Wyatt Technology Corporation. minidawn TREOS. The multi-angle light scattering detector for absolute macromolecular analysis

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1 Wyatt Technology Corporation minidawn TREOS The multi-angle light scattering detector for absolute macromolecular analysis

2 The minidawn TREOS The Multi-Angle Light Scattering Detector for Absolute Macromolecular Analysis The minidawn TREOS is a compact, simple-to-use multi-angle light scattering (MALS) detector for high performance liquid chromatography. It connects quickly to your existing chromatography equipment and allows you to determine the absolute molar masses and sizes of your polymers or biopolymers without the need for reference standards or column calibration. The TREOS patented, multi-angle detector array works for molecules of just a few hundred daltons to several million. It can even be used offline for stand-alone micro-batch measurements requiring only a couple hundred microliters of sample. The minidawn has the most sophisticated electrooptics of any light scattering instrument on the market. An exceptionally rigid glass-to-metal design, combined with an innovative vibration isolation feature, yields an optical bench of tremendous stability. The patented flow cell design contains windows that protrude into the eluent stream at the entrance and exit manifolds. This reduces beam/cell problems by keeping the cell The TREOS geometry, showing the laser light path, patented flow cell, and unique simultaneous triple photodiode detection. and its interfaces clean of precipitates, simplifies the measurement procedure, minimizes laser beam/interface flare, and ensures rapid sample-running. Because of the glass-to-metal design, the instrument can be used with aqueous or organic mobile phases and you can change solvents at any time without worrying about the chemical compatibility of the flow cell glass. The TREOS represents a major advancement in the sensitivity and dynamic range in the measurement of multi-angle light scattering for small molecules. It has over twice the signal to noise ratio and 100 times the dynamic range of t he minidaw N TriStar, allowing the measurement of smaller molecules at lower concentrations than ever before.the unique triple-angle configuration maximizes the range of angles and minimizes noise. This balance is the heart of the TREOS ability to characterize a wide variety of samples. Just by adding a TREOS to your existing chromatography line, you ll end your dependence on calibrationrelated techniques forever. 2 What does absolute mean? In solution, there are only three methods for making absolute measurements of molar mass. They are membrane osmometry, sedimentation equilibrium centrifugation, and light scattering. These three techniques provide absolute measurements because they are made without reference to molar mass standards, column calibration, or molecular conformation. In other words, an absolute measurement is one made from first principles; it is not a measurement derived from reference to other molar masses. Historically, absolute molar mass measurements were the domain of a very few laboratories, which had invested hundreds of thousands of dollars in elaborate equipment and tedious training. Because of the resources required to make these measurements, most people reverted to using analytical techniques based on calibration standards. But the accuracy and validity of the results obtained were always suspect. At best, they were only relative measurements. Since the absolute measurements made by light scattering instruments are the sine qua non of macromolecular characterization, the minidawn enables any laboratory to be able to afford the equipment and interface it to their existing HPLC apparatus. In order to determine an absolute molar mass from light scattering, there are three quantities that must be measured: The amount of light scattered at each angle, (the function of the minidawn TREOS or DAWN HELEOS ), The concentration of the polymer, (the purpose of an RI or UV detector), The specific refractive index increment, dn/dc, of the solute at the same wavelength as the light scattering instrument (this is an off-line, preparative determination for which our Optilab rex is ideally suited).

3 minidawn TREOS Advantages Produces absolute molar masses precisely from less than 10 3 to more than 10 6 g/mole Determines molecular radii from 10 nm to more than 50 nm Embedded COMET system automatically cleans the flow cell in situ. Just press a button, or schedule cleaning as part of an ASTRA sample set. This insures that your system produces the same high quality data day after day, with no downtime. The TREOS represents a major breakthrough in multi-angle light scattering (MALS) instruments. Embedded QELS allows simultaneous online measurement of hydrodynamic radius. This improves the resolution of size distribution measurements by over an order of magnitude compared to batch QELS. Determines conformation or molecular structure of polymers and biopolymers Reports branching ratios, average branching per molecule, and long chain branching Eliminates column calibration Simplifies chromatography Eliminates dependence on polymer standards

4 Optics & Electronics The first light scattering instruments to incorporate digital signal processing electronics The TREOS engineering elegance has been achieved by integrating its optics with the sample cell structure and the laser light source. There are no mirrors, prisms, or moving parts that could become misaligned, damaged, or dirty elements of particular concern when any measurements are made. Developed through years of research and analysis by the world leaders in light scattering instrument design, the kinematically-engineered flow cell, manifolds, read head, and laser mounts are unified on a single optical bench. Wyatt Technology s patented flow cell is the centerpiece of the TREOS, and its revolutionary design allows it to be cleaned and replaced without any instrument realignment. The flow cell minimizes sample volume and stray light but allows multi-angle measurements to be performed simultaneously a feature not found in any other instrument. The custom designed detectors incorporated in the mini- DAWN exceed the The integrated and patented manifold/ flow cell assembly insures highly reproducible measurements at all angles simultaneously. performance of similar devices available anywhere else. In fact, Wyatt Technology s staff pioneered the use of lasers in classical light scattering instruments more than three decades ago. Each analog signal (one for each angle) is processed by its own Digital Signal Processing (DSP) integrated circuit with 24-bit digital conversion for extremely high signal resolution. In addition, four auxiliary analog inputs (with their own DSP circuits) are available for interfacing to external detectors, such as DRI, UV, PDA, etc. We also record the temperature, laser power, and forward monitor signal a valuable extra for studying samples that absorb light. The data are then post-processed by a high speed, embedded PC using a proprietary algorithm to virtually eliminate noise caused by dust spikes or other contaminates, providing reliable results, even in the face of harsh chromatography conditions. The noise rejection algorithm gives the greatest possible noise rejection without peak distortion. Typically, other light scattering instruments use such long time constant analog filters that peak distortion becomes unavoidable. Since the analogto-digital conversion is performed onboard the TREOS, low light scattering signals are not prone to environmental noise or pick-up, which can cause erroneous data conversions. The TREOS digital output is transmitted from the instrument to a PC via ethernet for network operation, or USB when stand-alone operation is desired. Not only is digital data transfer the most reliable form of transmission, each packet includes a time stamp and checksum, guaranteeing no data are lost or corrupted. By virtue of its unique triple-angle geometry, the TREOS may be used to determine absolute molar masses from fewer than 1000 to more than a million g/mole (daltons) and radii from about 10 nm to more than 50 nm. How It Works... The minidawn TREOS utilizes a fixed, multi-angle detector array which simultaneously measures the light scattering data at three angles. Thousands of data are collected in less time than it takes a mechanically-driven device to make a measurement at even a single angle. In the limit of very small concentrations, as is invariably the case for liquid chromatography, the fundamental relationship linking the intensity of scattered light, the scattering angle, and the molecular properties is simply: K* c = A 2 c R(θ) M W P(θ) where: R(θ) is the excess measured Rayleigh ratio, c is the sample concentration (g/ml), Μ W is the weight-average molar mass (grams per mole), A 2 is the second virial coefficient, (a measure of the solvent-solute interaction) K* is an optical parameter equal to 4π 2 n 0 2 (dn/dc) 2 / [λ 0 4 NA ], n 0 is the solvent refractive index and dn/dc is the refractive index increment, N A is Avogadro s number, and λ 0 is the vacuum wavelength of the incident light. The function P(θ) describes the scattered light s angular dependence. At low angles, its variation depends only on the mean square radius <r g 2 > and is independent of molecular conformation or branching. A plot of K*c/R(θ) vs. sin 2 (θ/2) in the limit of very low concentrations, (e.g. chromatography experiments,) yields a curve whose intercept gives Μ W and whose slope at low angles gives <r g 2 >. K*c R(θ) intercept gives Μ W slope gives <r g 2 > 4 sin 2 (θ/2)

5 Chromatography ASTRA software is 21 CFR Part II compliant By coupling a minidawn TREOS to your existing chromatograph, you have a powerful system that can be used for routine on-line determinations of absolute molar masses, sizes, and conformations. ASTRA is our intuitive, Windowsbased chromatography software that permits the simultaneous collection and processing of different data files. Of course, ASTRA supports multitasking with other applications. In addition, ASTRA will: Determine absolute molar mass, size, and the second virial coefficient Integrate light scattering, QELS, RI, UV, and viscometry measurements Calculate M w based on column or universal calibration using a ViscoStar viscometer Measure intrinsic viscosities and Mark- Houwink parameters Determine polydispersity values (M w /M n) and (M z/m n) Count particle number densities Measure the molar mass and rms radius for each elution slice (and therefore the conformation) Determine protein mass and fraction for protein conjugates Correct for interdetector band-broadening Calculate the precision of each result it reports Run with Waters Empower software Comply with 21 CFR Part II regulationstion Besides controlling the data collection, ASTRA produces results from the chromatographic data without reference to standards, quasi-empirical calibration techniques, or a priori assumptions about molecular structure/conformation. With Wyatt Technology s software, every quantity required to make an unequivocal molar mass determination has its own independent means of verification from the dn/dc determination, to the concentration, to the absolute intensity of light scattered at each angle. ASTRA even reports the uncertainties of the measurements, so you can get an instant feel for the quality of your data. With the minidawn and ASTRA, pump speed fluctuations no longer play a role in determining molar masses: whenever the sample elutes, ASTRA will calculate its absolute molar mass at each elution volume. Changing your A typical minidawn chromatography layout. Just by adding a minidawn, you will be determining absolute molar masses and sizes without ever having to calibrate your GPC again. columns, your HPLC tubing connections, even your flow rate won t affect the minidawn s performance at all. The ASTRA analysis is based on rock-solid science developed at Wyatt Technology and is found in no other software package. For example, proprietary global fitting algorithms ensure that no extrapolation is needed in computing molar mass, size, or second virial coefficient. Branching ratios are computed directly, rather than from dubious empirical relations. The results of these calculations are displayed in intuitive graphical displays such as: Molar Mass and Molecular Size vs. Volume Differential Molar Mass and Molecular Size Distributions Cumulative Molar Mass and Molecular Size Distributions Long chain branching calculations and graphics Conformation plots of log M w vs. log r g Three dimensional plots displaying simultaneously each light scattering detector s response as a function of elution volume Custom plots of any measured or calculated quantity on x and y axes ASTRA's graphics capabilities overshadow its predecessor s in just about every way imaginable. Graphs can be exported to metafile, bitmap, JPEG, PNG, GIF, PCX, PDF, even Postscript format for the greatest possible compatibility with your reporting requirements. In addition, you can create Bar, Pie, Line, and Scatter plots from the data with just a few clicks of the mouse. Moreover, you have complete control over tick marks, scaling, line weight and widths, patterns, fills, etc. ASTRA has industrial strength graphing built right in. 5

6 Additional Products & Applications Wyatt Technology provides the ideal instruments for comprehensive characterization of macromolecules and nanoparticles. Multi-Angle Light Scattering (MALS) Wyatt s Optilab T-rEX has 256 times the detection power and up to 50 times the dynamic range of any other RI detector. and up to 50 times the dynamic range of any RI detector in existence today. The Optilab T-rEX may also be used to measure the absolute refractive index increment, dn/dc, at the same wavelength as the light scattering instrument. Finally, the Optilab may be used to measure the absolute refractive index of a solution. made of the highest quality silica with well-controlled pore size and highly reproducible surface chemistry. As a result, WTC columns provide high resolution, extended lifetime, excellent lot-to-lot reproducibility, and a full range of pore size selections. Moreover, these columns have exceptionally low light scattering baseline noise and better pressure shock resistance than any comparable columns on the market. Wyatt Eclipse The Eclipse system brings a powerful sub-micron particle separation technology to your lab in the form of Asymmetric-Flow Field Flow The DAWN HELEOS II is the ultimate in a research-oriented light scattering instrument. Its 18 angles of detection give it the widest angular range of any commercially-made light scattering detectors, and its numerous options enable it to be customized for practically any application. SEC Columns for Multi-Angle Light Scattering Wyatt s new silica based columns are specifically designed for SEC- MALS protein applications. They are The Wyatt Eclipse is the ultimate system for separating macromolecules, proteins, colloids, and nanoparticles. The MALS family of instruments includes the DAWN HELEOS and minidaw N TREOS. These instruments measure the absolute molar mass, size, and shape of macromolecules in solution. They may be used in batch mode (off-line) or connected on-line to an HPLC/FPLC/ AFFF, etc. Each instrument contains connections and on-board digital signal processing hardware for four external devices. The HELEOS II has 18 detectors for unparalleled multiangle measurements (from a low angle of about 10 to a high angle of almost 180 ), a 120mW GaAs linearly polarized laser, and options for a variety of thermostatic-control systems to go up to 210ºC or as low as -15ºC. Optilab T-rEX The Optilab T-rEX (refractometer with EXtended range) is an RI detector that has 256 times the detection power Wyatt SEC columns provide high resolution, extended lifetime, excellent lot-tolot reproducibility, and a full range of pore size selections. Fractionation (AF4). It integrates perfectly with Wyatt s DAWN HELEOS and Optilab T-rEX instruments to measure submicrometer particles and their distributions in solution. The AF4 system may also separate macromolecules, such as proteins. The AF4/ MALS technique is a key orthogonal technique (along with SEC-MALS and AUC) for identifying and quantifying protein aggregates in solution. 6

7 linear polyethylene branched metallocene ASTRA s Protein Conjugate Analysis reveals the amounts of protein and modifier in a PEGylated protein. The calculations are based on light scattering, refractive index, and UV absorbance data. Here the total mass of 60kDa for the monomer is comprised of 40kDA of PEG and 20kDa of protein. The ratio of protein:peg holds for the dimer. Multi-angles mean more information... ASTRA s powerful plotting capabilities allow you to see everything with the greatest possible precision. Here, the cumulative molar masses from a BSA mixture are shown with their underlying numerical particulars. The highlighted area illustrates the dimer peak. Of note as well is the fact that ASTRA s proprietary band-broadening correction has been applied to the calculations. ASTRA comes replete with extensive on-line help. Specific sections of the program can be highlighted and described by pressing the F1 key. Context-sensitive help screens appear, with related materials just a mouse-click away. With ASTRA you ll have copious amounts of assistance whenever you need it. Superb reproducibility is exemplified by these two runs of a gelatin sample. Although it appears that there is only one data file shown, there are actually two overlaid. In this instance, the fluctuations are well below 1%. Historically, all classical light scattering measurements have been made over a broad range of scattering angles, which is the origin of the term multi-angle light scattering (MALS). This is in contrast to scattering measurements produced at only one or two angles, which yield pseudo-light scattering results because they are calibrated to known standards. Multi-angle light scattering is the preferred analytical technique for determining molar masses and sizes without making assumptions. And because it is a straightforward, absolute method a significant advantage over most other analytical techniques it provides results that are independent of so-called standards or measurements that someone else made in another laboratory. The amount of light scattered by a suspension of molecules in excess of that scattered by the solvent is directly proportional to the product of their weight-average molar mass and the concentration. The angular variation of the scattering (as a function of angle) reveals the molecules mean square radius. If you want to determine molar masses and sizes over the greatest possible range, a MALS instrument is the ideal solution. Unlike empirical techniques, such as viscometry, MALS doesn t require questionable assumptions, which relate the measurements to the results. Furthermore, MALS makes no prior assumptions about molecular conformation (since MALS can measure it), nor does it depend upon specific polymer reference standards. All the constants required for determining absolute molar masses are measured experimentally from first principles they do not depend on vague empirical relations. 7

8 Additional Products The ViscoStar II represents a new generation of online differential viscometers. ViscoStar The ViscoStar represents a new generation of online differential viscometers that is designed with a traditional four-arm bridge arrangement. The uniqueness of the ViscoStar springs from its high accuracy and contemporary electronics. It achieves at least twice the signal-to-noise ratio of any commercial viscometer currently on the market, and it incorporates a high efficiency heat exchanger to provide extremely stable baselines. Dynamic Light Scattering (DLS) When you want to have the best of both worlds (classical and dynamic light scattering), our MALS instruments can be ordered with the embedded WyattQELS, or they can interface with our DynaPro NanoStar. The combined instruments allow simultaneous static and dynamic light scattering measurements from which both absolute molar masses and hydrodynamic radii can be obtained. DynaPro NanoStar The DynaPro NanoStar DLS instrument is used predominantly in batch mode for the analysis of the size, aggregation and thermal stability of protein solutions, biomolecules, colloids, small molecule aggregates, buffers or other macromolecules or nanoparticles in solution. New specially-designed cuvettes allow one to use as little as 1 μl of sample! The NanoStar produces more than twice the signal-to-noise of its predecessor. The DynaPro now has a built-in autoattenuator and temperature control up to 150ºC! Even a dedicated static light scattering detector has been built into the NanoStar, so that it can measure not only hydrodynamic radii of a given sample, but also its absolute molar mass. DynaPro Plate Reader The DynaPro Plate Reader incorporates state of the art technology, providing superior high throughput DLS performance. The non-invasive and dry technology measures The DynaPro NanoStar can be used for nanoparticle and protein size determination, with sample volumes down to 1μL and a temperature range from C. samples directly from industry-standard microplates (96, 384, or 1536 well plates) using as little as 1.5 μl of sample per well. After the plate is loaded into the DynaPro, the data are collected automatically in an unattended man-ner by the easy-to-use DYNAMICS Software, which also supports full integration with industry leading liquid handling robotics. Once the data are collected, the samples can be recovered and the plates dis- 8 DynaPro Plate Reader is the most popu-lar automated DLS instrument for the highest levels of reproducibility on the market. carded no cleaning required! The DynaPro Plate Reader will help you increase your productivity by a factor of 10 or more when sizing biomolecules and other nanoparticles compared to conventional batch DLS systems. Calypso The new Calypso system simplifies the task of preparing batch samples for light scattering analyses. The Calypso s proprietary software automatically prepares sequential dilutions required to measure the composition and concentration dependence of light scattering measurements for the analysis of self- and hetero-associations and the measurement of kinetics. Calypso may be used to study the equilibrium and kinetics of reversible self- and hetero-association of proteins, as well as to measure virial coefficients.

9 Service & Support Worldwide Distributors World-class service and support from the leading developer of light scattering instrumentation. The founders of Wyatt Technology commercialized the first light scattering instruments incorporating lasers more than 40 years ago. It s no surprise that today, we re the world leader in the field. No instrumentation developer has greater resources devoted to the manufacture, service, and support of light scattering instruments than we do. We have more post-graduate degreed scientists involved in the support of this type of equipment than all of the other firms in the business combined. In fact, our company has more than 300 person-years of cumulative experience in light scattering. We re proud to offer our unique Light Scattering University course that includes three days of intensive hands-on training in Santa Barbara, California. The curriculum covers light scattering theory, data collection and analysis, troubleshooting, and maintenance. During training, our customers interact with the people who develop our software and hardware. Customers learn not only about hardware and software operation but also about sample preparation and data interpretation. Our internationally-acclaimed immersion training also insures that operating the instruments will be second nature by the time the course is done. When questions do arise, our superb staff answers telephone, fax, and is the home of light scattering instrumentation. The site includes over 180 Application Notes, more than 6000 known peer-reviewed publications using Wyatt instruments, all upcoming Light Scattering University training schedules, a variety of Light Scattering Seminar schedules, where customers may view their current instrument service histories, download software updates, view both live and archived webinars on a variety of topics, read new FAQs, and order spare parts and supplies. inquiries quickly. By means of our interactive customer support, our service personnel are always just a phone call away, and a proprietary Internet link can bring us directly into your lab for hands-on help and guidance with our Remote Assistant support feature. Since the company has so many biotechnology, pharmaceutical, and polymer customers, we have developed complete IQOQ service packages, which are available with on-site support. Our International Light Scattering Colloquium is held once a year to stimulate the exchange of information among our customers and to promote greater understanding of the capabilities of MALS and other technologies developed by Wyatt. All of Wyatt Technology s customers are eligible to attend and participate in this colloquium that is often called the most interesting scientific conference around. The company also maintains an online Support Center where customers can view their instrument service status, download current software updates, read our Frequently Asked Questions, and order spare parts and supplies from the on-line store. Our Website provides frequent additions to its extensive bibliography of thousands of refereed scientific papers that have utilized Wyatt instruments, training schedules, and Application Notes. You can find them all at AUSTRALIA/NEW ZEALAND Tel: Fax: john.hewetson@shimadzu.com.au AUSTRIA/GERMANY/SWITZERLAND Tel: Fax: info@wyatt.eu CHINA Tel: Fax: daijie@wyatt.com.cn FRANCE Tel: 33(0) Fax: 33(0) nmignard@wyatt.com INDIA Tel: Fax: info@spincotech.com ISRAEL Tel: Fax: bensi@eldan.biz ITALY Tel: Fax: info@alfatechspa.com JAPAN Tel: Fax: ei.tsuruta@shoko-sc.co.jp KOREA Tel: Fax: is@insung.net NORTH/SOUTH AMERICA Tel: (805) Fax: (805) info@wyatt.com SPAIN/PORTUGAL Tel: 33(0) Fax: 33(0) nmignard@wyatt.com SWEDEN/NORWAY Tel: Fax: tsandberg@wyatt.com TAIWAN Tel: Fax: wp4713@ms8.hinet.net TURKEY Tel: Fax: guran@infokimya.com UNITED KINGDOM Tel: Fax: kjackson@wyatt.com 9

10 Specifications Light Source: Laser Wavelength: Laser Lifetime: Detectors: Detector type: Detector Resolution: 50mW GaAs linearly polarized 658nm (nominal) Each instrument is individually characterized. >10,000 hours 3 angles Custom-designed, ultra-quiet hybrid photodiodes with built in FET transimpedance amplifiers 24 bit With installations in more than 50 countries, Wyatt Technology is the world s leading manufacturer of instruments for absolute macromolecular characterization. It is the only company in the world focused exclusively on such systems, their design, and their application. Mobile phase compatibility: All-solvent compatible (aqueous and organic) Instrument Options: Embedded QELS at 90 degrees Embedded COMET flow cell cleaning system Scattering Volume: 0.02 µl Signal Processing: On chip sinc 3 filter with proprietary dust rejection filter Electronic Inputs: 4 differential analog inputs with 24 bit resolution. TTL inputs for Alarm In, Autoinject contact closure, Recycle in Electronic Outputs: 3 single ended analog outputs from (-10V, 10V) which retransmit any measurement signal. TTL alarm output, autoinject retransmit, Recycle valve power Molar Mass Range: 10 3 to 10 6 g/mole (daltons)* Molecular Size Range: 10 nm to 50 nm Computer Interface: Ethernet and USB 2.0 interface Processed Data Transmission Rate: Software selectable from 36.6 Hz to 0.2 Hz Front Panel Display: Power: AC Voltage: 162.5mm 16-bit high resolution LCD display with Graphical User Interface 960W max, 75W typical Hz, universal power input Dimensions (cm): 168 mm x 357 mm x 595 mm (H x W x D) Shipping Weight: 12 kg DAWN, HELEOS, TREOS, OPTILAB, VISCOSTAR, NANOSTAR, CALYPSO, ASTRA, DYNAMICS, Light Scattering University, Light Scattering for the Masses, Protein Solutions, Wyatt Technology, and the Wyatt Technology logo are registered trademarks of Wyatt Technology Corporation. Wyatt Technology instruments, components and software are covered by one or more of the following: U.S. Patent Nos.: 5,404,217; 5,475,235; 5,528,366; 6,411,383; 6,426,794; 6,452,672; 6,519,032; 6,651,009; 6,774,994; 6,819,420; 6,975,392; 7,027,138; 7,283,221; 7,331,218; 7,386,427; British Patent Nos.: EP ; EP ; EP ; EP ; EP ; German Patent Nos.: ; ; ; ; ; Japanese Patent Nos.: 3,580,380; 4,346,694; 4,439,211; 4,381,914; 4,426,951. Chinese Patent Nos.: ZL ; ZL ; Korean Patent No.: 794,478. Other patents pending. No part of this brochure may be reproduced in any way without written permission from Wyatt Technology Corporation Wyatt Technology Corporation. W1100D * depending on dn/dc, the sample concentration, and chromatography conditions, this is typical. depending on the sample, this is typical. Warranty: All minidawn TREOS instruments are guaranteed against fabrication defects for twelve months. Should any unit become defective due to normal use within the warranty period, we will repair or replace it at no charge. Wyatt Technology has a policy of continual improvement. Specifications subject to change without notice. Light Scattering for the Masses CORPORATION 6300 Hollister Avenue Santa Barbara, CA USA Tel: 805/ Fax: 805/ info@wyatt.com