Monitor UPC-900. Data File. Real chromatographic information at a glance three monitors in one unit for on-line measurement of UV, ph and conductivity

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1 Monitor UPC-9 Data File High performance liquid chromatography Real chromatographic information at a glance three monitors in one unit for on-line measurement of UV, ph and conductivity Accurate information for all liquid chromatographic techniques use from analytical to preparative scale select wavelength for high specificity UV detection measure low conductivity in reversed phase to high conductivity in hydrophobic interaction monitor ph during gradient or step elutions in ion exchange, chromatofocusing or affinity Reliable day to day performance automatic self diagnosis simple to use as a stand-alone unit Introduction Monitor UPC-9 is a high precision on-line monitor offering the unique possibility to measure UV, conductivity and ph using a single instrument (Figure 2). The monitor is used as a stand-alone instrument, or as a component in ÄKTA FPLC, to record accurate information during any liquid chromatographic separation. Temperature is monitored and displayed, but can also be recorded if Monitor UPC-9 is used in ÄKTAFPLC. ÄKTAFPLC is one of a series of ÄKTAdesign high performance systems for purification and analysis of biomolecules. Fig. 1. Monitor UPC-9 with UV, ph and conductivity flow cells. Real chromatographic information at a glance Clarified E.coli extract, enzyme (DAOCS) purification HiPrep 16/1 Q XL 1 ml/min Buffer A: 5 mm TrisHCl, 1 mm EDTA, 2 mm DTT,.2 mm PMSF Buffer B: Buffer A + 1 M NaCl UV 28 nm conductivity ph temperature ms/cm ml Fig 2. UPC-9 monitor records UV, ph, conductivity and temperature during an anion exchange purification using ÄKTAFPLC Edition AB

2 On-line monitoring of UV, conductivity and ph during a chromatographic separation shows the true conditions of each run. This information is essential for fast, efficient method development and optimisation as well as providing a reliable check on sample and buffer conditions throughout any separation. Consistency in system performance and sample or buffer preparation are easily assessed by comparison of ph and conductivity traces from different runs. Accurate information for all liquid chromatographic techniques Monitor UPC-9 is optimally designed for all liquid chromatographic techniques used in purification and analysis of biomolecules. The single control unit is used with up to three flow cells UV, conductivity and ph. A chromatographic column is connected directly to the UV flow cell and all flow cells are connected in series to minimise dead volumes and eliminate band broadening effects (Figure 3). A sensor inside the conductivity flow cell provides automatic temperature compensation for ph and conductivity measurements. The 5 mm UV flow cell is optimised for chromatographic separations up to 2 ml/min, covering most laboratory scale purification. The conductivity, ph and 2 mm UV flow cells are specified for use up to 1 ml/min to ensure that Monitor UPC-9 can be used in both analytical and preparative chromatography. UV-monitor The UV detection wavelength is selected using a Hg lamp with 254 or 28 nm filters for proteins or a Zn lamp with a 214 nm filter for peptides or when higher sensitivity is required (using 214 nm increases the sensitivity of detection by approximately 2 fold, Figure 4). For special applications the Hg lamp can also be used with 313, 365, 45, 436 and 546 nm filters. Human IgG Superdex 2 HR1/3.5 ml/min 2 µl sample Buffer: 5 mm Na 2 PO 4, 15 mm NaCl nm 28 nm ml Fig. 4. Detection at 214 nm increases sensitivity (UV traces superimposed from two separate runs). Fig. 3. Direct connection of the column to the UV flow cell. 2

3 Conductivity monitor The conductivity monitor, with integrated temperature sensor functions, performs excellently over a broad dynamic range (1 µs/cm to ms/cm. This gives reliable give reliable measurements over the entire range of values typically seen during purification of biomolecules. Gradient performance is monitored from the very high salt concentrations used in hydrophobic interaction down to the very low conductivity levels seen during reversed phase or desalting separations (Figure 5). Regeneration and equilibration of chromatographic columns is easy to follow. Protein standards: chymotrypsinogen, lysozyme, myoglobin, ribonuclease RESOURCE 15 ETH, 1 ml 1 ml/min Buffer A: 1 mm Na 2 PO 4, 2.5 M (NH 4 ) 2 SO 4, ph 7.5 Buffer B: 1 mm Na 2 PO 4, ph 7.5 ph monitor Control of ph is essential for good results in affinity, ion exchange and chromatofocusing separations. Accurate monitoring of ph steps in affinity chromatography, such as during the purification of monoclonal antibodies by Protein A or Protein G media (Figure 6), enables optimisation of binding and elution conditions to achieve consistent results and high recoveries. A typical ion exchange separation is run at constant ph. Monitoring of ph can detect ph changes caused by inconsistent buffer preparation or an increase in salt concentration during gradient elution. The monitor is ideal for following the formation of ph gradients during chromatofocusing. The ph flow cell is easily exchanged with a dummy flow cell if organic solvents (potentially harmful to ph electrodes) are to be used. ms/cm Cell culture supernatant, mouse IgG 2a HiTrap rprotein A, 1 ml 1 ml/min 1 mm Na 2 PO 4, ph 7.7 (binding) 1 mm Na citrate, ph 3.3 (elution) 2 2 ph ml Fig. 5. Monitor UPC-9 monitors conductivity even at the high salt concentrations used in hydrophobic interaction chromatography ml 2 Fig. 6. Accurate ph monitoring enables optimisation of a separation using affinty chromatography. 3

4 Reliable day-to-day performance For reliable results, top performance throughout day-to-day operations must be guaranteed. At each start up Monitor UPC-9 performs a self diagnostic test (approximately 1 minute) to confirm instrument status. Operation is simple and easy. All menus and settings are entered with one dial on the front panel, as shown in Figure 7. Instructions can be selected from several languages (English, German, Spanish, French, Italian). The main menu display shows absorbance, ph and conductivity values at all times, and three analogue outputs enable data to be recorded on a chart recorder. Event marks are set and recorded as spikes on the UV trace. Technical specifications Operating data UV measurement Wavelengths Hg lamp (using filter selection) Zn lamp Absorbance range Autozero range Baseline adjust Linearity, 5 mm flow cell Noise (at 254nm) Drift 254, 28 nm (standard) 313, 45, 436, 546 nm (optional) 214 nm.1 5 AU (full scale).2 2. AU Adjustable 1% of full scale <3% up to 2 AU at 254 nm <5% up to 1 AU at 28 nm AU ±1 1 6 AU/h at 254 nm UV flow cells Maximum pressure 4 MPa (4 bar, 58 psi) Liquid temperature range + 4 to + 6 C Flow rate Optical path length Cell volume 2 mm 1 ml/min 5 mm 2 ml/min 2 mm 5 mm 2 µl (3 µl detector volume) 6 µl (1 µl detector volume) Fig. 7. When used as a stand alone unit a single dial controls selection and acceptance of all instructions. ph measurement ph range Accuracy Stability maximum 14 (specifications valid between 2 12) ±.1 ph unit, temperature compensated.1 ph units deviation/1 h Flexibility for future needs Monitor UPC-9 is an integral component of the chromatography system ÄKTAFPLC. In this system the monitor is under direct control of UNICORN, the control platform of ÄKTAdesign systems. All functions, such as UV, ph, conductivity and temperature are stored permanently within the run log of each chromatographic separation and the audit trail of the system, to provide complete run details and support GLP requirements. Your local Amersham Biosciences representative will be happy to provide further information on ÄKTAdesign systems. ph flow cell Flow rate.1 1 ml/min Maximum pressure.5 MPa (5 bar, 72 psi) Cell volume 4 µl Conductivity measurement Conductivity range Reproducibility Noise 1 µs/cm ms/cm ± 3% maximum or ± 15 µs/cm whichever is greater maximum ±.5% of full scale calibrated range 4

5 Conductivity flow cell Cell constant 5 cm 1 ±2 Flow rate 1 ml/min Maximum pressure 5 MPa (5 bar, 725 psi) Cell volume 24 µl Wetted materials UV flow cells Conductivity flow cell ph electrode and flow cell Dummy electrode ph stability range Chemical resistance Quartz, ETFE, titanium (only 5 mm flow cell) Titanium, PTFE Glass, polyethylene, EPDM (ethylene polypropylene rubber), PEEK PTFE (polytetrafluoroethylene) 1 13, 1 14 (<1 days exposure) All solvents and buffers commonly used in chromatography of biomolecules, except 1% ethylacetate, 1% hexane and 1% tetrahydrofuran (organic solvents are potentially harmful to ph electrodes, use a dummy flow cell when running organic solvents) Environment Ambient temperature C Relative humidity 2 95% relative humidity Atmospheric pressure kpa (84 16 mbar) Physical data Control stand alone use: ÄKTAFPLC system Display Functions Power requirement Power consumption Analogue outputs Weight Dimensions W D H (mm) dial UNICORN (version 2.3 or higher) system control or dial 2 lines with 2 characters each Languages selectable: English, German, Spanish, French, Italian 1 24 V AC, 5 6 Hz 25 VA 1 V full scale 8.5 kg mm Ordering Information Designation Quantity Code No. Monitor UPC-9 control unit supplied with signal cable Monitor UPC-9 control unit supplied with Hg optical unit, 254 nm and 28 nm filters, 2 mm flow cell, conductivity flow cell Accessories Flow cell 2 mm Flow cell 5 mm Conductivity flow cell ph flow cell, includes ph dummy electrode ph electrode ph electrode complete, flat tip (includes electrode, flow cell, dummy and holder) ph electrode, flat tip Hg optical unit with 254 and 28 nm filters Zn optical unit with 214 nm filter Hg lamp and housing complete Zn lamp and housing complete Filter 214 nm Filter 254 nm Filter 28 nm Filter 313 nm Filter 365 nm Filter 45 nm Filter 436 nm Filter 546 nm Related Product Literature Designation Code No. ÄKTAFPLC Data File: UNICORN control system Data File:

6 ÄKTA, UNICORN, FPLC, HiPrep, HiTrap, Superdex and RESOURCE are trademarks of Amersham Biosciences Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them. A copy of these terms and conditions is available on request Amersham Biosciences AB 1998 All rights reserved Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE Uppsala Sweden Amersham Biosciences Inc 8 Centennial Avenue P.O. Box 1327 Piscataway NJ 8855 USA Produced by Wikströms, Sweden , Oct., 1998 Printed matter. Licence