Journal of Chromatography A, 1320 (2013) 130–137
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Accurate on-line mass flow measurements in supercritical fluid chromatography Abhijit Tarafder, Péter Vajda, Georges Guiochon ∗ University of Tennessee, Knoxville, TN 37996, USA
a r t i c l e
i n f o
Article history: Received 12 August 2013 Received in revised form 9 October 2013 Accepted 10 October 2013 Available online 22 October 2013
a b s t r a c t This work demonstrates the possible advantages and the challenges of accurate on-line measurements of the CO2 mass flow rate during supercritical fluid chromatography (SFC) operations. Only the mass flow rate is constant along the column in SFC. The volume flow rate is not. The critical importance of accurate measurements of mass flow rates for the achievement of reproducible data and the serious difficulties encountered in supercritical fluid chromatography for its assessment were discussed earlier based on the physical properties of carbon dioxide. In this report, we experimentally demonstrate the problems encountered when performing mass flow rate measurements and the gain that can possibly be achieved by acquiring reproducible data using a Coriolis flow meter. The results obtained show how the use of a highly accurate mass flow meter permits, besides the determination of accurate values of the mass flow rate, a systematic, constant diagnosis of the correct operation of the instrument and the monitoring of the condition of the carbon dioxide pump. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In a chromatographic process, the parameters that control the characteristics of a separation, the thermodynamic interactions between analyte molecules, mobile and stationary phases and the transport properties of analyte molecules through the mobile phase, must be carefully controlled and accurately measured. These characteristics depend on the temperature and the density profiles along the column, on the mobile phase composition, and on the interstitial velocity of the mobile phase [1]. So the reliability and repeatability of experimental results provided by supercritical fluid chromatography (SFC) depend on the reproducibility of the temperature, the density, and the composition profiles along the column and on the profile of the interstitial velocity of the mobile phase. Similarly, during the scale-up of chromatographic separations from analytical to preparative scale, the experimental conditions under which separations are performed should be the same in the preparative column as they were in the analytical column with which the method was developed. To ensure the degree of reproducibility that is necessary, it is critical that these parameters be accurately measured. We showed recently [2] that, although modern commercial instruments available for SFC provide reliable sensors allowing accurate and precise measurements of the column temperature and
∗ Corresponding author. Tel.: +1 8659740733; fax: +1 8659742667. E-mail addresses: [email protected], [email protected] (G. Guiochon). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.10.041
of the pressure at the inlet and/or the outlet of the column, they provide no direct, accurate measurement of either the mass or the volumetric flow rates of CO2 . We explained why the knowledge of both these flow rates is critical to characterize an SFC system. Accurate measurements of the mass flow rate is necessary to determine the exact mobile phase composition, that of the volumetric flow rate is necessary to determine the profile of the interstitial velocities along the column. In spite of the critical role of these two flow rates in SFC, commercially available instruments rarely provide any accurate method to measure either. They only allow the users to set the volumetric flow rate of the mobile phase at the pump outlet but do not provide real time measurements of either flow rate anywhere else in the instrument. This is not a satisfactory arrangement because it does not provide means to ensure that the flow rate along the column is the one desired. Most SFC instruments have two pumps, one for CO2 and one for the modifier. The performance of the modifier pump, which is practically a classical HPLC pump, can be conveniently tested. It is generally found to be stable over the long periods of time during which SFC instruments are operated and to accurately deliver the required flow rate. The performance of the CO2 pumps, on the other hand, should be monitored more regularly as there is a higher probability that the outlet valves of the pump leak [2,3]. The CO2 pump may fail to deliver the programmed flow rates, both in terms of mass and of volumetric unit, due to the inherent problems of pumping a lowviscosity, highly compressible fluid like CO2 through reciprocating pumps [2]. The purpose of this work was to investigate the accuracy of the flow rates of CO2 delivered by commercial SFC instruments in
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our laboratory, coming from different manufacturers and all procured within the last two years. The general operability of these instruments was satisfactory and none showed indications of any uncontrollable malfunctioning. To perform this study we needed to use a high accuracy flow meter. We chose one working on the principle of the Coriolis effect. The use of this flow meter permitted an investigation of the overall performance of the CO2 pump, which cannot be understood otherwise than from the results provided by this sensor. The results obtained highlight the widely different values of the CO2 flow rates pumped by different commercial instruments under the same operating conditions.
2. The measurement of the mobile phase flow rates in SFC This section discusses briefly different sources of variabilities of the mass and the volumetric flow rates of the mobile phase in an SFC instrument and the possible methods of measurement of flow rates. These issues were recently discussed in detail [2], so only a summary of the main points is presented here for convenience. Because all fluids are compressible and expand when their temperature increases or their pressure decreases, their volumetric flow rate vary along any channel, particularly along columns packed with fine particles, which have a low permeability. The situation is strongly relevant in SFC where the operating temperature may vary from sub-ambient [4] to well above 300 ◦ C and the pressure may vary from values below the critical pressure of CO2 (which is often the case when a modifier is used as in simplified fluid chromatography [5–9]) to above 350 bar. As the compressibility of CO2 varies considerably within these operating ranges, even when organic modifiers, e.g. methanol, are added to the mobile phase, the volumetric flow rate of the mobile phase may vary significantly in SFC between different parts of the instrument and along the column, even when the effective mass flow rate stays constant along the instrument. Although not as much as the volumetric flow rate, the mass flow rate of CO2 may also vary depending on the mode of the operation [2]. With a perfectly operating CO2 pump, which delivers the exact volume of CO2 as specified by the user, the net mass flow rate of the CO2 may vary as a function of the pressure at which the fluid is pumped because CO2 is significantly compressible even at the low temperatures at which it is pumped. During an isocratic mode of operation the mass flow rate of CO2 will not vary if the back pressure of the instrument is kept constant, because, the outlet pressure of the CO2 pump, which results from the combination of the instrument back pressure and the pressure drop along the column, stays constant. During a gradient operation, on the other hand, the modifier composition of the mobile phase is modulated as a function of time and the mass flow rate of the mobile phase varies during the analysis because the modifier composition varies, causing a variation of the pressure drop along the column, and leading to variations of the column outlet pressure. Accurate measurements of variations of the mass flow rates of the different components of the mobile phase and of the total volumetric flow rate of the mobile phase should be performed in any accurate analysis of the performance of an SFC operation [2]. From the mass flow rates of the mobile phase components we can accurately calculate the mobile phase mass (or molar) composition, a critical parameter to calculate mobile phase properties such as its density and viscosity. However, to know the distribution of the interstitial velocities along the instrument channel, we need to measure the volumetric flow rate of the mobile phase under all pressure–temperature conditions inside the column. This can be done either by directly measuring the volumetric flow rates inside the column, which is practically impossible, or by converting the mass flow rate into the local volumetric flow rates based on the
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density profile of the mobile phase along the column. This requires accurate measurements of the temperature and the pressure profiles along the column and an accurate equation of state (EOS) for the CO2 + modifier mixture used. Measuring the mass flow rate of organic solvents, as mentioned earlier, is relatively easy. Accurate data on their density at standard ambient temperature and pressure (SATP) is available and vary little with moderate fluctuations of these parameters. This allows the simple calculation of the modifier mass flow rate using the volumetric flow rate displayed by the modifier pump. Replicating the same procedure for CO2 is not useful because (1) the reliability of CO2 pumps to deliver a set volumetric flow rate is much less than that of organic modifier pumps [2]; (2) the CO2 density at the temperature(s) at which it is pumped, can vary considerably with the pump outlet pressure. Therefore, to know the CO2 mass flow rate accurately, we would need an accurate EOS and accurate measurements of the temperature, pressure and volumetric flow rate of CO2 at the pump outlet. Although we have a reasonably accurate EOS for CO2 (the Span and Wagner EOS), we need proper instruments to accurately measure all the temperature, pressure, and volumetric flow rate of CO2 at the pump outlet. This is not a trivial task. In summary, the accuracy of the measurements of the CO2 mass flow rate at the pump outlet and of the mobile phase volumetric flow rates along the column depends on the accuracy of the pressure, temperature, and flow rate measurements at multiple locations and on that of the EOSs of pure CO2 and of CO2 + modifier mixtures. Although the choice of suitable measuring devices for temperatures and pressures is straight forward, the choice of the flow meters is less so. There are various ways to directly measure flow rates in SFC. The volumetric flow rate of the mobile phase has been measured downstream the back pressure regulator, after its expansion to near atmospheric pressure, with a soap bubble flow meter adapted from gas chromatography [10] or with a rotameter. However, a rotameter provides only the volumetric flow rate and do so with quite a modest precision. A thermal mass flow meter was also used to measure the mass flow rate from the amount of heat carried away by the stream from a heated resistor [11,12], a gauge that was used in earlier studies of SFC. The derivation of the actual mass flow rate from the response of a thermal mass flow meter requires knowledge of the thermal properties of the fluid used; this value depends on the local volume flow rate and pressure. Thus, the accuracy of the thermal mass flow measurements depends on the accurate knowledge of the thermal properties of the fluid being handled. The thermal properties of gas mixtures are rarely known with a sufficient accuracy for the purpose, Although it may provide the mass flow rate, the thermal flow meter suffers from the same drawbacks as do volumetric techniques. Proper calibration is required. The response factor must be adjusted every time when the composition of the mixture used as the mobile phase is changed. The direct on-line measurement of the mass flow rate of the mobile phase at a location selected in the equipment, e.g., at the pump outlet, the column inlet or immediately downstream the detector, upstream the ABPR, would be more attractive and practical. In recent years, Coriolis flow meters (CFMs) have been designed and are now available. The CFM directly measures the true mass flow of a gas or liquid stream. The usefulness and robustness of these sensors have recently increased significantly, specially for applications needing highly accurate measurements [13,14]. Their primary advantage is that their signal provides directly the true value of the mass flow rate while other methods require the use of indirect techniques and perform ancillary measurements that let errors creep in [13]. The response of the CFM is a direct function of the mass flow rate. In this work, we used a CFM to measure the CO2 mass flow rate in four SFC instruments and present the results obtained. By using the CFM we could eliminate most inaccuracies in
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the measurement of the CO2 mass flow rate, hence of the total mass flow rate and the composition of the mobile phase. Regarding the accuracies of the volumetric flow rates inside the column, however, our measurements still depend on the accuracy of the pressure and the temperature gauges and, most importantly, on the accuracy of the mixed mobile phase EOS. 3. Experimental The main purpose of this study was to investigate the performance of the CO2 pumps of our SFC systems with a high accuracy CFM mass flow meter. More specifically we needed to ascertain the reliability and stability of their pumps and the accuracy and precision of the flow rates posted by the softwares of these different instruments. 3.1. SFC instruments The mass flow rates of the CO2 streams delivered by four SFC instruments were measured under different sets of experimental conditions, to determine what can be the range of variations of the mass flow rates obtained when different volume flow rates are set on the instrument software. These instruments are two instruments made by Thar (Pittsburgh, PA, USA) and two more recent instruments made by Waters and JASCO, respectively. The first instrument (MS1) was a TharSFC given by Waters (Milford, MA, USA) soon after the purchase of the Thar company by the Waters company. The second Thar instrument (MS2) was loaned to us by the department of Biosystems Engineering and Soil Science (BESS) of our University. The MS1 is an upgraded version of the older MS2 with a few design changes. Both systems include a fluid delivery module, containing three parallel reciprocating pumps for CO2 delivery and two parallel reciprocating pumps for modifier delivery. MS1 has an autosampler whereas MS2 does not. The oven of MS2 can accommodate several columns, the smaller one of MS1 can house only one column. MS1 has a photo diode array detector (Waters 2998) whereas MS2 has a UV/vis detector from Gilson. Both systems are equipped with automated back pressure regulators (ABPRs). The third instrument is the latest Acquity UPC2 . It was also given by Waters (Milford, MA, USA). It has a modular structure, slightly different from that of the TharSFC. A “convergence manager” module controls the inlet of the CO2 stream to the instrument and houses the ABPR. The inlet CO2 flows from the CO2 pump cylinders via the convergence manager to the “binary solvent manager”, which is the pump module of the instrument. Downstream the binary solvent manager comes the sample manager, followed by the column manager and the PDA detector. Downstream the detector, the solvent goes back to the convergence manager before exiting the instrument. The fourth instrument used in these measurements is from Jasco (Easton, MD, USA). It was equipped with a PU-2080-CO2 carbondioxide pump, a PU-1580 isocratic modifier pump, an AS-2059-SF autosampler with a 5 L sample loop, a CO-2060 column oven, an MD-2010 multi-wavelength detector and a 150-81 backpressureregulator. For additional analysis, the outlet temperatures and pressures of all the instruments were noted in run-time. For measuring the pump outlet temperatures of the instrument MS1 and MS2 high accuracy resistance temperature detectors (RTD), SA1-RTD (Class A Accuracy, ±0.15 ◦ C) bought from Omega Engineering (Stamford, CT, USA) were used. For the UPC2 and the Jasco system, the pump outlet temperature shown by the instrument was noted as is. In all cases the pump outlet pressure shown by the instrument software was noted directly.
3.2. Mass flow meter In all the experiments, the mass flow rate of the CO2 stream was measured with a mini CORI-FLOW instrument from Bronkhorst High-Tech B.V. (Ruurlo, NL, USA), with Model No. M13-ABD-11-0S, Serial No. B11200776A. This model is promised an accuracy of ±(0.2% of the read value + 0.5 g/h), which translates into a sensitivity of 0.01 g/min of CO2 . The pressure drop along the tubes is approximately 1 bar at a flow rate of 16 g/min of water but these figures are different for carbon dioxide, which has a much lower viscosity and a density that may vary widely, from ca. 0.1 to 1.1 g/mL. 3.3. Positioning the mass flow meter In principle, the mass flow meter can be installed in several possible places in the SFC system: (1) at the inlet line of the CO2 pump; (2) at the outlet of the CO2 pump, upstream the mixer; (3) downstream the mixer which mixes the CO2 with the organic modifier; (4) downstream the column; and (5) downstream the back pressure regulator. However, there is a restriction due to the pressure limitation of the CFM. So, we chose to install it at the inlet line of the CO2 pump mainly due to the following reasons: (1) the maximum pressure under which the CFM may be used is 150 bar, so installing it anywhere between the pump outlets and the ABPR inlet would limit our choice of operating pressure; and (2) putting it on the CO2 line and assuming that the modifier pump delivers an accurate flow rate (which turned out to be mostly true) helped us measure not only the net mass flow rate but also to determine the mass or mole fractions of the components in the mobile phase. 3.4. Mobile phase In all the experiments made, industrial grade carbon dioxide was used. It was bought from Airgas (Knoxville, TN, USA). Methanol was used as the modifier. It came from Acros Organics (New Jersey, USA). Two PNAs, anthracene (99.9%) and pyrene (99%), both from Sigma–Aldrich (Milwaukee, WI, USA), were used to measure their retention factors. Benzene was used as the tracer to measure the column void volume. 4. Results and discussion Several series of experiments were conducted to use the CFM and record on-line the mass flow rate delivered by the pumps of the SFC instruments. The general outline of a TharSFC Method Station is shown schematically in Fig. 1. The MS2 instrument has no Autosampler and a UV/vis detector instead of the PDA detector of MS1. As shown in the figure, the CFM was located between the CO2 source and the CO2 pump. As advised by Bronkhorst, a filter (SS-2TF-15) was installed upstream the CFM to block particles that could accidentally enter into the CFM. The CFM was strongly bolted to the ground, close to the SFC instrument, in order to prevent any possible vibrations from interfering with its response. The CFM was placed in the UPC2 between the CO2 cylinder and the convergence manager. 4.1. Influence of the CFM on the mobile phase stream After installation of the CFM, its possible influence on the flow rate of the mobile phase stream and on its properties was assessed. Because the CFM was installed upstream the CO2 pump, it was likely that it cannot affect the fluid behavior beyond the pump outlet but it was essential to check the possible consequences of installing the CFM. Possible causes of interferences of the CFM with the SFC instrument could be: (1) the CFM might cause a significant pressure drop, making the CO2 from the cylinder reach the pump inlet
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Fig. 1. Schematic diagram of a TharSFC Method Station setup.
under a lower pressure; and (2) the mechanism of the CFM could add a significant amount of enthalpy to the fluid, increasing the stream temperature to a value depending on the mass flow rate. These effects of lowering the inlet pressure of the stream and of increasing the temperature at the pump inlet would affect the inlet density of the fluid entering the pump. However, the CO2 pumps are all supplied with a cooling unit, either a chiller or Peltier cooling elements, as shown in Fig. 2. Before entering the pump, the CO2 stream flows through a heat-exchanger where the heat that could have been added by the CFM may dissipate. Thus, the installation of the CFM at the pump inlet would result almost only into a pressure drop but probably not in a temperature drop. One way to understand the effects of installing a CFM could have been to model the phenomenon, but this would be too time-consuming and complicated. A simpler, more practical way is to compare the chromatographic performance of the instrument with and without the CFM on-line. Experiments were done to compare the retention factors of anthracene and pyrene. Our results show that there is no variation in their retention data whether the CFM is on-line the instrument or not. The installation of the CFM did not affect any chromatographic property of the columns used. The CFM can be regarded as neutral toward the chromatographic measurements in the same way as are detectors. 4.2. Measurement of mass flow at different set flow rates Fig. 3a shows the mass flow rate recorded at four different volumetric flow rates, 2, 3, 4 and 5 mL/min of neat CO2 by the TharSFC
Method Station (MS1). Each change in the flow rate results into a sharp step increase of the CO2 mass flow rate. The most striking feature of this figure is the important noise of the mass flow signal observed when the CFM unit was placed before the pump. The source of this noise should be located before the data provided by the CFM can be accepted. To identify the source of this noise and to investigate if it takes place through the whole chromatographic system, further experiments were conducted. At first measurements were carried out with the carbon dioxide pumps turned off. Fig. 3b shows the record of the CFM signal when the pump is shut off. A gradual decrease of the CO2 flow rate took place before the outlet valve of the pump closed completely. The later steep flow rate hikes were caused by sudden openings of the outlet valve. Therefore, Fig. 3b shows that the CFM signal is not noisy when the CO2 pump does not operate, which shows clearly that the pump is at the origin of the noise observed. To verify if the pump is able to deliver a smooth flow rate to the column, the CFM unit was placed downstream the pumping unit. The disadvantage of this option was discussed in previous sections. However, this was necessary to measure the mass flow rate before and after the pump to check (1) whether the noisy mass flow recorded before the pump is unchanged when the mass flow meter is set after the pumping unit; and (2) whether the mobile phase flow pumped into the column fluctuates. The mass flow measured on 1, 2, 3, and 4 cm3 /min set volumetric flow rates before and after the CO2 pump of the Jasco system is shown by Fig. 4. It clearly shows that measurements made downstream the pump result into a smoother mass flow and
Liquefier unit
To the chiller Bronkhorst mini Cori-flow Mass flow meter
Pumping unit
Pump From the chiller Swagelok SS-2TF-15 Filter
Airgas Industrial Grade CO2 Fig. 2. Cooling arrangement of the incoming CO2 before entering the CO2 pump.
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Fig. 3. The results generate by the CFM (a) with varying set flow rates of 2, 3, 4 and 5 mL/min, and (b) flow without the pump running. The measurements were made with a TharSFC Method Station (MS1).
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clearly shows that the pump generates noise with higher and higher frequencies with increasing flow rate, making noisy the mass flow rate signal noisy upstream the pump. The frequencies corresponding to the maximum noise are those of the piston of the carbon dioxide pump at the corresponding set of volumetric flow rate. Similar results were noted with the other systems used in this study (results not shown). This behavior was not surprising due to the very nature of the operation of reciprocating pumps like those used for pumping CO2 in all the instruments used in this study. Reciprocating pumps do stop the inlet flow when the fluid inside the pump cylinder begins to be compressed or is pushed out the cylinder, which leads to flow discontinuities (hence pulsations) at the pump inlet. In further work we measured the mass flow rate upstream the pump and averaged the mass flow rate data in all the analyses.
after pump before pump
mass flow rate [g/min]
4 3.5 3 2.5 2 1.5 1 0.5 0
200
400
600
800
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1200
time [min] Fig. 4. The results generate by the CFM with varying set flow rates of 1, 2, 3 and 4 mL/min placed it before, and after the carbon dioxide pump of the Jasco system.
provide the same average value as measurements made at the pump inlet. Fig. 5 shows the Fourier analysis of the data gathered placing the CFM upstream the Jasco pump. The result of the analysis
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4.3. Influence of the temperature and back pressure on the mass flow rate The influence of the operating temperature and pressure on the true mass flow rate of CO2 during an SFC run were measured on the MS1 instrument. The results of these measurements are shown in Fig. 6. The closed symbols on this figure represent mass flow rates measured for different temperatures and back pressures, with a
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1 cm /min 2 cm3/min 3 3 cm /min 3 4 cm /min
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amplitude
0.08
0.06
0.04
0.02
0 0
0.5
1
1.5 frequency [Hz]
2
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Fig. 5. Fourier analysis of the data recorded with the mass flow meter connected before the pump of the Jasco system. The increase of the volumetric flow rate of the pump generates noise with a higher frequency as well.
Fig. 6. Variation of the mass flow rate of CO2 as a function of set flow rates, back pressure and column temperature. The close symbols represent a set flow rate of 3 mL/min, the open symbols a set flow rate of 4 mL/min, and the open symbols with crosses a set flow rate of 5 mL/min.
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pressure. However, in a recent publication [2] we showed that the effect of the column temperature on the mass flow rate is always minimal. 4.4. Variation of CO2 flow rates in CO2 –methanol mixture
Fig. 7. Variation of the mass flow rate of CO2 as a function of set flow rates and back pressure at 340 K on the Thar instrument (above), and on the UPC2 system (below). The legends in the figures show the set volumetric flow rates.
set volumetric flow rate of 3 mL/min. Similarly the open symbols without and with crosses represent the mass flow rates measured at 4 and 5 mL/min respectively. Similar experiments were also carried out at different flow rates, back pressures and temperatures with the UPC2 . The results obtained with both instrument demonstrate that the back pressure moderately affects the net mass flow rate at all volumetric flow rates but that the temperature does not affect them. Fig. 7 compares the MS1 and UPC2 pump performance at different volumetric flow rates when changing the back pressures. It may be surprising, however, that the mass flow rate decreased with increasing back pressure for the MS1 while it increased for the UPC2 . Increasing the instrument back pressure leads to a higher average fluid density and the net mass flow rate should increase if the volume swept by the pump head remains constant, as it should [2]. This is valid for the results of the UPC2 and the reverse trend of the MS1 was not expected. This phenomenon can only happen if there are instrumental problems, most likely in this case leaking pump outlet valves. If the outlet valve leaks, the net mass flow of CO2 leaking back to the pump increases with increasing pressure, which in turn allows a decreasing mass of CO2 to enter the pump from the cylinder. It is absolutely impossible to detect such leaks unless the mass flow rate is monitored online with an accurate mass flow meter. The set column temperature had almost no effect on the flow rate, which not surprising. The mobile phase viscosity decreases with increasing column temperature, which should affect the flow rate delivered by the pump in a way similar a change of the back
After measuring the mass flow rates of neat CO2 , we measured CO2 mass flow rates when a mixed mobile phase was used. The mass flow rate of CO2 was measured at different back pressures and at different set volumetric flow rates when 5 and 20% MeOH were added to the mobile phase. The results obtained with the instrument MS2 are shown in Fig. 8. The left hand side of the figure corresponds to a mobile phase with 5% MeOH, the right hand side to one with 20% MeOH. The solid symbols and the solid lines represent the flow rates measured whereas the open symbols and the dashed lines represent the expected or theoretical flow rates. The most striking feature of all these results is the significant difference between the CO2 mass flow rates achieved and those that were expected. The expected values were calculated as the products of the set volumetric flow rate and the CO2 density provided by the REFPROP software (NIST) at the temperature and pressure of the pump outlet. For all the experimental results, the pump outlet temperature was measured with a high accuracy RTD. For the outlet pressures, the data shown by the instruments were recorded. For both instruments, the actual CO2 flow rates were markedly below the estimated flow rates, which means that when a modifier is added to the mobile phase during any isocratic or gradient operation, the actual modifier concentration may be significantly different from the one reported by the instrument software. Again the most important point here is the complete absence of any feedback provided by the instrument, hence the lack of any warning regarding the deviations of the experimental conditions under which the actual results were obtained from those initially programmed. Fig. 9 shows similar results obtained with the UPC2 SFC chromatograph. The left hand side of the figure shows results obtained at a 2 mL/min flow rate, the right hand side results obtained at a 4 mL/min flow rate. The design and construction of this instrument are sophisticated so it was carefully scrutinized. The measurements were performed under four sets of experimental conditions: (1) with neat CO2 as the mobile phase; (2) with a mobile phase containing 5% MeOH in CO2 ; and (3) with 20% MeOH in CO2 . The mass flow rates calculated from the values of the CO2 volumetric flow rates are also shown, with the assumption that in all cases the pump outlet density was equal to 1 g/mL. This represents a situation in which, in the absence of any precise values of CO2 density under the conditions used, the analysts would assume that the mass flow rate is numerically equal to the volumetric flow rate in order to calculated other properties, e.g. the mass or the molar composition of the mobile phase. All our results show that the actual CO2 flow rate measured by the CFM is significantly higher than the values estimated from the temperature and the pressure provided by the data station of the UPC2 . This data station shows the outlet CO2 temperature, stable at 286 K, and a pump outlet pressure that depends on the back pressure and on the set flow rate. The measured flow rates of CO2 were between 4 and 14% higher than the flow rates calculated on the basis of the pressure and the temperature at the pump outlet given by the instrument. This means that, even if the analyst uses the best CO2 EOS available to calculate the density of CO2 and meticulously notes or records the pump outlet pressure and the temperature supplied by the instrument, the data provided might be off by 4–14%. Obviously, assuming that the outlet density of the CO2 stream at the pump exit is equal to 1 g/mL is generally incorrect. There might be several reasons for these differences. We do not know the mixing volume of carbon dioxide and methanol.
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Fig. 8. Variation of the neat CO2 mass flow rates as a function of back pressure when using (a) 5% MeOH in the mobile phase and (b) 20% MeOH in the mobile phase for the system MS2. The closed symbols with solid lines represent the measured results while the open symbols with dashed lines represent the expected or theoretical results.
Fig. 9. Variation of the mass flow rates as a function of back pressure using neat CO2 in the mobile phase having flow rates (a) 2 mL/min and (b) 4 mL/min for the UPC2 system. The closed symbols with solid lines represent the mass flow rate measured with the CFM, the open symbols represent the estimated mass flow rate assuming 286 K as the CO2 temperature just outside the pump, and the open symbols with cross represent the mass flow rate assuming 1 g/mL as the CO2 density.
However, the important fact that is demonstrated by these results is that the dependence on secondary measurements to estimate the mass flow rate of the mobile phase in SFC may lead to significant errors. Unfortunately, most users of SFC instruments ignore the exact density of the mobile phase that they use, its actual mass and volumetric flow rate and, in most cases, seem to ignore the problem completely. 5. Additional diagnostic service of the system Another significant advantage of using the high accuracy CFM sensor is that it provides a continuous accurate diagnosis of the CO2 pump. For example, the difference between the mass flow rate data of the CFM and the value calculated from the pump piston frequency (i.e., from the volumetric flow rate), informs on possible problems in the pump functioning. A pulse generated by the pump at the opening of the inlet valve clearly indicates a mismatch between the set value of the compressibility of CO2 and the actual value. The effect of this mismatch was described in detail earlier [2]. The fact that when the back pressure is increased, the net CO2 mass flow rate decreases, indicates possible leaks at the outlet valves of the pump as was reported in the case of MS 1 instrument in Fig. 7. As indicated by Berger [3] every CO2 pump leaks to a degree but a different degree for each pump. This observation is explained by the viscosity of CO2 being about ten times lower than that of organic liquids. So the advantage of employing CO2 as a mobile phase in chromatography causes a disadvantage to pump it. Such
a small leakage is impossible to detect without further instrumentation, but it seriously can affect the experimental parameters and the separations carried out using supercritical mobile phases. 6. Conclusion Our work demonstrates the usefulness of placing an accurate mass flow meter at the inlet of the CO2 pump. To understand and control the operation of an SFC instrument, accurate measurements of the CO2 mass flow rate are necessary. This mass flow rate depends on several external factors, some of which are easily measurable and others are not. Besides external factors, the mass flow rate can be affected by several instrumental problems. The most important problem with the current instrumentations, it that it seems impossible to estimate the true mass flow rate or even to understand whether there is a problem with the instrument and what is the possible extent of this problem. It seems that the only way to solve this situation is to install a high accuracy mass flow meter, e.g. a Coriolis flow meter at a suitable point in the system. Instrument manufacturers should investigate in which part of the line it should be attached. Placing it at inlet of the instrument pump may not be the best decision but it is the only one that it easy to use when we have an instrument that does not include it. Acknowledgements This work was supported in part by grant CHE-1108681 of the National Science Foundation, by the financial and technical
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support by Waters Technologies Corporation, and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory. We thank Martin Gilar (Waters Technology Corporation) for fruitful discussions and for his support. References [1] A. Tarafder, G. Guiochon, J. Chromatogr. A 1218 (2011) 4569. [2] A. Tarafder, G. Guiochon, J. Chromatogr. A 1285 (2013) 148. [3] T.A. Berger, Packed Column SFC, RSC Chromatography Monographs, Letchworth, United Kingdom, 1995.
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[4] A. Tarafder, K. Kaczmarski, M. Ranger, D.P. Poe, G. Guiochon, J. Chromatogr. A 1238 (2012) 132. [5] P. Sandra, A. Kot, A. Medvedovici, F. David, J. Chromatogr. A 703 (1995) 467. [6] A. Medvedovici, P. Sandra, L. Toribio, F. David, J. Chromatogr. A 785 (1997) 159. [7] Y. Zhao, G. Woo, S. Thomas, D. Semin, P. Sandra, J. Chromatogr. A 1003 (2003) 157. [8] C. Brunelli, Y. Zhao, M.-H. Brown, P. Sandra, J. Chromatogr. A 1185 (2008) 263. [9] P. Sandra, B. Szucs, R. Sandwich, Hanna-Brown, HPLC-2009, 2009, Communication L-1. [10] A. Wilsch, R. Feist, G.M. Schneider, Fluid Phase Equilib. 10 (1983) 299. [11] D.R. Gere, R. Board, D. McManigill, Anal. Chem. 54 (1982) 736. [12] P.J. Schoenmakers, L.G.M. Uunk, Chromatographia 24 (1987) 51. [13] M. Anklin, W. Drahm, A. Rieder, Flow Meas. Instrum. 17 (2006) 317. [14] K. Kolahi, T. Schröder, H. Röck, IEEE Trans. Instrum. Meas. 55 (2006) 1258.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Accurate on-line mass flow measurements in supercritical fluid chromatography Abhijit Tarafder, Péter Vajda, Georges Guiochon ∗ University of Tennessee, Knoxville, TN 37996, USA
a r t i c l e
i n f o
Article history: Received 12 August 2013 Received in revised form 9 October 2013 Accepted 10 October 2013 Available online 22 October 2013
a b s t r a c t This work demonstrates the possible advantages and the challenges of accurate on-line measurements of the CO2 mass flow rate during supercritical fluid chromatography (SFC) operations. Only the mass flow rate is constant along the column in SFC. The volume flow rate is not. The critical importance of accurate measurements of mass flow rates for the achievement of reproducible data and the serious difficulties encountered in supercritical fluid chromatography for its assessment were discussed earlier based on the physical properties of carbon dioxide. In this report, we experimentally demonstrate the problems encountered when performing mass flow rate measurements and the gain that can possibly be achieved by acquiring reproducible data using a Coriolis flow meter. The results obtained show how the use of a highly accurate mass flow meter permits, besides the determination of accurate values of the mass flow rate, a systematic, constant diagnosis of the correct operation of the instrument and the monitoring of the condition of the carbon dioxide pump. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In a chromatographic process, the parameters that control the characteristics of a separation, the thermodynamic interactions between analyte molecules, mobile and stationary phases and the transport properties of analyte molecules through the mobile phase, must be carefully controlled and accurately measured. These characteristics depend on the temperature and the density profiles along the column, on the mobile phase composition, and on the interstitial velocity of the mobile phase [1]. So the reliability and repeatability of experimental results provided by supercritical fluid chromatography (SFC) depend on the reproducibility of the temperature, the density, and the composition profiles along the column and on the profile of the interstitial velocity of the mobile phase. Similarly, during the scale-up of chromatographic separations from analytical to preparative scale, the experimental conditions under which separations are performed should be the same in the preparative column as they were in the analytical column with which the method was developed. To ensure the degree of reproducibility that is necessary, it is critical that these parameters be accurately measured. We showed recently [2] that, although modern commercial instruments available for SFC provide reliable sensors allowing accurate and precise measurements of the column temperature and
∗ Corresponding author. Tel.: +1 8659740733; fax: +1 8659742667. E-mail addresses: [email protected], [email protected] (G. Guiochon). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.10.041
of the pressure at the inlet and/or the outlet of the column, they provide no direct, accurate measurement of either the mass or the volumetric flow rates of CO2 . We explained why the knowledge of both these flow rates is critical to characterize an SFC system. Accurate measurements of the mass flow rate is necessary to determine the exact mobile phase composition, that of the volumetric flow rate is necessary to determine the profile of the interstitial velocities along the column. In spite of the critical role of these two flow rates in SFC, commercially available instruments rarely provide any accurate method to measure either. They only allow the users to set the volumetric flow rate of the mobile phase at the pump outlet but do not provide real time measurements of either flow rate anywhere else in the instrument. This is not a satisfactory arrangement because it does not provide means to ensure that the flow rate along the column is the one desired. Most SFC instruments have two pumps, one for CO2 and one for the modifier. The performance of the modifier pump, which is practically a classical HPLC pump, can be conveniently tested. It is generally found to be stable over the long periods of time during which SFC instruments are operated and to accurately deliver the required flow rate. The performance of the CO2 pumps, on the other hand, should be monitored more regularly as there is a higher probability that the outlet valves of the pump leak [2,3]. The CO2 pump may fail to deliver the programmed flow rates, both in terms of mass and of volumetric unit, due to the inherent problems of pumping a lowviscosity, highly compressible fluid like CO2 through reciprocating pumps [2]. The purpose of this work was to investigate the accuracy of the flow rates of CO2 delivered by commercial SFC instruments in
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our laboratory, coming from different manufacturers and all procured within the last two years. The general operability of these instruments was satisfactory and none showed indications of any uncontrollable malfunctioning. To perform this study we needed to use a high accuracy flow meter. We chose one working on the principle of the Coriolis effect. The use of this flow meter permitted an investigation of the overall performance of the CO2 pump, which cannot be understood otherwise than from the results provided by this sensor. The results obtained highlight the widely different values of the CO2 flow rates pumped by different commercial instruments under the same operating conditions.
2. The measurement of the mobile phase flow rates in SFC This section discusses briefly different sources of variabilities of the mass and the volumetric flow rates of the mobile phase in an SFC instrument and the possible methods of measurement of flow rates. These issues were recently discussed in detail [2], so only a summary of the main points is presented here for convenience. Because all fluids are compressible and expand when their temperature increases or their pressure decreases, their volumetric flow rate vary along any channel, particularly along columns packed with fine particles, which have a low permeability. The situation is strongly relevant in SFC where the operating temperature may vary from sub-ambient [4] to well above 300 ◦ C and the pressure may vary from values below the critical pressure of CO2 (which is often the case when a modifier is used as in simplified fluid chromatography [5–9]) to above 350 bar. As the compressibility of CO2 varies considerably within these operating ranges, even when organic modifiers, e.g. methanol, are added to the mobile phase, the volumetric flow rate of the mobile phase may vary significantly in SFC between different parts of the instrument and along the column, even when the effective mass flow rate stays constant along the instrument. Although not as much as the volumetric flow rate, the mass flow rate of CO2 may also vary depending on the mode of the operation [2]. With a perfectly operating CO2 pump, which delivers the exact volume of CO2 as specified by the user, the net mass flow rate of the CO2 may vary as a function of the pressure at which the fluid is pumped because CO2 is significantly compressible even at the low temperatures at which it is pumped. During an isocratic mode of operation the mass flow rate of CO2 will not vary if the back pressure of the instrument is kept constant, because, the outlet pressure of the CO2 pump, which results from the combination of the instrument back pressure and the pressure drop along the column, stays constant. During a gradient operation, on the other hand, the modifier composition of the mobile phase is modulated as a function of time and the mass flow rate of the mobile phase varies during the analysis because the modifier composition varies, causing a variation of the pressure drop along the column, and leading to variations of the column outlet pressure. Accurate measurements of variations of the mass flow rates of the different components of the mobile phase and of the total volumetric flow rate of the mobile phase should be performed in any accurate analysis of the performance of an SFC operation [2]. From the mass flow rates of the mobile phase components we can accurately calculate the mobile phase mass (or molar) composition, a critical parameter to calculate mobile phase properties such as its density and viscosity. However, to know the distribution of the interstitial velocities along the instrument channel, we need to measure the volumetric flow rate of the mobile phase under all pressure–temperature conditions inside the column. This can be done either by directly measuring the volumetric flow rates inside the column, which is practically impossible, or by converting the mass flow rate into the local volumetric flow rates based on the
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density profile of the mobile phase along the column. This requires accurate measurements of the temperature and the pressure profiles along the column and an accurate equation of state (EOS) for the CO2 + modifier mixture used. Measuring the mass flow rate of organic solvents, as mentioned earlier, is relatively easy. Accurate data on their density at standard ambient temperature and pressure (SATP) is available and vary little with moderate fluctuations of these parameters. This allows the simple calculation of the modifier mass flow rate using the volumetric flow rate displayed by the modifier pump. Replicating the same procedure for CO2 is not useful because (1) the reliability of CO2 pumps to deliver a set volumetric flow rate is much less than that of organic modifier pumps [2]; (2) the CO2 density at the temperature(s) at which it is pumped, can vary considerably with the pump outlet pressure. Therefore, to know the CO2 mass flow rate accurately, we would need an accurate EOS and accurate measurements of the temperature, pressure and volumetric flow rate of CO2 at the pump outlet. Although we have a reasonably accurate EOS for CO2 (the Span and Wagner EOS), we need proper instruments to accurately measure all the temperature, pressure, and volumetric flow rate of CO2 at the pump outlet. This is not a trivial task. In summary, the accuracy of the measurements of the CO2 mass flow rate at the pump outlet and of the mobile phase volumetric flow rates along the column depends on the accuracy of the pressure, temperature, and flow rate measurements at multiple locations and on that of the EOSs of pure CO2 and of CO2 + modifier mixtures. Although the choice of suitable measuring devices for temperatures and pressures is straight forward, the choice of the flow meters is less so. There are various ways to directly measure flow rates in SFC. The volumetric flow rate of the mobile phase has been measured downstream the back pressure regulator, after its expansion to near atmospheric pressure, with a soap bubble flow meter adapted from gas chromatography [10] or with a rotameter. However, a rotameter provides only the volumetric flow rate and do so with quite a modest precision. A thermal mass flow meter was also used to measure the mass flow rate from the amount of heat carried away by the stream from a heated resistor [11,12], a gauge that was used in earlier studies of SFC. The derivation of the actual mass flow rate from the response of a thermal mass flow meter requires knowledge of the thermal properties of the fluid used; this value depends on the local volume flow rate and pressure. Thus, the accuracy of the thermal mass flow measurements depends on the accurate knowledge of the thermal properties of the fluid being handled. The thermal properties of gas mixtures are rarely known with a sufficient accuracy for the purpose, Although it may provide the mass flow rate, the thermal flow meter suffers from the same drawbacks as do volumetric techniques. Proper calibration is required. The response factor must be adjusted every time when the composition of the mixture used as the mobile phase is changed. The direct on-line measurement of the mass flow rate of the mobile phase at a location selected in the equipment, e.g., at the pump outlet, the column inlet or immediately downstream the detector, upstream the ABPR, would be more attractive and practical. In recent years, Coriolis flow meters (CFMs) have been designed and are now available. The CFM directly measures the true mass flow of a gas or liquid stream. The usefulness and robustness of these sensors have recently increased significantly, specially for applications needing highly accurate measurements [13,14]. Their primary advantage is that their signal provides directly the true value of the mass flow rate while other methods require the use of indirect techniques and perform ancillary measurements that let errors creep in [13]. The response of the CFM is a direct function of the mass flow rate. In this work, we used a CFM to measure the CO2 mass flow rate in four SFC instruments and present the results obtained. By using the CFM we could eliminate most inaccuracies in
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the measurement of the CO2 mass flow rate, hence of the total mass flow rate and the composition of the mobile phase. Regarding the accuracies of the volumetric flow rates inside the column, however, our measurements still depend on the accuracy of the pressure and the temperature gauges and, most importantly, on the accuracy of the mixed mobile phase EOS. 3. Experimental The main purpose of this study was to investigate the performance of the CO2 pumps of our SFC systems with a high accuracy CFM mass flow meter. More specifically we needed to ascertain the reliability and stability of their pumps and the accuracy and precision of the flow rates posted by the softwares of these different instruments. 3.1. SFC instruments The mass flow rates of the CO2 streams delivered by four SFC instruments were measured under different sets of experimental conditions, to determine what can be the range of variations of the mass flow rates obtained when different volume flow rates are set on the instrument software. These instruments are two instruments made by Thar (Pittsburgh, PA, USA) and two more recent instruments made by Waters and JASCO, respectively. The first instrument (MS1) was a TharSFC given by Waters (Milford, MA, USA) soon after the purchase of the Thar company by the Waters company. The second Thar instrument (MS2) was loaned to us by the department of Biosystems Engineering and Soil Science (BESS) of our University. The MS1 is an upgraded version of the older MS2 with a few design changes. Both systems include a fluid delivery module, containing three parallel reciprocating pumps for CO2 delivery and two parallel reciprocating pumps for modifier delivery. MS1 has an autosampler whereas MS2 does not. The oven of MS2 can accommodate several columns, the smaller one of MS1 can house only one column. MS1 has a photo diode array detector (Waters 2998) whereas MS2 has a UV/vis detector from Gilson. Both systems are equipped with automated back pressure regulators (ABPRs). The third instrument is the latest Acquity UPC2 . It was also given by Waters (Milford, MA, USA). It has a modular structure, slightly different from that of the TharSFC. A “convergence manager” module controls the inlet of the CO2 stream to the instrument and houses the ABPR. The inlet CO2 flows from the CO2 pump cylinders via the convergence manager to the “binary solvent manager”, which is the pump module of the instrument. Downstream the binary solvent manager comes the sample manager, followed by the column manager and the PDA detector. Downstream the detector, the solvent goes back to the convergence manager before exiting the instrument. The fourth instrument used in these measurements is from Jasco (Easton, MD, USA). It was equipped with a PU-2080-CO2 carbondioxide pump, a PU-1580 isocratic modifier pump, an AS-2059-SF autosampler with a 5 L sample loop, a CO-2060 column oven, an MD-2010 multi-wavelength detector and a 150-81 backpressureregulator. For additional analysis, the outlet temperatures and pressures of all the instruments were noted in run-time. For measuring the pump outlet temperatures of the instrument MS1 and MS2 high accuracy resistance temperature detectors (RTD), SA1-RTD (Class A Accuracy, ±0.15 ◦ C) bought from Omega Engineering (Stamford, CT, USA) were used. For the UPC2 and the Jasco system, the pump outlet temperature shown by the instrument was noted as is. In all cases the pump outlet pressure shown by the instrument software was noted directly.
3.2. Mass flow meter In all the experiments, the mass flow rate of the CO2 stream was measured with a mini CORI-FLOW instrument from Bronkhorst High-Tech B.V. (Ruurlo, NL, USA), with Model No. M13-ABD-11-0S, Serial No. B11200776A. This model is promised an accuracy of ±(0.2% of the read value + 0.5 g/h), which translates into a sensitivity of 0.01 g/min of CO2 . The pressure drop along the tubes is approximately 1 bar at a flow rate of 16 g/min of water but these figures are different for carbon dioxide, which has a much lower viscosity and a density that may vary widely, from ca. 0.1 to 1.1 g/mL. 3.3. Positioning the mass flow meter In principle, the mass flow meter can be installed in several possible places in the SFC system: (1) at the inlet line of the CO2 pump; (2) at the outlet of the CO2 pump, upstream the mixer; (3) downstream the mixer which mixes the CO2 with the organic modifier; (4) downstream the column; and (5) downstream the back pressure regulator. However, there is a restriction due to the pressure limitation of the CFM. So, we chose to install it at the inlet line of the CO2 pump mainly due to the following reasons: (1) the maximum pressure under which the CFM may be used is 150 bar, so installing it anywhere between the pump outlets and the ABPR inlet would limit our choice of operating pressure; and (2) putting it on the CO2 line and assuming that the modifier pump delivers an accurate flow rate (which turned out to be mostly true) helped us measure not only the net mass flow rate but also to determine the mass or mole fractions of the components in the mobile phase. 3.4. Mobile phase In all the experiments made, industrial grade carbon dioxide was used. It was bought from Airgas (Knoxville, TN, USA). Methanol was used as the modifier. It came from Acros Organics (New Jersey, USA). Two PNAs, anthracene (99.9%) and pyrene (99%), both from Sigma–Aldrich (Milwaukee, WI, USA), were used to measure their retention factors. Benzene was used as the tracer to measure the column void volume. 4. Results and discussion Several series of experiments were conducted to use the CFM and record on-line the mass flow rate delivered by the pumps of the SFC instruments. The general outline of a TharSFC Method Station is shown schematically in Fig. 1. The MS2 instrument has no Autosampler and a UV/vis detector instead of the PDA detector of MS1. As shown in the figure, the CFM was located between the CO2 source and the CO2 pump. As advised by Bronkhorst, a filter (SS-2TF-15) was installed upstream the CFM to block particles that could accidentally enter into the CFM. The CFM was strongly bolted to the ground, close to the SFC instrument, in order to prevent any possible vibrations from interfering with its response. The CFM was placed in the UPC2 between the CO2 cylinder and the convergence manager. 4.1. Influence of the CFM on the mobile phase stream After installation of the CFM, its possible influence on the flow rate of the mobile phase stream and on its properties was assessed. Because the CFM was installed upstream the CO2 pump, it was likely that it cannot affect the fluid behavior beyond the pump outlet but it was essential to check the possible consequences of installing the CFM. Possible causes of interferences of the CFM with the SFC instrument could be: (1) the CFM might cause a significant pressure drop, making the CO2 from the cylinder reach the pump inlet
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Fig. 1. Schematic diagram of a TharSFC Method Station setup.
under a lower pressure; and (2) the mechanism of the CFM could add a significant amount of enthalpy to the fluid, increasing the stream temperature to a value depending on the mass flow rate. These effects of lowering the inlet pressure of the stream and of increasing the temperature at the pump inlet would affect the inlet density of the fluid entering the pump. However, the CO2 pumps are all supplied with a cooling unit, either a chiller or Peltier cooling elements, as shown in Fig. 2. Before entering the pump, the CO2 stream flows through a heat-exchanger where the heat that could have been added by the CFM may dissipate. Thus, the installation of the CFM at the pump inlet would result almost only into a pressure drop but probably not in a temperature drop. One way to understand the effects of installing a CFM could have been to model the phenomenon, but this would be too time-consuming and complicated. A simpler, more practical way is to compare the chromatographic performance of the instrument with and without the CFM on-line. Experiments were done to compare the retention factors of anthracene and pyrene. Our results show that there is no variation in their retention data whether the CFM is on-line the instrument or not. The installation of the CFM did not affect any chromatographic property of the columns used. The CFM can be regarded as neutral toward the chromatographic measurements in the same way as are detectors. 4.2. Measurement of mass flow at different set flow rates Fig. 3a shows the mass flow rate recorded at four different volumetric flow rates, 2, 3, 4 and 5 mL/min of neat CO2 by the TharSFC
Method Station (MS1). Each change in the flow rate results into a sharp step increase of the CO2 mass flow rate. The most striking feature of this figure is the important noise of the mass flow signal observed when the CFM unit was placed before the pump. The source of this noise should be located before the data provided by the CFM can be accepted. To identify the source of this noise and to investigate if it takes place through the whole chromatographic system, further experiments were conducted. At first measurements were carried out with the carbon dioxide pumps turned off. Fig. 3b shows the record of the CFM signal when the pump is shut off. A gradual decrease of the CO2 flow rate took place before the outlet valve of the pump closed completely. The later steep flow rate hikes were caused by sudden openings of the outlet valve. Therefore, Fig. 3b shows that the CFM signal is not noisy when the CO2 pump does not operate, which shows clearly that the pump is at the origin of the noise observed. To verify if the pump is able to deliver a smooth flow rate to the column, the CFM unit was placed downstream the pumping unit. The disadvantage of this option was discussed in previous sections. However, this was necessary to measure the mass flow rate before and after the pump to check (1) whether the noisy mass flow recorded before the pump is unchanged when the mass flow meter is set after the pumping unit; and (2) whether the mobile phase flow pumped into the column fluctuates. The mass flow measured on 1, 2, 3, and 4 cm3 /min set volumetric flow rates before and after the CO2 pump of the Jasco system is shown by Fig. 4. It clearly shows that measurements made downstream the pump result into a smoother mass flow and
Liquefier unit
To the chiller Bronkhorst mini Cori-flow Mass flow meter
Pumping unit
Pump From the chiller Swagelok SS-2TF-15 Filter
Airgas Industrial Grade CO2 Fig. 2. Cooling arrangement of the incoming CO2 before entering the CO2 pump.
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Fig. 3. The results generate by the CFM (a) with varying set flow rates of 2, 3, 4 and 5 mL/min, and (b) flow without the pump running. The measurements were made with a TharSFC Method Station (MS1).
4.5
clearly shows that the pump generates noise with higher and higher frequencies with increasing flow rate, making noisy the mass flow rate signal noisy upstream the pump. The frequencies corresponding to the maximum noise are those of the piston of the carbon dioxide pump at the corresponding set of volumetric flow rate. Similar results were noted with the other systems used in this study (results not shown). This behavior was not surprising due to the very nature of the operation of reciprocating pumps like those used for pumping CO2 in all the instruments used in this study. Reciprocating pumps do stop the inlet flow when the fluid inside the pump cylinder begins to be compressed or is pushed out the cylinder, which leads to flow discontinuities (hence pulsations) at the pump inlet. In further work we measured the mass flow rate upstream the pump and averaged the mass flow rate data in all the analyses.
after pump before pump
mass flow rate [g/min]
4 3.5 3 2.5 2 1.5 1 0.5 0
200
400
600
800
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time [min] Fig. 4. The results generate by the CFM with varying set flow rates of 1, 2, 3 and 4 mL/min placed it before, and after the carbon dioxide pump of the Jasco system.
provide the same average value as measurements made at the pump inlet. Fig. 5 shows the Fourier analysis of the data gathered placing the CFM upstream the Jasco pump. The result of the analysis
0.12
4.3. Influence of the temperature and back pressure on the mass flow rate The influence of the operating temperature and pressure on the true mass flow rate of CO2 during an SFC run were measured on the MS1 instrument. The results of these measurements are shown in Fig. 6. The closed symbols on this figure represent mass flow rates measured for different temperatures and back pressures, with a
3
1 cm /min 2 cm3/min 3 3 cm /min 3 4 cm /min
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amplitude
0.08
0.06
0.04
0.02
0 0
0.5
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1.5 frequency [Hz]
2
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Fig. 5. Fourier analysis of the data recorded with the mass flow meter connected before the pump of the Jasco system. The increase of the volumetric flow rate of the pump generates noise with a higher frequency as well.
Fig. 6. Variation of the mass flow rate of CO2 as a function of set flow rates, back pressure and column temperature. The close symbols represent a set flow rate of 3 mL/min, the open symbols a set flow rate of 4 mL/min, and the open symbols with crosses a set flow rate of 5 mL/min.
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pressure. However, in a recent publication [2] we showed that the effect of the column temperature on the mass flow rate is always minimal. 4.4. Variation of CO2 flow rates in CO2 –methanol mixture
Fig. 7. Variation of the mass flow rate of CO2 as a function of set flow rates and back pressure at 340 K on the Thar instrument (above), and on the UPC2 system (below). The legends in the figures show the set volumetric flow rates.
set volumetric flow rate of 3 mL/min. Similarly the open symbols without and with crosses represent the mass flow rates measured at 4 and 5 mL/min respectively. Similar experiments were also carried out at different flow rates, back pressures and temperatures with the UPC2 . The results obtained with both instrument demonstrate that the back pressure moderately affects the net mass flow rate at all volumetric flow rates but that the temperature does not affect them. Fig. 7 compares the MS1 and UPC2 pump performance at different volumetric flow rates when changing the back pressures. It may be surprising, however, that the mass flow rate decreased with increasing back pressure for the MS1 while it increased for the UPC2 . Increasing the instrument back pressure leads to a higher average fluid density and the net mass flow rate should increase if the volume swept by the pump head remains constant, as it should [2]. This is valid for the results of the UPC2 and the reverse trend of the MS1 was not expected. This phenomenon can only happen if there are instrumental problems, most likely in this case leaking pump outlet valves. If the outlet valve leaks, the net mass flow of CO2 leaking back to the pump increases with increasing pressure, which in turn allows a decreasing mass of CO2 to enter the pump from the cylinder. It is absolutely impossible to detect such leaks unless the mass flow rate is monitored online with an accurate mass flow meter. The set column temperature had almost no effect on the flow rate, which not surprising. The mobile phase viscosity decreases with increasing column temperature, which should affect the flow rate delivered by the pump in a way similar a change of the back
After measuring the mass flow rates of neat CO2 , we measured CO2 mass flow rates when a mixed mobile phase was used. The mass flow rate of CO2 was measured at different back pressures and at different set volumetric flow rates when 5 and 20% MeOH were added to the mobile phase. The results obtained with the instrument MS2 are shown in Fig. 8. The left hand side of the figure corresponds to a mobile phase with 5% MeOH, the right hand side to one with 20% MeOH. The solid symbols and the solid lines represent the flow rates measured whereas the open symbols and the dashed lines represent the expected or theoretical flow rates. The most striking feature of all these results is the significant difference between the CO2 mass flow rates achieved and those that were expected. The expected values were calculated as the products of the set volumetric flow rate and the CO2 density provided by the REFPROP software (NIST) at the temperature and pressure of the pump outlet. For all the experimental results, the pump outlet temperature was measured with a high accuracy RTD. For the outlet pressures, the data shown by the instruments were recorded. For both instruments, the actual CO2 flow rates were markedly below the estimated flow rates, which means that when a modifier is added to the mobile phase during any isocratic or gradient operation, the actual modifier concentration may be significantly different from the one reported by the instrument software. Again the most important point here is the complete absence of any feedback provided by the instrument, hence the lack of any warning regarding the deviations of the experimental conditions under which the actual results were obtained from those initially programmed. Fig. 9 shows similar results obtained with the UPC2 SFC chromatograph. The left hand side of the figure shows results obtained at a 2 mL/min flow rate, the right hand side results obtained at a 4 mL/min flow rate. The design and construction of this instrument are sophisticated so it was carefully scrutinized. The measurements were performed under four sets of experimental conditions: (1) with neat CO2 as the mobile phase; (2) with a mobile phase containing 5% MeOH in CO2 ; and (3) with 20% MeOH in CO2 . The mass flow rates calculated from the values of the CO2 volumetric flow rates are also shown, with the assumption that in all cases the pump outlet density was equal to 1 g/mL. This represents a situation in which, in the absence of any precise values of CO2 density under the conditions used, the analysts would assume that the mass flow rate is numerically equal to the volumetric flow rate in order to calculated other properties, e.g. the mass or the molar composition of the mobile phase. All our results show that the actual CO2 flow rate measured by the CFM is significantly higher than the values estimated from the temperature and the pressure provided by the data station of the UPC2 . This data station shows the outlet CO2 temperature, stable at 286 K, and a pump outlet pressure that depends on the back pressure and on the set flow rate. The measured flow rates of CO2 were between 4 and 14% higher than the flow rates calculated on the basis of the pressure and the temperature at the pump outlet given by the instrument. This means that, even if the analyst uses the best CO2 EOS available to calculate the density of CO2 and meticulously notes or records the pump outlet pressure and the temperature supplied by the instrument, the data provided might be off by 4–14%. Obviously, assuming that the outlet density of the CO2 stream at the pump exit is equal to 1 g/mL is generally incorrect. There might be several reasons for these differences. We do not know the mixing volume of carbon dioxide and methanol.
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Fig. 8. Variation of the neat CO2 mass flow rates as a function of back pressure when using (a) 5% MeOH in the mobile phase and (b) 20% MeOH in the mobile phase for the system MS2. The closed symbols with solid lines represent the measured results while the open symbols with dashed lines represent the expected or theoretical results.
Fig. 9. Variation of the mass flow rates as a function of back pressure using neat CO2 in the mobile phase having flow rates (a) 2 mL/min and (b) 4 mL/min for the UPC2 system. The closed symbols with solid lines represent the mass flow rate measured with the CFM, the open symbols represent the estimated mass flow rate assuming 286 K as the CO2 temperature just outside the pump, and the open symbols with cross represent the mass flow rate assuming 1 g/mL as the CO2 density.
However, the important fact that is demonstrated by these results is that the dependence on secondary measurements to estimate the mass flow rate of the mobile phase in SFC may lead to significant errors. Unfortunately, most users of SFC instruments ignore the exact density of the mobile phase that they use, its actual mass and volumetric flow rate and, in most cases, seem to ignore the problem completely. 5. Additional diagnostic service of the system Another significant advantage of using the high accuracy CFM sensor is that it provides a continuous accurate diagnosis of the CO2 pump. For example, the difference between the mass flow rate data of the CFM and the value calculated from the pump piston frequency (i.e., from the volumetric flow rate), informs on possible problems in the pump functioning. A pulse generated by the pump at the opening of the inlet valve clearly indicates a mismatch between the set value of the compressibility of CO2 and the actual value. The effect of this mismatch was described in detail earlier [2]. The fact that when the back pressure is increased, the net CO2 mass flow rate decreases, indicates possible leaks at the outlet valves of the pump as was reported in the case of MS 1 instrument in Fig. 7. As indicated by Berger [3] every CO2 pump leaks to a degree but a different degree for each pump. This observation is explained by the viscosity of CO2 being about ten times lower than that of organic liquids. So the advantage of employing CO2 as a mobile phase in chromatography causes a disadvantage to pump it. Such
a small leakage is impossible to detect without further instrumentation, but it seriously can affect the experimental parameters and the separations carried out using supercritical mobile phases. 6. Conclusion Our work demonstrates the usefulness of placing an accurate mass flow meter at the inlet of the CO2 pump. To understand and control the operation of an SFC instrument, accurate measurements of the CO2 mass flow rate are necessary. This mass flow rate depends on several external factors, some of which are easily measurable and others are not. Besides external factors, the mass flow rate can be affected by several instrumental problems. The most important problem with the current instrumentations, it that it seems impossible to estimate the true mass flow rate or even to understand whether there is a problem with the instrument and what is the possible extent of this problem. It seems that the only way to solve this situation is to install a high accuracy mass flow meter, e.g. a Coriolis flow meter at a suitable point in the system. Instrument manufacturers should investigate in which part of the line it should be attached. Placing it at inlet of the instrument pump may not be the best decision but it is the only one that it easy to use when we have an instrument that does not include it. Acknowledgements This work was supported in part by grant CHE-1108681 of the National Science Foundation, by the financial and technical
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support by Waters Technologies Corporation, and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory. We thank Martin Gilar (Waters Technology Corporation) for fruitful discussions and for his support. References [1] A. Tarafder, G. Guiochon, J. Chromatogr. A 1218 (2011) 4569. [2] A. Tarafder, G. Guiochon, J. Chromatogr. A 1285 (2013) 148. [3] T.A. Berger, Packed Column SFC, RSC Chromatography Monographs, Letchworth, United Kingdom, 1995.
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[4] A. Tarafder, K. Kaczmarski, M. Ranger, D.P. Poe, G. Guiochon, J. Chromatogr. A 1238 (2012) 132. [5] P. Sandra, A. Kot, A. Medvedovici, F. David, J. Chromatogr. A 703 (1995) 467. [6] A. Medvedovici, P. Sandra, L. Toribio, F. David, J. Chromatogr. A 785 (1997) 159. [7] Y. Zhao, G. Woo, S. Thomas, D. Semin, P. Sandra, J. Chromatogr. A 1003 (2003) 157. [8] C. Brunelli, Y. Zhao, M.-H. Brown, P. Sandra, J. Chromatogr. A 1185 (2008) 263. [9] P. Sandra, B. Szucs, R. Sandwich, Hanna-Brown, HPLC-2009, 2009, Communication L-1. [10] A. Wilsch, R. Feist, G.M. Schneider, Fluid Phase Equilib. 10 (1983) 299. [11] D.R. Gere, R. Board, D. McManigill, Anal. Chem. 54 (1982) 736. [12] P.J. Schoenmakers, L.G.M. Uunk, Chromatographia 24 (1987) 51. [13] M. Anklin, W. Drahm, A. Rieder, Flow Meas. Instrum. 17 (2006) 317. [14] K. Kolahi, T. Schröder, H. Röck, IEEE Trans. Instrum. Meas. 55 (2006) 1258.
