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In recent years, in addition to the basic activities surrounding the optimization of techniques and instruments for analysis, every area of the analytical laboratory needs to adopt modern management and productivity processes. Methods are increasingly developed using QbD (Quality by Design) principles and are constrained by active life cycle management; most of the instruments in the laboratory-from high-end high-performance liquid chromatography mass spectrometry (HPLC-MS) systems to more rudimentary Centrifuge, balance and pump-all integrated into the control software and laboratory management tools. Goal: Repeatable/certifiable performance, highest efficiency and cost-effective operation, and increased environmental awareness.
One result of this change is that as analyzers and laboratory equipment are updated, new models with technological innovation can be introduced that support this shifted analytical landscape. In this article, we look at the key part of any HPLC setup, the degasser, which is still out of this trend to this day. We showcased the results of years of development process to re-evaluate the design, performance and controllability of this key system component, and focused on the data of the new universal flat membrane degasser that has emerged. This new technology represents a shift, shifting the focus from "constant vacuum" to "constant performance" by allowing users to select and control a fixed degassing efficiency for any HPLC system or method.
Reducing the amount of dissolved air in the HPLC mobile phase has a significant impact on the stability of the system flow rate and mobile phase composition. Low-pressure mixing HPLC pumps rely only on the solvent entering the pump, and any outgassing that occurs during transfer from the proportional valve to the inlet check valve can cause multiple types of errors. First, because the volume in the transfer line contains air instead of a fluid mobile phase, composition errors can occur. As the bubbles in the transmission line stretch, the accuracy of the mixture continues to decline. Finally, air bubbles entering the pump interfere with the inlet check valve, so that the pump does not deliver the full volume of mobile phase to the column, but instead pushes a portion of the mobile phase back to the proportional valve. In addition, the pump must compress any air bubbles to system pressure before it can deliver the mobile phase to the column. In a high-pressure mixed HPLC system, the dissolved gas will affect the operation of the inlet check valve, resulting in the formation of microbubbles due to cavitation. As with low-pressure hybrid HPLC pumps, air bubbles can cause incorrect flow rates, which can affect retention time. This fluctuating flow rate may also increase the system noise in the detector, depending on the detector type and flow sensitivity. Therefore, dissolved air affects the accuracy and resolution of separation, and the ability to reliably identify separated compounds on the column. Therefore, for a long time, almost all HPLC systems have included some form of degassing, from vacuum batch degassing, helium jetting, sonication, to online methods using membrane technologies (including PTFE membranes and Teflon™ AF). Today's HPLC systems have one of two mobile phase mixing devices-solvents are mixed before entering the pump (low pressure mixing); or, mobile phase mixing occurs after the pump but before the injection valve (high pressure mixing). In both cases, efficient online vacuum degassing of the mobile phase mixture and its components helps avoid chromatographic problems. In 1975, Tokunaga published a data set that laid the foundation for the degassing of HPLC solvent mixtures [1]. He determined the Ostwald coefficient of the solubility of oxygen in various alcohol-water mixtures and proved the degree to which the mixture needs to be degassed to prevent the formation of bubbles. This seminal paper laid the foundation for the development of a pipe-based degassing system routinely used in most laboratories today. Figure 1 plots Tokunaga's data, recalculated in a way that the HPLC system mixes the mobile phase as a volume percentage. The difference between the solid red line above and the Ostwald coefficient data line represents the supersaturation of the dissolved air mixture. Three example lines for reducing the amount of air by degassing are also shown. According to this data, the actual air concentration in the mixture that will not degas under atmospheric conditions is 38%, which is the goal that most degasser designs must meet (under specific instrument specific flow rate and applied vacuum) design requirements ).
The currently commonly used online degasser uses tubular Teflon™ AF or polytetrafluoroethylene (PTFE) membranes. According to Henry's Law, Dalton's Law, and Raoult's Law, they allow air to pass through the membrane and leave the mobile phase. Operating at a constant vacuum level, they remove air more effectively from the mobile phase at low flow rates, but are less effective at high flow rates. This is due to the residence time in the tube. Solvent molecules may also move from the mobile phase to the vacuum side of the membrane. This effect is called pervaporation, and in some cases and in certain HPLC methods, the concentration of the mobile phase can be significantly changed. This is because when the vacuum is fixed at a pressure lower than the solvent vapor pressure, the pump will continue to remove dissolved air and solvent vapor. As long as the pump is active, the solvent supply bottle will replenish the system and solvent vapor will be pumped into the atmosphere. Therefore, it is desirable to use the vacuum side of the degasser to control the pervaporation and set the pressure as high as possible without reaching the level of degassing in the HPLC system. This will affect the efficiency of the pump and the inlet check valve, and may cause inaccurate mobile phase composition and pump system flow rate, and may cause the method to fail due to quantification and identification issues.
The ideal design features of any new degassing method should include: • Lower flow restriction than a pipe-based degasser • Small size and no internal pipe fittings will leak • Minimum vacuum volume to limit the initial pervaporation of volatiles • Constant flow Restrictions, no matter how vacuum is applied • Degas the mobile phase at the highest possible pressure without allowing the mobile phase to be supersaturated with air. Known here as "high pressure degassing", this technology can reduce or eliminate solvent vapor emissions into the laboratory atmosphere. • The degasser is integrated into the HPLC system control software, which can realize intelligent control of vacuum to ensure improved degassing efficiency.
In addition, a universally applicable degasser—flow rates up to 10 mL/min, depending on the type of HPLC system—and all common solvents, including hexafluoroisopropanol, will be a significant advantage. There is now a patented flat membrane membrane and a dedicated vacuum pump control algorithm (patent pending) that can solve these goals. Figure 2 shows a schematic diagram of the new flat membrane degasser. This is a simple design that can be directly applied to low-pressure and high-pressure mixed HPLC systems (Figure 3). This design allows the product to have minimal accessories and connections. Its high-efficiency membrane has sufficient surface area to degas solvents for analytical-scale HPLC systems (flow rates up to 10 mL/min). The unique flow channel layout provides low fluid resistance before the inlet check valve of the pump. The matching vacuum control algorithm provides integration with the separation method control protocol and allows the selection of a given degassing efficiency for any HPLC system. The vacuum pressure can be adjusted up or down to achieve the required accurate HPLC method specifications. The simplified interface accepts the degassing flow rate and required efficiency of each separation method from HPLC, and adjusts the vacuum to the highest possible pressure for effective degassing. This method can prevent the dissolved atmosphere from being oversaturated, while suppressing pervaporation and changes in mobile phase concentration. Regardless of the degree of vacuum applied, the flow restriction is constant.
An initial evaluation of the new degasser/control algorithm produced some encouraging data and positive reports on availability. To characterize the degasser, the mathematical model of performance and applied vacuum level is derived from the running HPLC separation and stored in the degasser controller or HPLC control system. In the first step, a 210 nanometer (nm) or 215 nm methanol-oxygen charge transfer complex was used to determine the efficiency of the chamber at different flow rates and applied vacuum. Figure 4 shows the efficiency and flow rate at four different vacuum pressures. Note that the maximum flow rate for 30% residual air (70% efficiency) at 50 mmHg is approximately 2.5 mL/min. This is sufficient to degas gradients or any isocratic mobile phase at speeds up to 5 mL/min, and is equipped with this degassing because it requires 62% efficiency (38% residual air, Figure 1) to prevent degassing The machine's HPLC system can be operated at 50 mmHg, and method flow rates up to 7 mL/min can be expected without the formation of bubbles. Figure 4. The characteristic curve shows the efficiency and flow rate of the film degasser at four different vacuum levels.
The subsequent steps plot the relationship between the efficiency of each flow rate and the vacuum level, and use the required efficiency and flow rate to solve the efficiency-vacuum curve equation. The formula for each curve relates the flow rate to the output vacuum level so that once the degas chamber is characterized, the vacuum level applied to the degasser is a function of the required efficiency and process flow rate. The vacuum control can then be used to adjust the degassing performance to cover the entire performance range of the HPLC system. Therefore, the target degassing efficiency can always be ensured at any flow rate, while the concentration change or pervaporation in the mobile phase is minimal. Figure 5 shows data from experiments to compare the degassing efficiency of flat membrane and tubular systems. It should be noted that the vacuum level is significantly different, but at the required flow rate (1 mL/min) and efficiency (70%), the performance of the new membrane degasser matches that of the tubular degasser. This shows that any degassing machine can be characterized, and then the resulting data set can be used to control the vacuum degassing system based on the input of efficiency and method flow rate.
In summary, compared with degassing under constant vacuum, the development of the flat membrane degasser and its supporting control algorithm described here provides chromatographers with improved degassing performance. These benefits will not only improve degassing efficiency, but most importantly, method reproducibility, laboratory proficiency and productivity. For more information, please visit idex-health.com
1. Tokanuga J (1975) The solubility of oxygen, nitrogen and carbon dioxide in aqueous alcohol solution, J Chem Eng Data, 20(1): 41-46
Carl Sims is the chief scientist at IDEX Health & Science in Rohnert Park, California, focusing on HPLC systems in the fields of membrane degassing, fluid optics, and UPLC valves. With 47 years of chemical experience in the instrument field, he has obtained 52 US patents and another 150 foreign patents, focusing on HPLC, ion chromatography, Teflon AF optics, and DNA synthesis early in his career. Carl is a Navy veteran with a bachelor's degree in chemistry from Lewisburg College in Durango, Colorado and a master's degree in chemistry from Northern Arizona University in Flagstaff, Arizona.
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