The Zetasizer µV is a light scattering system capable of simultaneous dynamic (DLS) and static light scattering (SLS) measurements of size and molecular weight. It can be used as a stand-alone batch instrument and can also be integrated into a size-exclusion chromatography (SEC) system. This allows the system to perform DLS and SLS measurements under chromatographic conditions for measurements of size and molecular weight independent of sample elution volume. The OmniFACE analogue/digital interface allows the connection of electrical signals from up to two concentration detectors and a trigger signal from an autosampler. The data is all collected and analyzed in OmniSEC, the dedicated SEC software package.
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This technical note describes how to integrate the Zetasizer µV and OmniFACE into a third-party SEC system. The fluidic connections are described, as are general electrical connections along with a few specific examples. Finally, instructions on how to connect OmniFACE and the Zetasizer µV to the PC and how to begin data collection are provided.
The flow cell may include opaque connectors and tubing that are not appropriate for chromatography. A second set of black PEEK connectors and 1/16” chromatography tubing should be provided with the cell and these should be used (figure 2). In a standard chromatography setup, the eluent will flow from a reservoir through a pump, an autosampler, a column, and finally one or two detectors before flowing to waste. The eluent generally travels through 1/16” tubing after the pump. The black headless PEEK nut should be pushed onto the 1/16” inch tubing. Then the blue or white flat bottom compression fitting provided should be pushed onto the tubing so it is flush with the end of the tube (figure 2).
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The Zetasizer µV can be placed at any point in the detector series after the column. This should be done in accordance with the requirements of other detectors which have back-pressure limits that should be carefully followed. The outlet from the column or another detector on the SEC system should be connected to the inlet on the flow cell and then from the outlet of the flow cell to the next detector or to waste (figure 3). The inlet is indicated by an arrow (figure 3A). The cell should be inserted and the lid closed without putting too much tension on the tubing (figure 3B).
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Adding the Zetasizer µV to a third party chromatography setup should have no effect on the system. The flow cell can be removed from the Zetasizer µV, leaving it free for batch use, without having to be disconnected from the fluid connections to other detectors unless desired.
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Analogue cables from SEC systems are often provided by the manufacturer. If unavailable, the appropriate connections can usually be identified and created from the user manuals.
On the back of each external detector should be connectors to output an analogue signal (e.g. 0-1V). The manual should help identify the ‘signal out’ and ‘ground’ connections from the external detectors. The ‘signal out’ or ‘+’ connection should be connected to the ‘UV in HI’ or ‘RI in HI’ on the OmniFACE depending on the detector used. The ‘ground’ or ‘–’ should be connected to ‘UV in LO’ or ‘RI in LO’ on the OmniFACE.
To test the connection, a run can be started in the OmniSEC software and the signal from the detectors monitored.
Many chromatography systems include an autosampler to enable multiple injections to be performed without the need for an operator to be present e.g. overnight. In order to synchronize the actions of the equipment used in this process, most chromatography equipment sends an electrical signal between injectors/pumps/detectors. The OmniFACE can recognize this signal in order to automatically start data collection. The OmniFACE requires that this signal is a contact closure
The connector that sends the start signal can be identified by looking at the back of the autoinjector and pump and in the manual. This will be labeled ‘inject start’ or ‘start signal’ or ‘trigger signal’ or similar. The ‘+’ or ‘signal’ socket is connected to ‘TRIG SW; on the OmniFACE and the ‘–’ or ‘ground’ to ‘TRIG GND’ on the OmniFACE.
To test this, a run with an autosampler should be started and then when OmniSEC is waiting for the trigger, an injection can be performed and the measurement should start.
The AKTA chromatography system is in common usage throughout the life sciences market. The connections on this system are not standard but can still be connected to the Zetasizer µV.
UV analogue signal
The analogue signal comes from a 6-pin mini DIN socket located at the back of the instrument (figure 5) labeled ‘Analogue out 0-1V’. This contains analogue outputs and grounds for UV, conductivity and pH depending on the configuration of the instrument. A cable may have been provided with the AKTA system but may also need to be created. This will have an appropriate 6-pin mini DIN plug at one end and loose wires at the other. The black wire (1) corresponds to the UV ‘signal out’ and the brown wire (2) corresponds to the appropriate ground. Connecting these two to the ‘UV in HI’ and ‘UV in LO’ sockets on OmniFACE should provide the analogue UV signal.
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When the signal becomes negative, the analogue output from the AKTA is reset to 1 V to show the decrease. To avoid this, an offset can be set into the instrument such that the output signal is 2% (0.02 V) above 0V. This is done on the Monitor UPC-900. Using the wheel select - Set Parameters: UV: Set UV Analogue Out: Set UV zero level. It is useful to include this step to avoid the analogue signal jumping to 1V at random intervals throughout the run.
Start signal
The start signal can be set to come from the ‘remote’ socket on the back of the AKTA instrument (figure 6). This is an RS-232 type socket. Pins 6-9 correspond to ‘AuxOut’ signals and pin 5 is a universal ground signal.
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A new cable should be made by connecting wires to pins 5 and 6 on a new RS-232 plug, or an existing cable can be cut and the wires connecting to pins 5 and 6 identified. The cable is plugged into the socket on the back of the AKTA instrument. The wire connected to pin 5 is connected to ‘TRIG SW’ on the OmniFACE and the wire connected to pin 6 to ‘TRIG GND’ on the OmniFACE.
The Unicorn software controlling the customer’s AKTA system is required to control the signal. Click on ‘manual’ – ‘pump’ in the menu of the ‘system control’ application. The menu should include a number of options including AuxOut 1 to 4. If the wire has been correctly connected, then AuxOut1 should act as the start signal. If the aux signals are not present, they need to be activated in the system settings.
Go to the ‘Unicorn manager’ application. Click on ‘admin’ - ‘system setup’. Click on the diagram representing the chromatography system and click ‘edit’ then on ‘components’. Check the box next to ‘Auxiliary equipment’ and click ‘Ok’. The AuxOut signals should now be available in the ‘manual’ – ‘pump’ options and in the method editor.
The following connections are made between the Zetasizer µV, the Waters Alliance separation unit and the Waters 2487 Dual Wavelength Absorbance Detector (figure 7).
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UV analogue signal
The detector measures at two wavelengths (A & B). One of these can be connected as the UV detector on the OmniFACE. The analogue outputs on this device are screw fittings. ‘UV in HI’ on the OmniFACE is connected to socket B1 and ‘UV in LO’ on the OmniFACE to socket B3 for the wavelength A.
Start signal
The start signal from the separations module can be connected to the Zetasizer µV. The 2695 module has 2 connections for an inject start output labelled B1 and B2. These should be connected to ‘TRIG SW’ and ‘TRIG GND’, respectively.
These HPLC systems are the white stackable systems. The individual units communicate with each other using CAN connectors that look like Ethernet connectors.
HP/Agilent’s Variable Wavelength Detector (VWD) and RI
At the back of the detector a BNC connector is used to output an analogue signal. The outside is the ground. This should be connected to ‘UV in LO’ on the OmniFACE and the core of the BNC connector to ‘UV in HI’ on the OmniFACE as desired. Using the software, the BNC analogue output can be configured to send either the VWD or the dRI signal for the RI detector.
HP/Agilent’s pump
The pump has a DB-9 connector at the back labeled REMOTE. Pin 1 connects to ‘TRIG GND’ on the OmniFACE, while pin 3 from the APG remote goes to the ‘TRIG SW’ on the OmniFACE. Figure 8 shows the two relevant sockets.
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The Zetasizer µV connects directly to the PC via USB while the OmniFACE connects via a RS-232. The drivers for the Zetasizer µV will be installed with either the Zetasizer or the OmniSEC software. In OmniSEC, the OmniFACE should be connected first. This is done in the ‘Acquire-Search for Devices’ menu, found under the “?” menu. ‘TDA 302 Family’ should be selected and the search started. The software should identify the com port to which the OmniFACE is attached. Click OK (figure 9A).
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Connection of the Zetasizer µV is performed in the ‘Acquire-Configure’ menu; found under the “?” menu. ‘Configure’ is selected next to the ‘in-line DLS’. The ‘use DLS’ box must be checked in order to run the Zetasizer µV. The other options in this box allow the DLS settings and thresholds to be set. Click OK (figure 9B).
To start a run, the ‘quick-run’ button with the ‘play’ symbol is clicked. The details of the run are filled in and the ‘quick-run’ button clicked. The software will initialize the Zetasizer and the measurement will be started. If an autosampler is connected, the system will wait for the trigger signal before starting (figure 9C).
ÄKTA is a trademark of GE. Healthcare Bio-Sciences AB.
Waters Alliance is a registered trademark of Waters Corporation.
Agilent, HP and 1050, 1100, 1200 are trademarks of Agilent Technologies Corp.
Malvern Zetasizer Nano is a trademark of Malvern Instruments.
All other trademarks are acknowledged.
