing Automatic sample list generation for post collection fraction processing such as pooling or re-injection. Automatic sample re-injection based on condition such as area size. Graphical Tracking of Sample & Fractions. Visual Operation List Auto sample list generation. Use of variables for easy scale-up from Analytical to Preparative runs. Real Time Viewing Real time chromatogram and fraction collection. Real Time Action Full access to all instruments during the run to: Change gradient profile. Activate fraction collection. Stop the pumps. Graphical Sample Tracking Identify fraction from chromatogram or injection vial. Open Access Module Administrator control & permission-setting capabilities. Walk-up user access. Apex: Collects the entire peak with subdividing at the Apex using a combination of slope settings and Apex throughput collection settings. Graphical tasks Drag-and-drop to specific time points for fast and easy method creation. Injection Task Fraction Collection Settings Task Start Fraction Collection Task Stop Fraction Collection Task Rinse Probe Task Detector Auto-Zero Channel Task Switch VALVEMATE Task 11

12 Fraction Collectors FC 203B/FC 204 Fraction Collectors Stand alone, compact Fraction Collectors. Collect by time, drop or peak in tubes, vials or microplates. 3-way diverter valve for collection by signal. Up to 10 programmable time windows can be added for collecting only samples of interest, while discarding the column s void volume, areas of non-interest and equilibration volumes. Contact closure inputs are available to remotely start, advance or stop fraction collection. Multi columns adapter up to 18 channels available. Specifications Dimensions (w x d x h) FC 203B: 32.4 x 29.2 x 26.7 cm (12.8 x 11.5 x 10.5 in) FC 204: 47.9 x 46.4 x 33 cm (18.9 x 18.3 x 13 in) Liquid Contact Material PTFE and 316 stainless steel Valve Switching Speed 100 ms Max Flow Rate FC 203B: 20 ml/min. FC 204: 20 or 200 ml/min. GX Fraction Collector in Closed Vials High mount fraction collection valve for fraction collection into microplates or into closed vials. HPLC Pumps 30X Series HPLC Pumps Stand alone 30X High Pressure Pumps can be used for: Repetitive injection of the same sample. Isocratic or Gradient pumping system. Up to 200 ml/min. with exchangeable pump head. Easy maintenance. The 306 model functions as a slave unit. The 305 and 307 Pumps are programmable through the incorporated keypad. Specifications Flow Rate 305/306: 10 μl/min. 200 ml/min. 307: 10 μl/min. 10 ml/min. Accuracy < ± 1% Reproducibility < 1% Dimensions (w x d x h) 33 x 33 x 15 cm (13 x 13 x 6 in) 12

13 Service Contracts Choosing a Maintenance Contract from your instrument manufacturer is an evidence of: Maximize Investments Increase longevity and reliability of equipment. Benefit of the expertise of the manufacturer for update. Control Costs Budget & plan maintenance. Preferential and attractive prices. Guarantee Quality Certified service engineers performing IQ/OQ. Comply with manufacturer specifications. Resources Services & Resources Gilson Publications Gilson HPLC Troubleshooting Guide Troubleshooting High Pressure Liquid Chromatography (HPLC) systems can be straightforward, despite the multiple components. With our guide that contains helpful forms and checklists, troubleshooting Gilson HPLC systems is simpler than ever! Included in this guide are real-life examples using HPLC systems with real problems and solutions organized into a spiral bound guide to eliminate problems that may be related to software, hardware, column, solvent, or any combination of these. Simple tests to prove purification and reduce or eliminate carryover are included! Technical Notes Handbooks Trilution LC v2.1 Fraction Collection Fundamentals. Methods for Verifying Fraction Recovery in Preparative HPLC Systems. Gilson Quarterly Application Notes Handbooks The complimentary quarterly Gilson Application Notes Handbook allows you to page through scientific advances using Gilson automation and Gilson pipettes in a number of different markets from other chemists and laboratories who have published applications. We welcome you to submit your application to us for publishing in our quarterly handbooks. Published applications offer everyone a chance to learn about the recent scientific highlights and new technologies. Attend our complimentary G.E.T. LIVE Web Seminars to learn about more scientific advances with industry leading applications and technologies from TODAY! Gilson is the host for our guest speakers, who lead the industry with their innovative methods and techniques. Contact [email protected] or visit to learn more. It s FREE! 13

14 Resources Optimizing Preparative HPLC There are numerous parameters and techniques to optimize preparative HPLC. Optimization will improve chromatography and percent recovery of collected fractions. Separation Scale-up Equations When scaling up from analytical scout runs to preparative purification the main parameters to scale up are: Flow Rate Fc2=Fc1 x D²c2 x Pc1 D²c1 Pc2 Fc1= Flow rate on column 1 (ml/min.) Dc1= Internal Diameter of column 1 (mm) Dc2= Internal Diameter of column 2 (mm) Pc1= Particle size of column 1 material (μm) Pc2= Particle size of column 2 material (μm) Injection Volume D²c2 x Lc2 Vc2 = Vc1 x D²c1 x Lc1 Vc1= Injection volume on column 1 (μl) Dc1= Internal Diameter of column 1 (mm) Lc1 = Length of column 1 (mm) Dc2= Internal Diameter of column 2 (mm) Lc2 = Length of column 2 (mm) Gradient Duration length preparative (diameter preparative) 2 flow rate analytical GD preparative = GD analytical x x x length analytical (diameter analytical) 2 flow rate preparative Pressure Equation In preparative chromatography the back pressure generated by the column and the various tubing can become a constraint. At constant mobile phase viscosity, the pressure drop can be estimated: Pc2 = Pc1 x Fc2 x Lc2 Pc1x Dc1 x( ) ² Pc1= Pressure on column 1 (psi) Pc2= Pressure on column 2 (psi) Fc1 x Lc1 Pc2x Dc2 Tubing & Column Back Pressure Tubing Back Pressure - for 1 meter length Pressure (psi/bar) Tubing ID (in/mm - Color coded) Flow (ml/min.) 0,005/0,13 - Red 0,010/0,25 - Natural 0,020/0,50 - Or 0,030/0,75 - Green 0,040/1,00 - Natural 1 695/48 43/ /60 55/4 11/ /9 24/ /22 65/5 20/ /7 30/2 Typical Volume (μl) Basic Column Back Pressure - Solvent: Methanol/Water - Solid phase: C18-5 μm Pressure Column ID (mm) Column Length (mm) 4, psi / 130 bars psi / 213 bars Typical flow (ml/min.)

15 Concentration Overloading on PLC2020 Saturation of solid phase from on column front change peak shape. Late eluting peak is accelerated by earlier co-eluting compound. Resources Solid Injection on PLC2020 FLASH FLASH Dry Load Run Method - FLASH Dry Load To FLASH Waste Waste Mobile Phase To FLASH Mobile Phase Detector Detector Automatic Purity Checking on Gilson System Advantage of dual injection paths and high pressure pumps for analytical and preparative works: Gilson Preparative Solutions: Automatic Re-injections on short column for purity check Pumping System Type Column Flow Pressure Quantity Purification 30 x 250 mm - 5 μm 45 ml/min. 230 bars Up to 6 g 331/332 Purity check 2,1 x 50 mm - 2,7 μm 0,8 ml/min. 430 bars Inject several milliliters or do multiple injections on a preparative column. Then automatically switch to a very selective short analytical column and inject few microliters to quickly check purity of collected fractions. Pure fractions can be pooled to reduce evaporation time. 15

16 Gilson International B.V. Laan van s-gravenmade AJ DEN HAAG The Netherlands Gilson International B.V. Specifications subject to change without notice.