A Novel Inexpensive Pumping System for Continuous Operation at Low Noise Levels with High Sensitivity Detectors in Liquid Chromatography Barry L. Karger and Laverne V. Berry Department of Chemistry, Northeastern University, Boston, Mass. 02115 A new pumping system is presented for high speed liquid chromatography, a continuous gas displacement pump (CDP), operating on a new principle. The system pressure is maintained constant with a gas regulator, and a pulsating piston pump supplies the flow. This flow is split between the main flowstream and a controlled leak section which recycles the excess solvent to a reservoir. The CDP controls pressure to +0.5% which is equivalent to the performance of a gas displacement pump and a pulsating piston pump with pressurized gas ballast. High frequency noise (in a UV detector) for the CDP and the above other two pumping systems is equivalent to that of a trapped solvent in the sample cell. Short term noise is also comparable for the three systems but a factor of ten times higher than the trapped solvent case. Both high frequency and short term noise are significantly less than those obtained with undampened pulsating piston pumps. The influence of flow and pressure on high frequency and short term noise for both the CDP and a pulsating piston pump is also examined. Suggestions are presented for several of the origins of high frequency and short term noise in high speed liquid chromatography using an ultraviolet and refractive index detector. Finally, the use of a liquid ballast to minimize high frequency noise is presented.
gas displacement pumps makes them attractive for LC at low to moderate pressures. This paper describes a novel pumping system, a continuous gas displacement pump (CDP), that offers the advantages of the classical gas displacement pump : no pulsations, simple, inexpensive construction, and an equivalent detector noise level. Further, the CDP offers these advantages: compact size, continuous operation, and absence of bubble formation in the detector cell. This pumping system will be experimentally compared to the classical gas displacement pump, piston pump with pressurized gas ballast, and two commercial pumps (Milton Roy and Orlita) operating directly. Although reciprocating pumps such as the Milton Roy operating directly with no ballast devices are used only at very low flow rates in chromatography, the direct pumping mode serves as a basis of comparison of the efficiency of the other systems under study. Several criteria will be presented, one of which is the detector noise levels obtained with each pumping system. In the course of this comparison, we shall discuss some aspects of the general problem of noise in high speed liquid chromatography.
IN RECENT YEARS there has been increased interest in high speed, high efficiency, column liquid chromatography (LC) involving high inlet pressures to achieve the required flows. The pumps necessary for these high pressures may operate at either constant flow or constant pressure ( I ) . Constant flow is desired for qualitative analysis, as well as quantitative analysis when using concentration sensitive detectors ( 2 ) . In many types of chromatographic systems, the column permeability and viscosity of the eluent are maintained constant so that inexpensive and simple constant pressure devices can be successfully used. Of the constant flow devices, pulseless, motor driven displacement pumps are expensive, and reciprocating piston pumps introduce pulsations which, if not removed, can seriously affect the limits of detectability of a solute. One way in which the pulsations can be removed is by using a pressurized gas ballast; however, start up is complicated, ballast capacity changes with time, and dissolved gas may bubble out in the detector (detector outgasing). Of the constant pressure devices, gas displacement pumps are frequently used at pressures up to 5000 psi to produce constant flow if the permeability of the column and connecting lines is maintained constant (3, 4 ) . However, these simple pumps require a bulky reservoir, have limited operating time, and have detector outgasing problems. Despite these disadvantages, the low detector noise, simplicity, and low cost of
Figure 1 shows a schematic diagram of the entire liquid chromatographic apparatus which can be used either as the CDP, classical gas displacement pump, or piston pump with pressurized gas ballast. Summarized below are the specific details of the three modes. For clarity, the total apparatus using the CDP will be described first. Some parts, as indicated in the figure, will be common to all three modes. Continuous Gas Displacement Pump. The liquid chromatographic apparatus, incorporating the CDP system, consists of all the components illustrated in Figure 1 except those in Section A. The controlled leak section of the CDP, which is critical to the operation of this pump, is simply two segments of vertical stainless steel tubing (30 cm long, 6-mm 0.d.) connected with a tee (Type 400-3-316, Crawford Fitting Co., Solon, Ohio). A needle valve (Type 22RS4-316, Whitey Research Tool Co., Emeryville, Calif.) is connected to the horizontal outlet of this tee and serves as the adjustable controlled leak (the operation of this controlled leak is explained later). The top of this section is connected to a 3500-psi nitrogen tank (Type l H , Matheson Gas Products, East Rutherford, N.J.) through a spring loaded regulator (Model 4, Matheson Gas Products). An on-off valve (Type 2RS4, Whitey Research Tool Co.) is placed at the bottom of the controlled leak section. This valve is not necessary for the operation of the CDP, but is essential for the operation of the gas ballast system. The recycling piston pump (Model INMMl-B-29R, Milton Roy Co., St. Petersburg, Fla.) has a pulsation frequency of 29 strokes per minute, a maximum flow rate of 5 ml/min, and a rated maximum pressure of 1000 psi. (The pump has been operated over 2000 psi.) The pump ball valves are protected with a 7-p filter (Type SS-2FT7, Nupro Co., Cleveland, Ohio). The pressure relief valve (Type 78051, Sprague Engineering, Gardenia, Calif.) is of the spring-
EXPERIMENTAL
(1) R. A. Henry, “Modern Practice of Liquid Chromatography,’’ J. J. Kirkland, Ed., Wiley-Interscience, New York, N.Y., 1971. (2) I. Halasz, ANAL.CHEM., 36, 1428 (1964). (3) L. R. Snyder, J. Clirornatogr. Sci., 7, 595 (1969). (4) R. E. Jentoft and T. H. Gouw, ANAL.CHEM., 38, 949 (1966).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
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loaded type that vents pressure in excess of the set pressure. Safety valves that take the system to zero pressure when exceeded (e.g., rupture disks), should not be used with systems employing pressurized gas since the safety valves would empty the gas cylinder. The Bourdon pressure gauge (Type 47-18205, 0-5000 psi., American Instrument Co., Silver Spring, Md.) is installed upside down to eliminate trapped gas and unknown ballasting from the gauge. T o minimize further the effects of the Bourdon gauge on the noise levels of the system, the gauge was connected to the main flow stream by a long narrow tube (30 cm long, 0.76-mm i.d.). In order to distinguish detector noise effects arising from the pressure of the system and the flow of solvent through the detector cells, two adjustable needle valves (Type 22RS4-316, Whitey Research Tool Co.) are used. As indicated in Figure 1, one needle valve is connected to the ultraviolet (UV) detector and the other leads directly to the reservoir. This arrangement allows considerable flexibility in adjustment of the flow rate through the detectors. For example, identical flow rates through the detectors can be achieved for different system pressures by adjusting both needle valves. Conversely, for the same system pressure, different flow rates can be achieved through the detectors. The UV detector (Type 1205, Laboratory Data Control, Riviera Beach, Fla.) is connected directly to the refractive index (RI) detector (Type R-401, Waters Associates, Inc., Framingham, Mass.) by means of a short length of Teflon (Du Pont) tubing. Both detectors are operated a t minimum attenuation: 0.02 OD full scale for the UV detector and 6 X R I unit full scale for the R I detector. The outputs of the detectors are displayed on the same potentiometric recorder (Model PSOl W6A, 0-10 mV, Texas Instruments Inc., Houston, Texas) operated at a chart speed of 0.5 inch/ min. Except that of the controlled leak, all tubing in the system is stainless steel (3-mm 0.d.). The temperature of all parts is maintained at 20 ZIZ 0.1 "C by circulating water from a constant temperature bath (Model NBE, Haake, PolyScience Corp., Evanston, Ill.) through a horizontal aluminum trough made from sheet aluminum. The reservoir is a 1-liter three-neck round bottom flask immersed in the water bath and stirred with a magnetic stirrer. Start up of the CDP is exceedingly simple. The Milton Roy pump is set at maximum output for any pressure setting of the CDP. The gas regulator is then set to the desired pressure as indicated on the Bourdon gauge. Then the needle valve in the controlled leak section is adjusted so that a small stream of gas continually flows through the side arm. The desired pressure is achieved immediately and is maintained constant. Start up at either low pressure (-200 psi) or high pressure (-2000 psi) is equally easy. The upper pressure limits of the system depend on the availability of a suitable gas regulator. In addition, changing miscible solvents is simple and rapid by switching solvent reservoirs. The piston pump is already operated at maximum flow, and thus the system is rapidly flushed. For solvent change between immiscible solvents, an intermediate flush with a mutually miscible solvent is required. Piston Pump with Pressurized Gas Ballast. As noted previously, the same apparatus as described for the CDP can be operated to achieve a pressurized gas ballast of known and reproducible volume at any operating pressure. After closing the on-off valve connecting the controlled leak section to the system, the Milton Roy pump is adjusted t o the desired flow and the average pulsating pressure is noted on the Bourdon gauge. The on-off valve is then opened and solvent is allowed to exit through the CDP leak. Subsequently, the gas regulator is carefully opened t o bleed gas into the system until the pressure is just below the pump solvent pressure. When gas begins to exit from the CDP leak, the leak valve and gas regulator are shut off, trapping a known vohme of pressurized gas in the vertical arm. During 94
I I I I
I I I I
I I I
needle
IT
E detector
1
I ddetectore
I
t
e
u
p
solvent
1
I
1; I
I
. _ _ _ . . . I I I
8
I
1 - _ - _ - - - _ _ _ -L_ - _ Figure 1. Block diagram of the entire apparatus Section A represents the classical gas displacement pump. Section B represents either the continuous gas displacement pump (CDP) or the piston pump with pressurized gas ballast. Parts outside of Sections A and B are common to all three systems. Specific components and surces are listed in the text
start up, care must be exercised to prevent gas from entering the flow system. A half hour or longel is often required for the ballast to reach a stable pressure. Classical Gas Displacement Pump. The apparatus may be used as a gas displacement pump by replacing the controlled leak section (B in Figure 1) with a high pressure cylinder (75 ml, Type DOT-3B-400, Hoke Mfg. Co., Cresskill, N.J.) (A in Figure 1). Note that the Milton Roy pump is not used. The classical gas displacement pump is operated by first filling the cylinder with solvent, connecting the nitrogen tank to the top, and then adjusting the gas regulator t o give the desired system pressure as indicated on the Bourdon gauge. A needle valve is inserted in the line connecting the pressure regulator to the top of the high pressure cylinder. This valve is adjusted to allow a slow leak of nitrogen (ca. 20 ml/min) in order to improve the pressure constancy of the system. In some of this work, we tested a three-head membrane Orlita M3S 4/4 Pump (Orlita KG, Giessen, Germany) with a net pulsation frequency of 493 strokes per minute. This pump has a rated discharge pressure of 5000 psi and a maximum flow rate of 20 ml/min. Chemicals. Throughout this study, heptane was used as solvent, The heptane (highest purity, 96-97 "C, Eastman Kodak Co., Rochester, N.Y.) was treated and distilled as previously described (5). RESULTS AND DISCUSSION
The CDP, shown in Figure 1, produces pulseless, highly regulated pressure much like a gas displacement pump; however, a totally new principle allows truly continuous operation. Thus, a chromatographic column can be equilibrated overnight or run for extended time periods without changing ( 5 ) B. L. Karger and L. V. Berry, C[in. Chem., 17, 757 (1971).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
NZ
N2
-1L* on-off volve
Figure 2. Enlarged views of Figure 1, Section B, illustrating the action of the CDP as the piston of the Milton Roy pump is emptying (a) and filling (b) See text for details
operating controls. In this section, we will first explain the operation of the C D P and then discuss some of its characteristics by a comparison with other pumping systems. Of special interest in this comparison will be a study of the detector noise levels produced by each system. We shall also describe the influence of several experimental parameters affecting noise in high speed liquid chromatography. Operation of the Continuous Gas Displacement Pump. Figures 2a and 2b, which are enlarged views of section B of Figure 1, illustrate the operating principles of the C D P pumping system. The on-off valve is maintained in the on position in this mode of operation. During the first halfcycle of the Milton Roy pump, as the piston chamber is emptying (Figure 2a), the exiting solvent is split between the flow stream to the detector and the controlled leak section. The solvent level in the vertical arm of the controlled leak section rises slightly. Upon reaching the side arm, the solvent exits with gas from the controlled leak tee and passes back to the reservoir for recycling. Since the full-cycle pulsing time of the pump is 2 seconds, solvent is observed to exit in short pulses every second. On the other half-cycle, as the piston chamber of the pump is refilling (2b), the flow stream receives solvent from the vertical tube. Hence, only gas exits from the side arm during this half-cycle. The Milton Roy pump flow output, of course, must exceed the flow through the detectors at all operating pressures. The excess flow exits through the side arm in the controlled leak tee. At no time during the piston cycle is the gas in the controlled leak tee compressed (cf. gas ballast). In order that no compression occur, the leak valve must be opened sufficiently to allow rapid exit of the excess liquid. Gas compression is indicated on the Bourdon gauge if the valve is not open sufficiently. Proper setting of the valve is just the point where gas is observed to exit through the controlled leak tee. If the needle valve is opened too much, the C D P operates successfully but more gas than necessary is lost. As mentioned in the experimental section, start-up of the pumping system is exceedingly simple and rapid. The Milton Roy pump is set at maximum output and the gas regulator then determines the pressure of the system.
Changes in the pressure, and thus flow, can be rapidly made. Pressure programming can be easily achieved with this set-up, using a mechanical drive to change the gas regulator (6) or a pneumatic system (7). Formation of gas bubbles is not a problem with the C D P since the nitrogen-saturated solvent layer in the pressurized controlled leak section is continually exiting out of the controlled leak valve, before any dissolved nitrogen at high pressures has sufficient time to diffuse into the main flow stream. From the leak valve, the nirogen gas and excess solvent (containing dissolved nitrogen) travel directly to the solvent reservoir. By this procedure, nitrogen is swept over the solvent reservoir, producing two advantages. First, in any LC system, outgasing the detector occurs only if solvents in the detector are supersaturated with Nz. Supersaturation in part may result if the solvent reservoir is at a lower temperature than the detector or if gas dissolves in the solvent at a higher pressure than in the detector. However, with the CDP the detector and reservoir are saturated with nitrogen at the same temperature and pressure and no outgasing occurs. Second, explosive vapor in the reservoir or vapors from very volatile solvents are vented, and dissolved oxygen is displaced from the solvent to minimize oxidation (8). In the next section, we cite some of the characteristics of the CDP by comparing it with other pumping systems. Pressure Control. Two characteristics of importance in any pumping system are constancy of pressure (and flow) and the noise level produced in the detector. These two characteristics will be examined for the C D P and will be compared with other common pumping systems. First, let us discuss the constancy of pressure for the CDP. The Bourdon gauge, 0-5000 psi, can be read to 1 5 psi. At 1000 psi, the gauge reading remained constant over at least a full day of operation. Thus, pressure control for the C D P was approximately 10.5%:. In one experiment, we substituted the three-head Orlita pump and found, as expected, equivalent pressure control for the CDP. By the criterion of pressure control, this means the more expensive Orlita pump offers no advantage over the Milton Roy pump with the CDP system (however, a much higher flow rate can be achieved with the Orlita pump). Assuming that the permeability of the system does not change, the flow constancy would also be equivalent to that of pressure. Clearly, the constancy of pressure control will be a function of the constancy of the pressure regulator. When the spring loaded regulator, used in Figure 1, was replaced with a gas domeloaded regulator [Type 7-(CGA), Matheson Gas Products], the pressure slowly drifted at the rate of 25 psi/hr. This was traced back to leakage in the dome-loaded regulator. Thus, it is very important that a regulator be selected that keeps the pressure constant. We also examined the constancy of pressure for four other pumping systems: gas displacement pump (Figure 1, section B); Milton Roy pump in direct mode; Orlita pump in direct mode; Milton Roy pump with gas ballast (Figure 1, section A). For the gas displacement pumping system, pressure control was equivalent to that obtained with the CDP (10.5z) The . Milton Roy pump (direct pump mode) pulsation read at the Bourdon gauge was 1 2 0 psi at 1000 (6) L. S. Ettre, L. Mazor, and J. Takacs, “Advances in Chromatography,” Vol. 8, J. C . Giddings and R. A. Keller, Ed., Marcel Dekker, New York, N.Y., 1969. (7) I. Halasz and G. Deininger, Z . Anal. Cliem., 228, 321 (1967). (8) J. J. Kirkland, “Modern Practice of Liquid Chromatography,” J. J. Kirkland, Ed., Wiley-Interscience, New York, N.Y., 1971. ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
95
A HFN (0 D) Drift
STN (OD)
(0 D./hr)
ti
d
k-4
0
q 0
I min
-
V
01 rnin.
8 Figure 3A. Hypothetical base-line trace to illustrate the measurement of detector noise HFN (greater than 1 cycle/5 sec) is the average peak-topeak distance of this component of noise (in OD units); STN (from 1 cycle/5 sec to 1 cycle/5 min) is the envelope of the peaks with the HFN subtracted out (in OD units); drift (greater than 1 cycle/5 min) is the base-line shift (in OD/hr)
3B. Rapid chart speed showing the HFN in a typical experimental case P = 600 psi, F = 3.5 ml/in., T = 20 “C psi and i.50 psi at 400 psi. In addition, at 1000 psi, the pressure dropped at 800 psi randomly as often as once every 2 minutes and required about 30 seconds to regain the average 1000-psi pressure. These latter pressure fluctuations are likely to originate from mis-seating of the pump ball-valves and seriously affect pressure and flow constancy. No such gauge pressure fluctuations were observed with the Orlita pump which maintained a gauge pressure control comparable to the CDP (i0.5%). When the gas ballast was placed in the system along with the Milton Roy piston pump (Figure l), the pressure control was i.2z over 8-hour periods of time, with the disappearance of the 200-psi pressure fluctuation. In summary, the CDP was as good in terms of pressure control as any of the pumping systems tested. Detector Noise Levels. A second characteristic of pumping systems is the noise levels they produce in the detector signal. To study this, both the UV and RI detectors were used. However, the RI detector was significantly more sensitive to flow and temperature, in agreement with the conclusion of others (9, IO). Consequently, the UV detector provided a more critical test of the noise characteristics of the various pumping systems. Therefore, we will first discuss in detail the noise levels produced in the UV detector, and then mention briefly results with the RI detector. We shall divide detector noise into three components (11, 12) as illustrated in Figure 3A by a hypothetical chart re(9A) R. D. Conlon, ANAL.CHEM.,41(4), 107A (1969). (9B) H. Veening, J. Chem. Educ., 47, A549 (1970). (10) M. N. Munk, J. Chromatogr. Sci., 8, 491 (1970). (11) Chromatronix, Inc., Lab Notes, 4, Jan. 1971. (12) S. H. Byme, Jr., “Modern Practice of Liquid Chromatography,” J. J. Kirkland, Ed., Wiley-Interscience, New York, N.Y., 1971.
96
VI
Figure 4. Comparison o f recorder tracings (noise levels) for various pumping systems using the U V detector with a slow flow of N1 (5 ml/min) through the reference cell Nitrogen flow reference; P = lo00 psi; F = 3.5 ml/min; T = 20 “C; (I) control case with static solvent in the sample cell; (11) CDP; (111) classical gas displacement pump; (IV) Milton Roy pump with 15-ml pressurized gas ballast; (V) direct pump, Milton Roy; (VI) direct pump, Orlita
cording. The second-to-second high frequency noise (HFN) appears as a rapidly oscillating signal and can be quantified readily as the average peak-to-peak distance (in optical density units, OD). Figure 3B shows this H F N more clearly using a faster chart speed. (This HFN is taken directly from a typical experiment.) A lower frequency minute-to-minute short term noise (STN) appears as a random fluctuating signal superimposed on the H F N and can be quantified (in OD units) by the envelope of the peaks with the H F N subtracted out, as shown in Figure 3A. A still lower frequency noise, drift, is ascribed to a shift in the base line over a period of minutes to hours and is quantified by the change in base line per unit time (e.g., OD/hr.). Figure 4 illustrates the experimental H F N and STN obtained with various pumping systems at the same pressure (1000 psi) and flow (3.5 ml/min). The needle valve leading directly to the solvent reservoir was shut off for this work and all the flow passed through the UV and RI detectors. We shall later discuss the influence of pressure and flow on STN and H F N for several of the pumping systems. The reference cell of the UV detector was first operated in four different ways: slow nitrogen flow; static nitrogen; slow solvent flow; and static solvent. Table I summarizes the results in terms of drift, using the CDP (1000 psi, 3.5 mljmin) for flow through the sample cell. There is a substantial difference between the four modes with the slow bleed of N1 (ca. 5 mlimin) through the reference cell producing the least drift. It is believed that the Teflon (Du Pont) gaskets sealing the reference and sample cells are sufficiently permeable to allow solvent from the sample side to slowly leak into the Table I. Comparison of Drift with the U V Detector for Various Reference Cell Conditions Sample Cell Condition, CDP Pump with P = 1000 psi, F ml/min, T = 20 “C UV reference cell condition OD/hr Slow nitrogen flow O.OOO4 Static nitrogen 0,001 Slow solvent flow 0.02 Static solvent 0.12
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
=
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0.006
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0 Flow, I 2 m l l m i n
0004
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Table 11. Comparison of HFN and STN for Various Pumping Systems Using the UV Detector (see Figure 4) Conditions, P = 1000 psi, F = 3.5 ml/min, T = 20 "C, Drift = 0.0004 OD/hr, Reference Cell = Nitrogen Flow (5 ml/min) Pumping modes HFN, ODa STN, OD" I Trapped solvent control 0 0001 0 0001 I1 CDP 0 OOOl 0 0009 III Gas displacement pump 0 0001 0 0009 IV Pressurized gas ballast with Milton Roy pump 0 0001 0 0009 V Direct pump mode, Milton ROY 0 0006 0 0020 VI Direct pump mode, Orlita 0 0001 0 0009 IS
8bO
Id00
I,bO
la00
I6bO
'
Other conditions as in Figure 5
Flow through the detector and system pressure were controlled independently by means of two split valves (see text)
1 mm on the recorder chart
6;O
Figure 6. STN cs. system pressure at various flows for the piston pump (Milton Roy) and the CDP
Figure 5. HFN os. system pressure at various flows for the piston pump (Milton Roy) and the CDP
a
4W
PRESSURE (p s I 1
1400
equivalent to 0 0001 OD.
~~~
reference side. Apparently, the slow flow of N:! is able t o sweep this solvent out of the reference cell. The H F N and STN were identical for the four reference cell conditions. As we return to the experiments of Figure 4, Table I1 presents the quantitative results for H F N and STN for the various pumping modes that were tested. In this table the STN is least for the static solvent control case (I) in which solvent is trapped in the sample cell. For the CDP (11), the gas displacement pump (111), the Milton Roy pump with a 15-ml pressurized gas ballast (IV), and the Orlita pump operating directly (VI), the STN is the same, but almost an order of magnitude greater than the control case. O n the other hand, the H F N for the four pumping systems is equal to that of the control. It is further interesting t o note that the Milton Roy pump (with no ballasting devices) gives significantly greater HFN and STN. Since STN frequency is of the same order of the frequency of chromatographic peaks, these results indicate that the sensitivity of the U V detector is much less for the Milton Roy, ' i no ballasting devices are used. It is to be concluded that at this operating pressure and flow rate, the CDP gives equivalent noise levels to the gas displacement pump and the piston pump with pressurized gas ballast. The third component of noise drift, was the same for all pumping modes (0.0004 OD/hr) which was slightly better than the specifications of the detector. We next examined the influence of flow and pressure on the H F N and STN by the different pumping modes. For the constant pressure pumps (CDP and gas displacement pump), the experiment was performed by setting the pressure (read on the Bourdon gauge) and varying the two needle valves shown
in Figure. 1. The pressure was thus maintained constant while the flow through the detector line (measured volumetrically) changed. For the constant flow pumping systems (Milton Roy or Orlita direct pump and Milton Roy pump with pressurized gas ballast), changes in the settings of the needle valves resulted in changes in the pressure on the Bourdon gauge. Thus, by changing the two needle valves in opposite directions, the same flow rate through the detectors can be obtained for different system pressures. In both cases, the influence of the two variables, system pressure and detector flow, on H F N and STN could be ascertained, since the permeability of the line to the detector was changed. Figures 5 and 6 show the results obtained for the Milton Roy pump in the direct pumping mode and the CDP. For the CDP, Figure 5 shows that the H F N is independent of pressure and flow, the H F N over the whole range being equal to the H F N found for the static solvent control case. This residual level of H F N is equivalent to that specified by the manufacturer. Similar results were obtained for the gas displacement pump and the Milton Roy pump with pressurized gas ballast. With the Milton Roy pump alone, on the other hand, the HFN is dependent on pressure and flow. For a given flow rate through the detector, a higher pressure gives a lower HFN. Also at a given pressure, higher flow rates produce lower H F N . Indeed, at a flow of 4.7 ml/min and a pressure of 1400 psi, the H F N becomes almost equivalent to that for the CDP. Contrary t o the results with H F N , using the direct Milton Roy pump, the STN in Figure 6 increases with increased flow rate at a given pressure. In addition, for a constant flow rate, the STN increases with increasing pressure. The same trends were observed with the CDP. While not shown on the figure, a lower flow rate, a t a given pressure, produced a lower STN for the CDP. The results of this noise study provide us with some understanding of the possible sources of noise using the U V detector. First, the constancy of drift for all pumping modes, a t all pressures and flows, indicates that the drift arises from temperature or electronic effects in the detector (12, 13). Of course, when a chromatographic column is inserted in the system, the drift can be larger due to changes in the mobile phase (e.g., bleeding from the stationary phase, build-up of samples in recycled solvent, temperature changes from the column to the detector, etc.). The fact that such widely different pumping systems as the CDP, classical gas displacement pump, piston pump with (13) G. Brooker, ANAL. CHEM., 43, 1095 (1971).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
97
d
Table 111. Comparison of H F N and S T N for Various Pumping Systems Using a Waters Associates Model 401 RI Detector Conditions, P = 1000 psi; F = 3.5 rnl/min; T = 20 "C; Drift 4X RI Unit/Hr HFN, STN, Pumping modes RI unitsa RI unitsa Trapped solvent control 1 x 10-7 1 . 2 x 10-7
0
-
=
I min
Figure 7, Effect of liquid ballast on H F N using a pulsating piston pump (Milton Roy)
CDP 1 x 10-7 1 . 5 x 10-7 Direct pump mode, Milton Roy 1 x IO-' 8 x lo-' a 1 mm on the recorder chart is equivalent to 2.4 X R1 unit.
pressurized gas ballast, and the Orlita pump, all produce the same level of H F N for all pressures and flows indicates that this residual H F N is not due to the method of pumping. In addition, the fact that this H F N from all three pumping systems is the same as the H F N for the static solvent control case suggests that the origin of this residual H F N is electronic in nature. The increased H F N of the system when the Milton Roy pump is used directly, indicates that, in addition to this electronic component, a high frequency component can be attributed to the pumping system when good pressure regulation is not maintained. Figure 4 indicates that this additional H F N decreases with flow and pressure. We suggest that this added high frequency component occurs because the ball valves of the pump d o not snap shut as rapidly at low pressure differentials across the valves. Thus during the filling cycle of the piston stroke, more solvent may back-flow through the valves at low pressures and therefore produce greater pulsations, since the ball valves are effectively open longer. It is not clear whether the behavior in Figure 5 for the Milton Roy pump is general for all pulsating pumps. For the static solvent control case (I), the residual STN is equivalent to that of the H F N . The STN is the same for the CDP, classical gas displacement pump, and Milton Roy pump with pressurized gas ballast and this STN level is greater than the static solvent control. This latter effect suggests that a component of the STN is associated with complex flow patterns of solvent through the detector cell [causing Schlieren effects ( I d ) ] . The fact that the STN of the Milton Roy and Orlita pumps operating directly is larger than the other three pumping systems, indicates a component of the STN is also associated with undampened piston pumping systems. As previously noted, some gauge pressure fluctuations (and thus flow changes) with the Milton Roy pump operating directly, were observed to correspond to the STN. As already suggested, this component of STN observed with the direct piston pumps may be associated with mis-seating of the pump ball valves. Brooker has recently shown that a marked reduction in STN can be obtained by thermostating the column effluent when a heated column is used (in this case T = 80 "C) with a detector at ambient temperature (13). He finds a STN of 0.0001 OD at a flow of 0.4 ml/min using a Milton Roy piston pump and a Varian, Model 02-001428-00, UV detector. Although most of our work was done at much higher flows (thermostated at 20 "C), we find comparable results. It is interesting to point out that Brooker showed that H F N can be markedly decreased using a n R C circuit to filter the detector signal.
P
= 800 psi, F = 3.5 ml/in., T = 20 "C. Left side, liquid ballast in;
right side, liquid ballast out
As mentioned previously, the R I detector was influenced by flow to a greater extent than the UV detector. The results for H F N and STN are shown in Table I11 for the CDP, Milton Roy direct pump, and trapped solvent control. In this case, a trapped solvent reference was used since the drift (4 x lo-' RI/hr) was ten times less than drift with a flowing reference. Of course, gas cannot be used in the reference side because the R I difference between gas and liquid is too great. It is first noted that the C D P and the Milton Roy pump gave the same H F N as the trapped solvent control. This level of H F N corresponds to the rated value. This result indicates that H F N originates in the detector electronics. The STN for the direct Milton Roy pump is much worse than for the CDP, as expected from the well known flow dependence of the RI detector ( 9 , 10). It is to be remembered that the RI detector is two orders of magnitude less sensitive to concentration than the UV detector. Additional Detector Noise Factors in LC. An interesting question that often arises concerning liquid chromatographic equipment is the influence on detector noise of the volume (mass) of solvent between the pump and the injector. To examine this question, we placed a liquid ballast in the system as follows: the controlled leak section of the C D P was eliminated from the pumping system by operating the on-off valve (Figure 1, Section B); and the 75-ml pressure vessel, previously used as the gas displacement pump, was totally filled with solvent and sealed at the top (Figure 1, Section A), Thus, by means of the on-off valve a t the bottom of this pressure vessel, the liquid ballast could be introduced into or isolated from the Milton Roy direct pumping system. This changed the net liquid volume of the system from 20 ml to 95 ml. Figure 7 shows the change in H F N obtained with the liquid ballast in (left side) and the liquid ballast out (right side). When the Milton Roy direct pumping system was used, the H F N was a factor of ten less with the liquid ballast in. We also found that the STN was unchanged when the liquid ballast was incorporated in this system. The change in H F N apparently arises from the fact that the inertia of the increased mass of solvent is able to act like an electrical inductor (15) to dampen out the high frequency noise component coming from the Milton Roy pump. Since there was much less H F N with the CDP, classical gas displacement pump, and Milton Roy pump with pressurized gas ballast, no effect could be seen for the liquid ballast with these systems. Thus, a static solvent volume in the system between the pump and the chromatographic column will decrease the H F N discernible at the detector when using pulsating pumps. However, it should be noted that such a configuration should be used with care with chromatographic systems involving gradients. (15) B. Hutchins, Waters Associates. Framingham. Mass., private
(14) C . Horvath and S. R. Lipsky, ANAL.CHEW, 41, 1227 (1969). 98
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communication, June 1971.
The Bourdon gauge (15-ml volume) in the normal upright position can also act as a gas ballast of the same volume as the gas ballast used in these experiments. As a gas ballast, the Bourdon gauge is often operated (sometimes unknowingly) with an undetermined volume of trapped gas. Many outgasing detector problems may originate from the gas trapped in the Bourdon gauge slowly dissolving in the solvent. The Bourdon gauge used in this work was used upside down to eliminate accidental volumes of trapped gas, and therefore unknown ballasting. In addition, the gauge was isolated by means of a long narrow tube.
of pressures ( 1 0 . 5 %) and detection noise levels (HFN, STN), equivalent to those obtained with a gas displacement pump and a piston pump operated with a pressurized gas ballast. In addition, the CDP is relatively inexpensive, can be easily operated in pressure programming, requires minimum start-up time, and can be operated in a truly continuous manner. Finally, recent work has shown the CDP performs at the same level as discussed in this paper with water and aqueous acid acetate buffers up to 6 M as solvents. ACKNOWLEGMENT
CONCLUSION
The authors acknowledge the helpful comments of Drs. Erwin Dallmeier and Harry E. Keller.
This paper has presented a novel pumping system for high speed liquid chromatography, a continuous gas displacement pump (CDP). The C D P has been shown to give constancy
RECEIVED for review July 12, 1971. Accepted August 16, 1971. Work supported by NIH Grant G M 15847.
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