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BECKMAN COULTER INC.

PARTICLE CHARACTERIZATION

The DelsaMax system

Pre-Course Reading material

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Light scattering is a non‐invasive technique for characterizing macromolecules and a wide range of particles in solution. In contrast to most methods for characterization, it does not require outside calibration standards. In this sense it is an absolute technique. DelsaMax instruments make two different types of light scattering measurements for absolute molecular and particle characterization:

• Classical Light Scattering / Static Light Scattering: Here, the intensity of the

scattered light is measured as a function of angle. For the case of macromolecules, this is often called Rayleigh scattering and can yield the molar mass, rms radius, and second virial coefficient (A2). For certain classes of particles, classical light scattering can yield the size, shape, and structure.

• Quasi-elastic Light Scattering (QELS) or Dynamic Light Scattering (DLS): In a QELS

measurement, time‐dependent fluctuations in the scattered light signal are measured using a fast photon counter. QELS measurements can determine the hydrodynamic radius of macromolecules or diameter of particles.

Understanding Laser Light Scattering Theory

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The DelsaMax family is a premium nanoparticle characterization tool for the measurement of particle size and zeta potential. The DelsaMax family allows users to make measurements extremely fast with very low sample volumes (as low as 1 microliter volume in 1 second).

DelsaMax CORE DelsaMax PRO DelsaMax ASSIST

The DelsaMax family

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The DelsaMax PRO Light Scattering Analyzer

The DelsaMax PRO combines a unique 31 detector diode array with an advanced Avalanche Photodiode Detector (APD) which enables analysis of both zeta potential and size virtually simultaneously. Each measurement takes approximately one second to complete. The optical design is patent pending. Key differentiators include optical design, easy injection port, and low sample volumes. The Pro is the top of the line, most capable instrument and includes both flow cells and cuvette based measurement options. DelsaMax PRO

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Intended Use / Overview

Intended Use:

The DelsaMax PRO is a bench top analyzer that provides non‐destructive electrophoretic mobility measurements and hydrodynamic radius of precious proteins, particles and macromolecules as small as 1 nm under dilute conditions, without perturbing these fragile species.

Overview:

The DelsaMax PRO incorporates an optical bench where the laser light source is split into a Sample Beam and a Reference Beam. The sample beam is scattered by the moving particles, collimated, and recombined with the reference beam onto an array of 30 photo detectors. Each of these photo detectors makes an independent measurement of the sample mobility in a process referred to as Massively Parallel Phase Analysis Light Scattering (MP‐PALS). These signals are then processed in real time with a proprietary demodulation algorithm. The Flow Cell has 1.6 mm spacing of the electrodes. This small gap reduces the required sample volume, decreases the required applied voltages, and further reduces potential sample damage. The simultaneous measurement of the hydrodynamic radius (for molecules) and diameter (for particles) are available with the embedded Quasi‐elastic light scattering (QELS). The integration of the QELS into the instrument utilizes proprietary optics, high quantum efficiency detection, and data processing to analyze backscattered light DelsaMax PRO

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DelsaMax PRO Optical Bench Block Diagram

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Optical Bench:

The laser system, optics and piezo modulator, flow‐ cell assembly, and multi‐channel photodiode array are all anchored to the base plate to provide a single, stable optical bench. The 50 mW diode pumped solid state (DPSS) single‐ longitudinal‐mode narrow beam laser (diameter 0.08 mm) vertically polarized provides the light source for the system. Laser monitor signals are incorporated into the laser assembly (Laser Power and Forward Monitor). The output of those monitors can be displayed on the Main panel on the instrument’s display . The Laser Power measures the intensity of the beam at the laser assembly before it enters the cell while the Forward Monitor measure the transmitted light through the sample cell and

• sample. The primary usage of the Forward Monitor is

simply as a diagnostic tool to determine if there are bubbles or other foreign particles in the cell.

PALS photo detectors assembly Laser assembly Cell block Piezo assembly

Optical bench

DelsaMax PRO

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The DelsaMax PRO light scattering analyzer implements two separate optical systems based on:

• PALS (Phase Analysis Light Scattering).
• QELS (Quasi-Elastic Light Scattering).

and can collect both QELS and PALS data at the same time on the same sample.

Principles of Operation

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Phase Analysis Light Scattering (PALS):

The laser beam is split into a Sample Beam and a Reference Beam. The sample beam is scattered by the moving particles, is collimated, and then mixed/heterodyned with the reference beam directly onto an array of 31 photo detectors that make independent measurements of the electrophoretic mobility. This technique is called Massively‐Parallel Phase Analysis Light Scattering (MP‐PALS). The signals from the array elements are processed in real time with a proprietary demodulation algorithm. The detectors used in the PALS array are linear and robust over the dynamic range. The multiple detectors used in the DelsaMax PRO analyzer efficiently average away the effect of Brownian motion and reveal the electrophoretic velocity, even with low applied voltages.

Principles of Operation

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Dynamic Light Scattering (DLS):

Sometimes referred to as Quasi‐Elastic Light Scattering (QELS), is a non‐invasive, well‐established technique based on Photon Correlation Spectroscopy (PCS) for measuring the size and size distribution

• f molecules and particles typically in the submicron region, and with the latest technology lower than

1nm. QELS is an internally installed function that measures time‐dependent fluctuations in the back scattered light signal using a fast photon counter. The fluctuations will occur rapidly for smaller, faster moving particles and more slowly for larger, slower moving particles. QELS measurements can determine the hydrodynamic radius of macromolecules or diameter of particles. The fluctuations of the scattered light are analyzed using the autocorrelation function.

Principles of Operation

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Technical Specifications

Measurement range: Size: 0.2 nm to 5000 µm, hydrodynamic radius Mobility: No practical limits Zeta Potential: No practical limits exist Cell Voltage range: 0 to 3V typical. Max 600 V/cm. Molecular weight: < 5 x 107 Daltons (g/mol) Light source: Laser Diode 532 nm (green), 50 mW DPSS (diode-pumped solid state) Laser classification: Class 1 for operation (no access to radiation) Class 3B for service and maintenance (trained personnel only). Laser lifetime: 10,000 hours Detection system:

• PALS (Phase Analysis Light Scattering) photodiode array for multi-

angle detection and mobility measurements. 31 detectors for analysis - 1 detector for Laser monitor.

• Single photon counting module for QELS option. 1 detector.

Instrument options: QELS (Embedded Quasi-Elastic Light Scattering) for size measurements.

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Scattering angle: Mobility (Zeta Potential): 4° to 15° (MP PALS) Hydrodynamic radius (Size): 171° (DLS - QELS) Correlator: Multi-Tao function by hardware for correlation curve Sample volume: Hydrodynamic radius (Size): 45 μL Mobility (Zeta Potential): 170 μL Sample concentration: Up to 40 % w/v Operating Temperature: 10°C to 35°C (50°F to 95°F), Analysis temperature range: 4°C to 70°C (39.2°F to 158°F), regulated by a Peltier. Power Supplies: +5V, +12V, ± 15V and +48V Power Voltage: 90 ~ 250 VAC. Universal Power Input. Frequency: 50 to 60 Hz inclusive. Consumption: 200 W (VA) typical. Fuses: Two fuses 4A 250V Slow Blow Dimensions: 59 cm (23”) x 36 cm (14”) x 21 cm (8”) Weight: 19 kg (41 lbs.)

Technical Specifications

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Controls, Connectors & Indicators

LCD display

Fluid connectors cover: In/Out fluid connectors for plumbing the instrument are behind this cover. LCD display (touchscreen ): The LCD display allows you to monitor, control, and configure the

DelsaMax PRO light scattering analyzer.

Keypad: The keypad allows you to control the LCD display. On/Off switch: Main power on/off switch.

Front view Fluid connectors cover Power On/Off switch Keypad

DelsaMax PRO

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Cell block

Cell block: Comes with a pop up door to allow access to the flow cell.

Flow Cell Front view Cell block

Controls, Connectors & Indicators

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Leak Sensor Rinse Port OUT connector IN connector

IN and OUT fluid connectors: Used to plumb the Flow cell from the front panel by using a syringe. Leak Sensor Rinse Port: Use the leak sensor rinse port to empty the leak sensor reservoir after a leak alarm.

Front view

Controls, Connectors & Indicators

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Ethernet / USB connectors: To connect the instrument to the computer via Ethernet or through USB. Nitrogen purge connector: Needed to prevent condensation when operating below ambient temperature. QELS connections: Provides a correlator input for the QELS option. Cooling Fan: Cooling fan to keep the temperature stable.

Rear view Ethernet connector USB connector Nitrogen purge connector Cooling fan QELS connections

Controls, Connectors & Indicators

Mains socket Fuse holder

DelsaMax PRO

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Sample Cells

Flow Cell

Cuvette Cell adapter Quartz Cuvette 45 µL

Flow cell:

The reusable PEEK (polyetheretherketone) flow cell supports simultaneous mobility and QELS measurements (size). It comes with replaceable platinum‐coated electrodes and optical quality

• windows. This cell can be used with aqueous and organic solvents. It is possible to use the flow cell

in a batch configuration.

Quartz Cuvette:

The reusable 45 µL Quartz batch cuvette can be used only for QELS measurements. It cannot be used to measure mobility. This is the same cuvette used with the DelsaMax CORE light scattering analyzer. An adaptor is used with the cuvette to fit the DelsaMax PRO cell holder.

QELS measurements (Size) only Mobility and QELS measurements DelsaMax PRO

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The DelsaMax ASSIST Cell Pressurization System

The DelsaMax ASSIST (optional) is a unique device which can pressurize the liquid in the Flow cell for the DelsaMax Pro only. By pressurizing the sample chamber, the DelsaMax ASSIST will dissolve the bubbles which can cause problems when measuring nanoparticles with dynamic light scattering and phase analysis light scattering (for zeta potential). DelsaMax ASSIST

DelsaMax PRO

DelsaMax ASSIST

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Intended Use / Overview

Intended Use:

The optional DelsaMax ASSIST cell pressurization system is an accessory that can be used to pressurize the liquid in the cell of the DelsaMax PRO light scattering analyzer.

Overview:

Pressure up to 500 psi (35 bar) enters the DelsaMax ASSIST accessory through the Pressure Port. An Input valve controlled by the ASSIST tab on the front panel of the pressurized instrument determines whether pressure connects to the sample line, and thus pressurizes the cell in the instrument being pressurized. The impedance on the pressurized line ensures that the system pressurizes slowly. It takes approximately 10 ~ 15 seconds to pressurize a sample and dissolve bubbles in the cell for a mobility measurement. A Pressure Transducer measures the pressure that enters the DelsaMax ASSIST accessory. Sample enters the DelsaMax ASSIST accessory through the Inject Port. A Check Valve prevents back flow in the

• system. The sample flows to the instrument being pressurized through the To Instrument port. After being

measured in the cell, the sample exits the instrument being pressurized and enters the DelsaMax ASSIST accessory through the From Instrument port. Within the DelsaMax ASSIST accessory, the sample flow is controlled by the Output Valve. If the sample pressure exceeds 725 psi (50 bar), a Mechanical Relief Valve allows sample to flow to the Drain Port in the bottom of the DelsaMax ASSIST accessory (even if the DelsaMax ASSIST accessory is powered

• ff).

DelsaMax ASSIST

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Pressure Port Input Valve Output Valve Inject Port

DelsaMax ASSIST Block Diagram

DelsaMax ASSIST

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Controls, Connectors & Indicators

Inject: Load the sample here via capillary tubing, syringe or a programmable auto injection system. Load: This light indicates that the line is not pressurized, and sample can be loaded. Pressurized: This light indicates that the line is currently pressurized. Flush: This mode is used briefly when switching from Pressurized mode to Load mode. Leak Check. This mode can be selected only from the front panel display on the DelsaMax PRO to run a leak

check.

Pressure Alarm: A red light comes on and an audio alarm is sounded if the pressure in the accessory’s lines

exceeds 580 psi (40 bar). If the pressure exceeds 725 psi (50 bar), a mechanical overpressure relief valve vents the system to the drain outlet for safety.

Power: This light is ON if the On/Off switch of DelsaMax ASSIST accessory is turned on.

Front view

DelsaMax ASSIST

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Controls, Connectors & Indicators

Rear view

Mains socket: Universal power supply, inlet voltages between 90 ~ 250 V, frequencies from 50 ~ 60 Hz. Fuse holder: The fuses in the AC power module are 4 Amp. Slow Blow. USB1 to USB4: Use USB1 to connect the DelsaMax ASSIST to the DelsaMax PRO. Fan and Air Filter: The air filter should be cleaned on a regular basis.

USB Ports Fan and Air filter Mains socket Fuse holder

DelsaMax ASSIST

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Pressure Source: Connect here the line from the gas cylinder or fluid pressurization system. To Instrument: Use the provided connection tube to connect the DelsaMax ASSIST to the input port of the

instrument that is to be pressurized.

From Instrument: Use the provided connection tube to connect the DelsaMax ASSIST to the output port of

the instrument that is to be pressurized.

Waste: Sample exits here. Plumb this outlet to a drain or waste reservoir.

Left side view

Controls, Connectors & Indicators

DelsaMax ASSIST

Left side view

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The DelsaMax CORE Light Scattering Analyzer

The DelsaMax CORE is a combination of Dynamic Light Scattering (DLS) and Static Light Scattering (SLS) instrument. The DLS detector operates at 90 degrees to measure the size distribution of the hydrodynamics radius, whereas the independent SLS detector, which also operates at 90 degrees, can measure the molecular weight of small molecules. DelsaMax CORE can be used for a broad range of applications requiring both accuracy and high sensitivity. DelsaMax CORE

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Intended Use / Overview

Intended Use:

The DelsaMax CORE is a particle sizing instrument that uses Dynamic Light Scattering (DLS) for measuring hydrodynamic radius of molecules as well as size and size distribution for particles typically in the submicron

• region. Also, a dedicated Static Light Scattering detector has been added to measure absolute molecular

weights as well.

Overview:

The sample is illuminated by a 120 mW air launched laser of ~ 658 nm wavelength. After laser intensity stabilization, 100 mW is delivered to the sample cell. The light scattered by the sample is collected by two independent detectors: one for Static scattering and one for Dynamic scattering. The Static scattering detector is a silicon PIN photodiode, whereas the DLS detector is an actively quenched, solid state Single Photon Counting Module (SPCM). The photons are converted to electrical pulses and sent to a high speed multi‐tau autocorrelator. DelsaMax CORE

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DelsaMax CORE Optical Bench Block Diagram

DelsaMax CORE

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Optical Bench

Top view (uncovered)

Optical Bench: The 100 mW air launched narrow beam laser (diameter 0.08 mm) vertically polarized provides the

light source for the system. This small beam diameter also helps reduce the noise contributions of larger particulate contaminants (such as dust). A Laser monitor signal are incorporated into the laser assembly (Laser Monitor and Forward Monitor). The output of those monitors can be displayed on the Main panel on the instrument’s display . The Laser Monitor measures the intensity of the beam at the laser assembly before it enters the cell while the Forward Monitor measure the transmitted light through the sample cell and sample. The primary usage of the forward monitor is simply as a diagnostic tool to determine if there are bubbles or other foreign particles in the cell. DelsaMax CORE

DelsaMax

Service Training Last update: July, 2013

The DelsaMax CORE particle sizing instrument implements two separate optical systems based on:

• SLS (Static Light Scattering).

The Static scattering detector is a silicon PIN photodiode at 90°.

• DLS (Dynamic Light Scattering) also known as: QELS

The DLS detector is an actively quenched, solid state Single Photon Counting Module (SPCM). The photons are converted to electrical pulses and sent to a high speed multi-tau autocorrelator. The DelsaMax CORE can collect both SLS and DLS data at the same time on the same sample.

Principles of Operation

DelsaMax CORE

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Technical Specifications

Size Range (Radius): 0.2 to 2500 nm Minimum Concentration (lysozyme)*: 0.1 mg/mL Scattering Angle Detected: 90° Laser Wavelength: 658 nm; optional: 785nm Laser Power: 10-100 mW, programmable Sample Cell Options: 1.25 μl quartz cuvette, 4 μl disposable cyclic olefin copolymer (COC) cuvette with adapter. Temperature Range:

• 15°C to 150°C †. Dry air or nitrogen required for
• peration below 20°C.

Correlator: 512 channels, multi-tau, 100 nsec sampling time. Data Acquisition Time: 1 to 3600 s Power Voltage: 90 ~ 250 VAC. Universal Power Input. Frequency: 50 to 60 Hz inclusive. Optical Fiber: Multi-Mode PC ports: Ethernet connectivity Weight: 13.5 kg * 14 kDa Lysozyme, PBS, 100 seconds data acquisition † Absolute accuracy of +/– 0.3ºC

DelsaMax CORE

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Cuvette storage: Cuvettes can be stored behind this cover. LCD display (touchscreen ): The LCD display allows you to monitor, control, and configure the

DelsaMax PRO light scattering analyzer.

Keypad: The keypad allows you to control the LCD display. On/Off switch: Main power on/off switch.

Controls, Connectors & Indicators

Cuvette storage LCD display Keypad On/Off switch

DelsaMax CORE

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Cell block: There is a sliding door to allow access to the flow cell. The door includes a magnetic proximity

switch which disables the laser when the door is open.

Cell block Sliding door Keyed Cuvette Receptacle Cuvette

Controls, Connectors & Indicators

DelsaMax CORE

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Nitrogen purge connector Correlator input Mains socket Ethernet connector Fuse holder Fan & Filters

Ethernet / USB connectors: To connect the instrument to the computer via Ethernet or through USB. Main AC input filter: Contains the power plug connector as well as the fuse holder. The Fuse holder

contains two fuses (4 Amp 250 V) for ALL AC input voltages (95V to 250V AC).

Nitrogen purge connector: Needed to prevent condensation when operating below ambient temperature. Correlator Input: Provides a correlator input for the QELS option. Cooling Fan: Cooling fan to keep the temperature stable.

Controls, Connectors & Indicators

DelsaMax CORE

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