News. Academia. Understanding Ecosystems Using Multiparameter Analysis. Edition No. 1 / February 2013

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1 Academia Weighing Analytical and solutions Analysis in the Laboratory laboratory 12 News Edition No. 1 / February 2013 Understanding Ecosystems Using Multiparameter Analysis In a country full of large forests and beautiful lakes, the research departments of the Swedish University of Agricultural Sciences are developing expertise in biological resources and production. At Umeå, they analyze the dynamics and management of forest ecosystems by measuring ph, conductivity, acidity and alkalinity of field samples by titration. Each week, 50 to 300 samples from various locations throughout the country are analyzed for any combination of the required parameters in the Department of Forest Ecology and Management. The result quality must remain high, which means that it is crucial for any influences that affect the analyses, i.e. temperature change during a conductivity or ph measurement, to be excluded. Furthermore, measurements, such as CO2 degassing, must be in place to avoid ph alteration. Last, but not least, the results have to be available within the shortest time possible to reflect the conditions at the sample s lake origin. A titration automation station meets the challenge The incoming samples are placed onto a Rondo 30 sample changer. The individual sample is pumped by peristaltic pumps through two heat-jacketed flow-through cells. These cells contain the conductivity and ph sensors. Both parameters are measured separately at a constant temperature of 25 +/- 0.5 C in order to avoid cross-contamination by the electrolyte flow from the ph sensor during the conductivity measurement. The sample is then transferred into the titration beaker where the volume is leveled exactly to a pre-defined quantity. Depending on the ph, the T90 Excellence Titrator decides whether alkalinity or acidity shall be analyzed by automatically selecting the appropriate titrant. Sample specific method selection, sample identification and result storage are all reliably conducted by LabX titration software. Field sample

2 Tips & Tricks Multiparameter Titration Peter Blomqvist of Umeå saves analysis time with the Rondo30 for ph and conductivity analysis. containers, closed with self-adhesive aluminum foils, can be directly placed on the sample changer tray. Sample transfer and CO2 degassing, with inert gas, is achieved by penetrating the foil with a needle. Any ambient contamination is completely avoided using this method. Thanks to the thorough cleaning of the system, carry-over from previous samples is non-existent. SLU Umeå system contributes to environmental research The workstation has been yielding results that perfectly meet the high expectations of Umeå for more than a year now. Thanks to the clever system setup and a complete sample analysis, including cleaning, requires a maximum of 10 minutes. The daily sample throughput can be easily analyzed. The important result trend evaluation of these samples is reliably achieved by LabX titration. The system performance can be easily controlled by standards or check samples integrated into the workflow. The scientific employees operating the system are very pleased with the simple shortcut operation set-up of the T90 and LabX, especially concerning the simple execution of sensor calibration or titrant standardization routines. In conclusion, this automatic workstation contributes to the improved understanding of Swedish ecosystems by providing the reliable measurement of important environmental parameters with minimal effort. Text: Hans-Joachim Muhr, Manager Market Support Group SBU AnaChem Download everything you need to know about titration Education is key to understanding your titrator and applications. The Basics of Titration guide covers the following areas: Theory of Titration Chemistry and Equations Automated Titrator Functions Instrument, Reagent and Sensor Maintenance and Care Sing up and receive a free copy of the comprehensive booklet. Publisher Mettler-Toledo AG Laboratory Division Im Langacher CH-8606 Greifensee, Switzerland Production LAB Division Marketing Global MarCom Switzerland Subject to technical changes. Mettler-Toledo AG 02/13 Printed in Switzerland. 2 METTLER TOLEDO Academia News 12

3 Easy Solution to Ensure Pipette Accuracy Small pipetting errors can have a big impact on research results. A balance that easily converts for quick pipette checks, such as METTLER TOLEDO s NewClassic MS, means the difference between first-time accuracy and costly rework. Pipette accuracy checks reduce errors. But they can also be time-consuming and resource intensive. The new convertible semi-micro NewClassic MS Balances from METTLER TOLEDO combine weighing and pipette accuracy checks in the same easy-to-use instrument. The twoin-one instrument not only provides better results, but also cost savings. Easy conversion The NewClassic MS converts easily into a stand-alone pipette checking station. Firmware guides users through the pipette check process. An evaporation trap is the only external device needed to keep humidity levels stable. This minimizes sample evaporation for accurate results. Automatic calculation Very small volumes of liquid, such as those dispensed from pipettes, cannot be accurately measured directly. Therefore, ISO recommends gravimetric methods for pipette testing: The test water dispensed from the pipette is weighed on a balance and the weight value is converted to a volumetric value. In the NewClassic MS, proprietary embedded firmware automatically calculates systematic and random pipetting errors in absolute and relative values. Results are displayed and can be printed out for reference. Assurance between calibrations Convertible balances, such as the New- Classic MS, are ideal for life science industries where more frequent accuracy checks than the periodic ISO standard calibrations provide added security that pipettes are still delivering exact results. For more about how METTLER TOLEDO s NewClassic MS can help eliminate errors and save time with easy pipette accuracy checks, visit Text: Joanna Imschweiler, Marketing Manager, Classic Balance The balance is quickly converted into a pipette check-station whenever necessary. An evaporation trap keeps humidity levels stable to minimize evaporation and ensure accurate results. METTLER TOLEDO Academia News 12 3

4 Pipetting Streamline Effort for DNA Sequencing Workflow As an integral part of the International Barcode of Life Project, the Canadian Center for DNA Barcoding (CCDB) produces a barcode reference database for standardized gene regions in order to discover new specimens from plant/animal species. The Liquidator 96 is utilized in crucial steps along the DNA barcoding process. DNA Barcoding Process Dr. Natalia Ivanova, lead DNA scientist at the CCDB, confirms that the Liquidator 96 plays an important role in four major steps of the DNA barcoding: DNA extraction from plant/animal tissue, PCR amplification of extracted DNA, cycle sequencing extension and cycle sequencing cleanup. The Liquidator 96 is a 96-channel manual pipetting system that accommodates high-throughput sample setup. The increased throughput, provided by the simultaneous pipetting of the 96-channels, makes the Liquidator 96 particularly useful for genomics applications, such as DNA and RNA purification, restriction digestion of DNA, reverse transcription and enzymatic processing of RNA, PCR amplification of DNA templates, ligation of sequences to cdna and sequencing reaction setup. Dr. Ivanova uses the Liquidator 96 differently depending on whether DNA is extracted from animal or plant tissue. With animal tissue, the Liquidator 96 is used to deliver lysis buffer, containing protease, to samples. After overnight sample incubation, Ivanova s team uses the Liquidator to transfer lysate to a micro-plate for robotic DNA extraction. With plant samples, the Liquidator 96 is used to purify DNA using glass fiber membrane binding. Firstly, plant extract in lysis buffer is transferred from a tube rack to a 96-well plate containing binding buffer using the Liquidator 96. The resulting sample is transferred to a 96-well glass fiber membrane purification plate. The Liquidator 96 is then used after sample binding to carry out two wash cycles. After sample drying, the Liquidator 96 is used to pipette 4 METTLER TOLEDO Academia News 12

5 Know-how See how Liquidator 96 processes DNA samples water to the plate, which elutes the DNA from the plate after centrifugation. Reliable DNA amplification using PCR After extraction, DNA is amplified using PCR. Here, the Liquidator 96 is used to deliver two microliters of purified DNA from the extraction step into a 96-well plate containing PCR mastermix. After sealing and centrifugation, the DNA is amplified using a thermocycler. into a thermocycler for DNA extension. This protocol processes 90 sequencing samples in parallel. After DNA extension, the reactions need to be cleaned up. With the Liquidator 96, the entire volume of the sequencing reaction can be simultaneously transferred to a Sephadex G-50 plate. After centrifugation, the samples are transferred with the Liquidator 96 to a plate containing 0.1 mm EDTA. The resulting plate is sealed and placed on a sequence assembly plate prior to loading onto a sequencer for DNA barcode analysis. The final results are imported to the CCDB information management system for accurate tracking. Follow Dr. Natalia Ivanova, lead DNA scientist, in the 4 short videos to see the fundamental roles that Rainin s Liquidator 96 plays in processing her DNA samples. See application videos and download the white paper at our CCDB microsite: After DNA amplification, the PCR products are prepared for cycle sequencing using the Liquidator 96. Here, it is used to deliver water to the PCR plate in order to dilute the amplified DNA three-fold. Two microliters of the diluted DNA are delivered by the Liquidator to 96-well plates containing sequencing mastermix. The reaction is then centrifuged and placed The protocol above demonstrates the Liquidator 96 s significant value to laboratories requiring accurate highthroughput data collection. In Dr. Ivanova s case, the Liquidator 96 most certainly facilitates her workflow. Text: Rishi Porecha, Ph.D., Applications Specialist METTLER TOLEDO Academia News 12 5

6 Powder Dosing Secure Handling of Nanoparticles with Automated Dosing System The French Alternative Energies and Atomic Energy Commission (CEA) is active in areas such as low-carbon energies, information technologies and health technologies. The Laboratoire Capteurs Diamant (LCD) of CEA, called on Quantos to optimize its nanoparticle suspension processes in order to guarantee the quality of the diamond deposits it produces. Some of the Quantos QB5-L users from LCD (from left to right): Hugues Girard (engineer researcher chemist), Céline Gesset (engineer researcher electronics), Bertrand Bazin (engineer researcher co-installation manager) and Jacques de Sanoit (engineer researcher electrochemist). Quantos: a vital link in the process In suspension, the size of certain nanoparticles, as well as their concentration and dispersion, changes their properties and especially their toxicity (e.g., metal nanoparticles). "Then we have to control the suspension of nanomaterials, the essential element of which is placing them in suspension, splitting the nanoparticle aggregates and measuring the size and dispersion of aggregates/particles," continues Hugues Girard. "Previously, we were using a balance and a volumetric flask to place particles in suspension, even though this initial dosage phase is critical. It is a little-known fact that diamond is an extreme material. Bertrand Bazin, lab manager of LCD in Saclay, France, explains that, "In addition to its hardness, diamond has some surprising properties, especially with regard to thermal conductivity (5 times better than copper), propagation of vibrations (15 times better than water) and electrical insulation (7 times better than glass). In its natural state, it is rare, expensive, small, and chemically impure. Happily, LCD has a routine method for synthesizing them: The CVD (Chemical Vapor Deposition) process produces wide film sheets of controlled purity diamonds. This process uses microwaves to excite a carbonated gas above a silicon sheet on which a suspension of diamond nanoparticles has previously been deposited in order to assist the creation of the diamond film. The team has acquired recognized expertise in nanoparticles but also in numerous other disciplines, such as electrochemistry and materials etc. LCD, therefore, works as much with manufacturers as with other research centers in France and overseas. Mr. Bazin explains, "For example, we have worked on perfecting dosimeters for radiotherapy, new-generation electronic components and an ultra-sensitive electronic nose (for explosives, toxins and drugs etc.) and, currently, industrial and agribusiness environment monitoring is a boom market. Céline Gesset adds, "Thanks to Quantos, we are now working in complete safety. The QB5-L ensures that we can handle toxic products without risk of exposure and there is no spatula, cupel or cleaning required. In addition, automatic dissolution ensures the repeat accuracy and precision of the concentrations of suspensions used. "Several operators, handle the QB5-L on a daily basis, creating and testing up to 30 suspensions a day, concludes Jacques de Sanoit. Text: Joël Goursot, Communication Manager, METTLER TOLEDO France 6 METTLER TOLEDO Academia News 12

7 Monitoring Cell Culture Bio-Processes in Situ and in Real-Time ReactIR and Focused Beam Reflectance Measurement (FBRM ) are proven in situ technologies for realtime monitoring of principle bioprocess components and changes in cell density. This article highlights the results of two applications, both demonstrating the value of in situ ReactIR and FBRM in bioprocess monitoring. With the increase of biopharmaceutical drugs on the market in recent years, research focus has turned to optimizing bioprocesses in order to maximize product recovery and improve product quality though the use of Process Analytical Technologies (PAT). Off-line techniques for monitoring cell cultures are time consuming, may require sample preparation and do not provide real-time bioprocess information, which restricts the ability to control the process. Non-invasive and non-destructive in situ probe-based technologies monitor processes in real-time by providing information about the bioprocess as it exists in the bioreactor. Monitoring carbon source and metabolites ReactIR is an ATR mid-ir spectrometer equipped with a DiComp probe. The ability of ReactIR to monitor the concentrations of 4 principle analytes, glucose, L-glutamine, lactose and ammonia in a mammalian cell culture was determined by comparison to offline enzymatic assays (Megazyme, Ireland). The data in figure 1 demonstrates how ReactIR was able to monitor component concentrations at both high and low concentrations. As cells metabolize glucose and glutamine, lactate and ammonia are produced, respectively, meaning there is a strong correlation between the metabolically related components. To confirm that ReactIR monitors each component independently of the other, further experiments tested the calibration models. In the example shown in figure 2, glucose was spiked into a solution of lactate and ReactIR spectra were collected. The data confirms that changes in glucose concentration did not affect the measurement of lactate even when the sample was spiked with a 30 mm of glucose. Monitoring cell density The in situ monitoring of cell density changes was proven using FBRM which counts cells and measures cell dimensions. Cell counts determined by FBRM demonstrated a strong linear relationship with cell density with a coefficient of determination greater than 0.99 (figure 3). Conclusions The non-invasive and non-destructive in situ, real-time monitoring of principle components and changes in cell density, as they exist in the reactor, is possible with ReactIR and FBRM, respectively. ReactIR and FBRM eliminate the need for sampling and sample workup, while reducing the risk of contamination. The real-time information provided by ReactIR and FBRM enables bioprocess understanding and immediate process control. Text: B. Rice, University College Dublin Total Counts 1 Total Counts 2 m = 3.22x10-5 R 2 = Total Counts (#/s) m = 1.36x10-5 R 2 = e+6 4e+6 6e+6 8e+6 1e+7 Cell Density (cells/ml) Figure 1. Actual and predicted values for 4 principle components of a cell culture. Figure 2. Changing glucose concentration does not affect the lactate concentration as measured by ReactIR. Figure 3. Effect of cell density on total cell count as measured by FBRM. METTLER TOLEDO Academia News 12 7

8 Good Measuring Practices Good Measuring Practices Reliability Throughout the Lifecycle Good Measuring Practices by METTLER TOLEDO is a global program supporting you in laboratory and production environments with quality assurance recommendations for weighing, pipetting and chemical analysis. The five steps of every Good Measuring Practices guideline cover the entire lifecycle of your instruments. By focusing on your processes and its associated requirements and risks, we provide you with effective measures to maximize operational security and minimize testing efforts and process risk. GWP Good Weighing Practice GTP Good Titration Practice GPP Good Pipetting Practice Guaranteed better weighing results GWP helps ensuring consistent product quality and avoiding outof-spec results or bad production batches. With GWP you comply with all regulations relevant to your industry for a minimal time and financial investment. GWP provides documentation to verify the accuracy of your balances and recommends optimal testing procedures. Reliable results and dependable titration in practice GTP forms the basis of dependable and risk-free titration. The appropriate titration system is selected based on user requirements, and professionally commissioned and installed. GTP helps protecting your investment far into the future, supporting you with tailored training and equipment qualification. It also proposes schedules for regular inspection and preventive maintenance. Optimize workflows for more reliable and repeatable results Improve your data quality with GPP our comprehensive, systematic approach to maximizing pipetting accuracy and repeatability. GPP is grounded in 40+ years of expertise working side-by-side with researchers in order to achieve the highest level of accuracy and precision across all applications METTLER TOLEDO Academia News 12

9 5 Routine Operation 1 Evaluation GDRP Good Density and Refractometry Practice GEP Good Electrochemistry Practice 4 Calibration / Qualification Good Measuring Practices 2 Selection 3 Installation / Training Minimize risk and improve performance GDRP is based on our expertise in density and refractive index measurement. Understanding the reasons and sources of measurement errors helps improving quality while reducing risks and costs. Professional commissioning, training, qualification and seamless documentation guarantee compliance with your process requirements from the very first step. Optimized ph results from the beginning GEP assists with professional tools and documentation to get reliable results for your laboratory ph, conductivity, DO, ion and redox applications. Use GEP to select the correct calibration solutions to comply with regulations, perfectly maintain your sensors to optimize product lifespan and reduce costly measurement mistakes to a minimum. Check your risks in weighing, titration and chemical analysis Risk Check All of the Good Measuring Practices guidelines start with an evaluation of your measurement processes and an assessment of their associated risks. Based on this information, you are provided with straightforward recommendations for selecting, installing, calibrating and operating your devices. For more Information: Good Measuring Practices METTLER TOLEDO Academia News 12 9

10 Product Highlight Simple and Flexible Solution for Dynamic Mechanical Analysis Dynamic Mechanical Analysis (DMA) is the method used to measure the mechanical and viscoelastic properties of materials as a function of temperature, time and frequency when subjected to periodic stress under a defined environment. The analyzed materials encompass thermoplasts, thermosets, composites, elastomers, ceramics and metals. In addition to operation in DMA mode, the DMA 1 permits measurements in TMA mode and under various environments. Controlled humidity experiments The humidity generator and humidity chamber allow samples to be analyzed under varying relative humidity conditions at a given temperature. Immersion measurements The fluid bath option permits the user to perform immersion studies on a sample. All DMA modes are supported by the immersion option. TMA experiments In addition to the established DMA modes, the DMA 1 permits operations to be performed under constant force (TMA or creep), in stress-strain or stress relaxation mode METTLER TOLEDO Academia News 12

11 Reliable and Intuitive ph Measurement with New SevenCompact For research groups and laboratory courses alike, the intuitive operation and versatility of SevenCompact series fulfil the needs of lab researchers and students. Perfect ergonomics with uplace The electrode arm can be operated with one hand, moving straight up and down to the position where it works best for your sample. One single keypress By switching to ufocus view, you will not be distracted by information of less importance. Broad sensor offering Find the best sensor for your application with tips and tricks from: METTLER TOLEDO Academia News 12 11

12 Everything in Our Lab Works With Just One Click Joanne, laboratory assistant: "Thanks to my personalized Homescreen, I can perform all the different analyses very quickly and efficiently." Bob, production assistant: "All I have to do to analyze the intermediate product every hour is press a Shortcut." Clark, quality assurance: "The individual user rights feature means that I can make sure that users stick to our quality guidelines." The One Click operation provides unsurpassed user guidance, secure navigation and easily selectable and adaptable methods. Learn more and see One Click in action: Key benefits of One Click operation: Know one know all One Click is a common interface for all your lab instruments. This means, if you can program and run analyses on a balance, you can also do it on a titrator, without extra information required. See only what is relevant to you User screen individualization permits you to adapt your home screen to your requirements: You only see what you need and avoid confusion by unneeded functionalities. Play by the rules Adherence to SOPs is now a functionality of your instrument via the pre-programming of methods. Transcription and calculation errors a thing of the past. Mettler-Toledo AG Laboratory Division Im Langacher CH-8606 Greifensee, Switzerland For more information