Real-time ph and Conductivity Data Acquisition with the ACQUITY UPLC H-Class Bio System, Auto Blend Plus, and the GE Healthcare Monitor ph/c-900 INTRODUCTION The ACQUITY UPLC H-Class Bio System with Auto Blend Plus Technology can deliver buffers with accurate ph and salt content using the familiar vocabulary of ph and salt concentration, rather than requiring the calculation of pump percentages. Constant levels, steps, or gradients for both the ph and salt concentrations of mobile phases are independently controlled and can be delivered simultaneously. The performance of the system has been extensively tested and validated. The algorithms used in Auto Blend Plus, coupled with the performance of the ACQUITY UPLC H-Class Bio System, accurately and precisely deliver the programmed ph and salt concentrations. For some applications, it can be useful to supplement control functions with real-time ph and conductivity monitoring. This can be helpful during methods development to identify the conditions where a particular protein elutes. Monitoring has also revealed ways in which the delivered ph is altered while passing through an ion exchange column. It is also a convenient way to determine when a column is equilibrated with the buffered mobile phase. Finally, observation of ph and conductivity can be one of the system suitability tests that effectively measures if the system is functioning properly in the mechanical sense and that stocks of buffers and salts have been correctly prepared. Monitoring is not mandatory in any of these examples, but it can occasionally be beneficial. Waters laboratories have extensive experience with the Amersham Pharmacia (GE Healthcare) Biotech Monitor ph/c-900. This system allows the acquisition of real-time ph and conductivity data using Empower 2 or Empower 3 Software. The mv analog output from the meter can be digitally converted by a Waters esat/in module and collected as data channels in Empower. Creating a derived channel in Empower can further facilitate the visualization of this mv data as the actual ph and conductivity levels (µs/cm). Herein, we describe a simple procedure to organize the hardware and software of the ACQUITY UPLC H-Class Bio System, the esat/in module, and the Monitor ph/c-900. Some suggested settings are included. Directions for the creation of a channel derived from Empower and examples of the output possible from this configuration are also provided. 1
MAT E R IA L S System configuration Waters ACQUITY UPLC H-Class Bio System Waters Empower 2 or Empower 3 Software Waters esat/in (P/N 668000230) with accompanying data cables GE Healthcare Monitor ph/c-900 with the accompanying analog cables (2), GE Healthcare conductivity flow cell (P/N 18-1111-05) and GE Healthcare ph electrode (P/N 18-1111-26) One Ethernet cable of appropriate length One I/O injection start cable A dual-row barrier strip with at least four positions (for example RadioShack P/N 274-658) P RO C E DU R E Before beginning this procedure, all units must be unplugged and powered down. This procedure assumes that both the Monitor ph/c-900 and the ACQUITY UPLC H-Class Bio System have been set up and calibrated as directed by the manufacturer. 1. At the rear of the esat/in module, connect both the power and the Ethernet cables. The Ethernet cable is plugged into the connector marked Data. Connect the other end of the Ethernet cable to any position of the Ethernet switch located on the rear of the ACQUITY UPLC H-Class Bio System as shown in Figure 1. 2. Connect the two esat/in input cables at the front of the esat/in module into the Channel 1 and Channel 2 receptacles displayed in Figure 2. 3. Attach the inject start cable (I/O) to the inject start connector of the esat/in. Wire the plug for positions 1 and 2 as seen in Figure 3. 4. The other end of the I/O cable is connected to the event-in connector on the rear of the system s biosample Manager-FTN as shown in Figure 4. 5. On the rear panel of the Monitor ph/c-900, connect the analog signal cables to the plugs labeled ph and Cond as shown in Figure 5. 6. The individual wires at the ends of each of the ph/c-900 analog signal cables are plastic-capped, colored, and bear a plastic number label. Uncap wires #1 (brown) and #2 (red) for each cable. The ends of the input cable for the esat/in are a ground, a white (+), and a black (-) wire as shown in Figure 6. 7. Connect the ph/c-900 analog signal cables to the esat/in data cables using a dual-row barrier strip. Specifically, connect the wires as shown in Figure 7; the brown (#1) to the white (+), and the red (#2) to the black (-). The barrier strip makes this very simple. For the purpose of this set-up, make sure that the ph analog signal cable is connected to the Channel 1 data cable, and the conductivity analog signal cable is attached to the Channel 2 data cable. 8. Plug in and power on the units to complete the physical set-up. Figure 1. Rear of esat/in. Power, Ethernet connection. Corresponding Ethernet connection to biosample Manager-FTN. 2
Figure 2. esat/in front panel. Data cables installed in the positions for channels 1 and 2. Figure 3. esat/in front panel. Inject start cable (I/O) installed. Figure 4. Rear of biosample Manager-FTN. Inject start cable installed. Figure 5. Rear panel of ph/c-900. ph and Cond analog signal cables installed. Figure 6. The (1) capped wires of the ph/c-900 analog signal cable, and (2) the end of the esat/in input cable. The set up will require two identical cable sets: one for ph and one for conductivity. 3
ph Analog Cable esat/in channel 1 Conductivity Analog Cable esat/in channel 2 Figure 7. The completed connection between the esat/in and the ph/c-900 using a 10-position barrier strip. Setting the analog ph range of the Monitor ph/c-900 The Monitor ph/c-900 has a feature that allows the user to define the range of the analog out signal of the meter, specifying mv/ph units. This setting is independent of that of the calibration ph range. Setting the analog range wide or narrow will change the mv/ph unit relationship of the analog output of the meter. Table 1 gives examples of the difference in mv/ph for two analog ranges. Note: Using a derived channel in Empower 2 will necessitate a ph 0 to 14 analog range setting. Analog ph range mv/ph unit 0 to 14 71 6 to 8 460 8 to 10 460 3 to 7 230 Table 1. Analog ph range settings and the corresponding mv/ph units. 4
Derived channels for ph and Conductivity in Empower 2 Software The direct analog output from both the ph and conductivity meters of the ph/c-900 will be in millivolts (mv). While this is useful, it is more desirable to convert this data into meaningful units of ph and ms/cm values that are familiar to users. This can be accomplished by creating a derived data channel in Empower 2 or Empower 3 Software. To create derived channels, follow this procedure: 1. With all units powered and recognized by Empower, enter the project view and open a new method set. Do not enter the method set editor wizard. 2. Right click the Derived Channels in the navigation tree and select New, then Derived Channel as shown in Figure 8. 3. The Edit Derived Channel window is now visible as shown in Figure 9. 4. From the drop-down Channel menu, select esat/in Channel 1 as displayed in Figure 10. 5. In the Constant box, enter the conversion factor to convert the analog mv output to ph. The conversion factor is a single number that will convert the mv output from the meter to ph using the inverse of the mv/ph unit determined for the analog range set by the user. As an example, for the analog range ph 0 to 14, the mv/ph unit was 71 so the conversion factor is 1/71 or 0.014 ph/mv as shown in Figure 11. 6. Click OK and then name the channel as shown in Figure 12. 7. Repeat the procedure for esat/in Channel 2, entering the conversion constant for the conductivity data, and naming the channel appropriately. The conductivity flow cell was calibrated following the procedure in the manufacturer s user manual. For a 1.00 M NaCl solution, the full scale conductivity was 76.11 ms/cm. The analog output range is from 0 to 1000 mv. The conductivity unit (ms/cm) per mv for the flow cell is then 76.11 ms/cm/1000 or 0.07611 ms/cm/mv. This is the conversion constant for the mv output of the conductivity flow cell. 8. An example of a completed method set editor window with two derived channels can be seen in Figure 13. This method set can then be used to process the multiple channel mv data from the meter, yielding results in the appropriate ph and conductivity values. Figure 8. Creating a derived channel. 5
Figure 9. The Edit Derived Channel window. Figure 10. esat/in channel selected. Figure 11. Conversion factor for mv to ph entered. Figure 12. Naming the derived channel. Figure 13. Example of a final Method Set editor window with the two, named derived channels. 6
Examples Following the procedure outlined here will allow the real-time acquisition of ph and conductivity data as channels in Empower. This data can be visualized as the direct mv output of the meter or as ph and conductivity levels in a derived channel. Two examples of this mv output and the respective derived channels are shown for ph in Figure 14, and conductivity in Figure 15. This capability also allows the simultaneous collection and visualization of more complex data. For example, a ph gradient comparison demonstrates the derived channel conversion from mv output to ph as shown in Figure 16. More complex data can also be collected, such as the acquisition of ph and conductivity channels simultaneously with the output of an optical detector yielding meaningful and valuable information as shown in Figure 17. mv ph 700.00 650.00 600.00 550.00 500.00 450.00 400.00 9.00 8.00 7.00 A B Figure 14. A linear ph gradient using Auto Blend Plus as (A) mv data acquired directly from the meter, and (B) as the more meaningful ph values from the derived channel. The ph gradient ranged from 5.26 to 9.61, beginning with a 10-minute hold, then the gradient from 10 to 40 minutes, followed by a hold from 40 to 60 minutes. No column was used. A flow restrictor was in place to ensure proper gradient delivery. 6.00 5.00 mv 350.00 300.00 250.00 200.00 150.00 100.00 50.00 25000.0 A B Figure 15. A linear NaCl gradient using Auto Blend Plus as (A) mv data acquired directly from the meter, and (B) as the more meaningful µs/cm values from the Empower-derived channel. The NaCl gradient was from 0 to 250 mm NaCl, beginning with a 10-minute hold, then the gradient from 10 to 40 minutes, followed by a hold from 40 to 60 minutes. No column was used. A flow restrictor was in place to ensure proper gradient delivery. 20000.0 µs/cm 15000.0 10000.0 5000.0 7
CONCLUSION ph 9.50 9.00 8.50 8.00 7.50 7.00 6.50 6.00 5.50 5.00 Figure 16. Linear ph gradient profile comparison between a Waters Protein-Pak Hi Res CM cation exchange column, 4.6 x 100 mm, 7 µm (blue) and no column (black). The ph gradient for both ranged from 5.26 to 9.61, beginning with a 10-minute hold, then the gradient from 10 to 40 minutes, followed by a hold from 40 to 60 minutes. The ACQUITY UPLC H-Class Bio System with Auto Blend Plus Technology can deliver ph and salinity mobile phases within the limits of the buffer stocks used. The ph levels and saline concentrations can be independently controlled and simultaneously delivered. Connecting a Monitor ph/c-900 meter through an esat/in module enables the acquisition of real-time ph and conductivity data in Empower Software. Utilizing the derived channel feature of Empower, this real-time data can be visualized as the correct ph and conductivity. This type of real-time monitoring is not required but can be very beneficial. 0.16 0.14 0.12 0.10 AU 0.08 0.06 0.04 0.02 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 Figure 17. Example of a chromatogram showing the separation of a protein mixture with three types of data: UV absorbance at 280 nm (black), ph profile (blue), and conductivity (green). The method was 0 to 20 minute(s) simultaneous ph 5.26 to 9.61, and 0 to 250 mm NaCl linear gradients, followed by a 20 to 24 minute hold; then from 24 to 25 minutes a step down to ph 5.26 and up to 500 mm NaCl, a hold from 25 to 27 minutes; then from 27 to 50 minutes a return to initial conditions and re-equilibration. The sample was analyzed on a Protein-Pak Hi Res CM cation exchange column, 4.6 x 100 mm, 7 µm. Waters and ACQUITY UPLC are registered trademarks of Waters Corporation. The Science of What s Possible, Auto Blend Plus, Protein-Pak, and Empower are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. 2011 Waters Corporation. Produced in the U.S.A. December 2011 720004149EN LB-PDF Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com