ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
50
-
40 -
-2
.->
-
30-
--C
c
.-01 2 0 A
~~
10
20
30
40
50
60
Diethyiirolurninol (pM)
Flgure 4. Comparison of the RMF system with the SI system for monitoring chemiluminescence reactions. Diethylisoluminol at the indicated final concentrations in the cell was oxidized with the RMF system and the light intensities were recorded as described under "Experimental" (A). Each data point is the result of one measurement. Reaction mixtures (150 pL) containing indicated levels of diethylisoluminol received a single automatic injection of H,Op to produce light as described in the Experimental section. The peak light intensities from three individual reactions were averaged for each data point (0)
Table I. Reproducibility of Chemiluminescence Measurementsa light standard 0.9
single injection system automatic rapid mixing hand flow system injection injection 2.0
38
8
a Each value represents the coefficient of variation (%) of the mean light intensity obtained with 18 identical reactions. The concentrations of chemiluminescent com-
pound were chosen t o produce similar light intensities with each method of measurement (also the light standard) to allow direct comparison of reproducibility. strument variability of hO.970 (Table I). The light standard described in the Experimental was measured repeatedly and the coefficient of variation is presented for reference. Chemiluminescent reactions were carried out with diethylisoluminol (30.8 pM final concentration) in the RMF system as described in the Experimental and 18 ten-second measurements were obtained. The mean light intensity was 22.6 and a background signal (absence of chemiluminescent compound) was generally 0.5. Similar chemiluminescence measurements were performed with the S I system utilizing automatic addition of HzOzas described previously ( 4 ) or by hand from a 25-pL Hamilton syringe as described in the Experimental.
1585
The enhanced reproducibility may partially be based on reaction mechanism. Since microperoxidase is destroyed by H,02, true catalysis by the haem moiety is observed only in the initial part of the reaction (10). Therefore, at steady-state chemiluminescence, sufficient rapid displacement of cell content may eliminate destruction of microperoxidase within the flow cell and thereby reduce variability in measurements. The residence time of reagents in the flow cell was about 6 ms. Other flow cells typically have usually greater than 50 ms (5). Such residence times are too long to take advantage of potential sensitivity and reproducibility of flow system with fast reactions. Precision in chemiluminescence measurements is related to speed of light production and to the reagent mixing system. The SI system gives poor reproducibility when light is produced rapidly because bubbles form in the solution and scatter light. Measurement of total light does not improve reproducibility in this case ( 4 ) . An instrument with high speed injection below the reaction surface may provide rapid mixing and improved reproducibility. We have taken an alternative approach and shown that variability in highly sensitive chemiluminescence measurements can be greatly reduced with an instrument based on rapid mixing and short cell residence time; e.g., at maximum light intensity, values became independent of flow rate. Only small quantities of commercially available, inexpensive reagents are required since the flow time for steady-state chemiluminescence is short (less than 10 5). Furthermore, detection reactions at pH 8.6 to 13 possess similar sensitivity ( 4 ) and the RMF system was also successful in brief tests in 7 5 mM barbital, pH 8.6. Although the system described here was designed for the specialized purpose of monitoring competitive protein binding reactions, other types of chemiluminescence measurements may benefit from a similar approach.
ACKNOWLEDGMENT We thank Frances M. Yeager for performing some of the chemiluminescence measurements.
LITERATURE CITED Schroeder, H. R.; Vogelhvt, P. 0.; Carrico, R. J.; Boguslaski, R. C.; Buckler, R. T. Anal. Chem. 1976, 4 8 , 1933-1937. Schroeder, H. R.; Bogushski, R. C.; Carrico, R. J.; Buckler, R. T. Methais Enzymol. 1978, 57, 424-445. Schroeder, H.R.; Yeager, F. M.; Boguslaski, R. C.; Vogelhut, P. 0. J . Immunol. Methods 1979, 25, 279. Schroeder, H. R.; Yeager, F. M. Anal. Chem. 1978, 50, 1114-1120. Seitz, W. R.; Hercules, D. M. Anal. Chem. 1972, 4 4 , 2143-2149. Seitz, W. R.; Neary, M. P. In "Methods of Biochemical Analysis", Vol. 23, Glick. D., Ed.; John Wiiey and Sons: New York, 1976; Vol. 23, pp 161-186.
Stieg, S.; Nieman, T. A. Anal. Chem. 1978, 50, 401-404. Sheehan, T. L.; Hercules, D. M. Anal. Chem. 1977, 49, 446-450. Bostick, D.T.; Hercules, D.M. Anal. Chem. 1975, 4 7 , 447-452. Kremer, M. L. Natufe (London) 1965, 205, 384-385.
RECEIVED for review March 7,1979. Accepted April 11, 1979.
Pulse Dampener for High Pressure Liquid Chromatography John G. Nikelly" and Dominic A. Ventura Department of Chemistry, Philadelphia College of Pharmacy and Science, Philadelphia, Pennsylvania
In the past two years mechanical reciprocating piston pumps have become the most widely used type of pump in high pressure liquid chromatography, owing to several advantages such as having a small internal volume and placing no restriction on the size of the solvent reservoir.
19 104
However, it should be noted that virtually all of the new pumps that are specifically designed for HPLC are of the multiple-head or multiple-action type. In addition, many of the multiple pumps are equipped with expensive features designed to further reduce or totally eliminate the pulsations
0003-2700/79/035 1-1585$01. O W 0 @ 1979 American Chemical Society
1586
ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
Table I. Dimensional Data for Tubing Dampener
tubing circular moderately flattened drastically flattened
aspect ratio 111 311
6/ 1
crosssectional area ratio 1.0 0.7 0.4
of the mobile phase. For example, in one approach the velocity of the piston is linearized during the pump portion of the stroke while the fill stroke is very fast and independent of the flow rate ( I ) . Another approach uses the concept of compression feedback (2). More conventional methods of pressure dampening involve a mechanical device, usually a flexible metal vessel (bellows) ( 3 ) ,or Bourdon tube ( 4 , 5 )which takes up about half of the energy during the pressure stroke and releases it during the intake stroke (6, 7). However, these devices are generally restricted to operation at specified pressure ranges ( 3 , 5 )and are relatively expensive. Furthermore, it appears that many of the dampeners which operate on the Bourdon tube principle are not totally effective and have a limited operating lifetime (8).
More recently, a relatively inexpensive dampener was reported (9) comprising a conventional pressure gauge in series with a commercially available stainless steel connector (annularly corrugated), capable of operating at pressures up to 2660 psi. The main drawback of this dampener is that it increases the total mobile phase volume ofthe HPLC system by 20 to 40 mL, thus increasing the time required to reach equilibrium when the mobile phase is changed (about 10 min). Moreover, such a highly flexible dampener has a variable volume which depends on the pressure, and therefore, when first starting up the pump, it requires additional time to reach operating pressure. This paper describes a highly effective, low cost, dampener comprising, in addition to the obligatory pressure gauge, a few feet of specially flattened stainless steel tubing used in a flow-through configuration, Le., as part of the mobile phase line between the pump and the analytical column. The dampener is based on the principle, illustrated in Table I, that the cross-sectional area of the flattened tubing is significantly smaller compared to the more rounded shape resulting from the pressure peak. The proposed dampener has major advantages over existing dampener systems. First, it is very inexpensive in that the pressure gauge and tube fittings (Swagelok) are the principal cost items. Second, i t has a very low volume, only a few milliliters. This allows rapid establishment of operating pressure (on starting the mobile phase pump) and it also substantially reduces the time required to change from one solvent to another. Third, it reduces the pressure pulses to very low values so that in most cases the base-line fluctuations are indistinguishable from the detector noise. Finally, the same dampener is equally effective over a wide range of operating pressures. While the proposed dampener may appear to be somewhat similar to other mechanical dampeners which are commercially available or which are proprietary component parts of HPLC instruments, there is little doubt that it is different in many respects. For example, there is a flattened stainless tube component in the new duPont Model 850 liquid chromatograph, but rather than pulse dampening, its primary function is the protection of low-backpressure columns (glass) or columns packed with soft, compressible gels (IO). Another dampener which has recently been described in the literature consists of a Teflon tube with an oval cross section sealed in a chamber containing a fluid which acts as a “liquid spring”
Figure 1. Pulse dampener, made from 4 m X ’ / e in. 0 . d . tubing
( I ) . Still another mechanical dampener, which is available commercially, appears to be similar to perhaps 10 to 12 pressure gauge Bourdon tubes in series and is engineered for specific pressure ranges, e.g., 100 to 1000 psi ( 5 ) .
EXPERIMENTAL Apparatus. In addition to the usual fittings, capillary tubing, and pressure gauge (Bourdon tube, 0 to 5000 psig range), the dampener itself, shown in Figure 1, consists of a few feet of stainless steel tubing, Type 316, which is tempered and shaped to a cross section approaching an ellipse or parallelogram with rounded corners. The tempering and shaping procedures were carried out by Handy and Harman Tube Company, Norristown, Pa. 19401. The dampener assembly was used as part of an HPLC system comprising a synchronous reciprocating pump, Model 396, and UV monitor, Model 1205, Laboratory Control Division, Milton Roy Company, Riviera Beach, Fla. 33404. Several tests were also conducted with a Model 5000 pump, Eldex Laboratories, Inc., Menlo Park, Calif. 94025. Samples were injected with a Valco Sample Injection Valve, Series SVOV-6-1(Glenco Scientific, Inc., Houston, Texas 77007). The analytical column was 25 cm X 3.0 mm i.d., packed with 10-wm particles of reverse phase CI8 Bondapak (Waters Associates, Inc., Milford, Mass. 01757). Procedure. The optimum design of the dampener obviously depends on many variables including length, diameter, wall thickness, shape, temper, relative position in the assembly, and configuration (in series, in parallel, flow-through, etc.). Several of these factors were tested under various operating conditions by noting the range of pressure pulses on the pressure gauge and the fluctuations on the recorder base line. RESULTS AND DISCUSSION Design Considerations: Diameter, Length, Temper, a n d Wall Thickness. Based on the typical flow rate of mobile phase, 1 mL/min, and considering that the reciprocating rate of single piston pumps is generally about 30 strokes/min, it follows that each pressure stroke delivers about 0.03 mL of solvent. A fraction of this volume, about one half, must be taken up by the ballast for delivery during the intake stroke, Le., the volume of the ballast should be able to expand elastically by approximately 0.015 mL. Considering that it is desirable that the ballast volume be kept reasonably small, e.g., about 2 mL, it follows that pulsations of 0.015 mL represent less than 1%of the total volume. It would appear likely that such a relatively low distortion may be easily elastic. Preliminary tests were run using dampeners of different diameters and wall thickness. These tests established that for the pressure range of interest, from 500 to 2000 psi, the largest dampening effect (largest elasticity) is realized with relatively small wall thicknesses: 0.006 in. for tubing originally 1/16-in.0.d. and 0.010 in. for 1/8-in.tubing. The larger of these two sizes (1/8-in. 0.d.) was found to produce the largest dampening effect per unit length-as expected from its larger area for flexing (larger capacity).
ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
1587
- A dampener
>
A
B
C
-
Figure 3. Effect of dampener on gauge pressure and on recorder base line at 0.01 AUFS and 1500 psi operating pressure. (A) WAh proposed dampener and gauge, 20 psi, peak to peak. (B)With gauge only, 400 psi, peak to peak. (C) With flexible connector (9)and gauge, 150 psi, peak to peak
C
I
t
D
Flgure 2. Pulse dampening system, four basic arrangements of the dampener components
The optimum length of the dampener was first estimated from the following calculations and then confirmed experimentally. The shaped tubing may be considered to have a cross section approaching the shape of an ellipse in which the minor axis is the difference between the outside thickness of the flattened tubing and the wall thickness. The approximate magnitude of the major axis (inside axis) is then calculated by the mensuration formula for the circumference of an ellipse,
2.rrl/(a2 + b 2 ) / 2 where a and b are the semi-axes. (The circumference of the ellipse as determined by measurement was found to be about 8% larger than the circular circumference of the original tubing.) Accordingly, the major and minor inside axes of a dampener made from tubing that was originally l/s-in. 0.d. is 0.08 and 0.01 in., respectively, with a cross section of 0.00063 in2 or 0.0040 cm2. Thus, in order to obtain a dampener of 2-mL internal volume (mentioned earlier), the required length would be 2 cm3/0.004 cm2 or 5 m. Several dampeners were made and tested using Type 316 stainless steel tempered or annealed to various degrees of hardness. For pressures up to 2000 psi it was found that the dampening effect was about the same regardless of temper. But dampeners of hard temper are definitely preferable since they can be taken up to 2500 psi without loosing any dampening effect upon returning to lower pressures, 1000 to 1500 psi. Dampener Configuration. The dampener may be used in the HPLC system in any one of several configurations (relative position in the system) but it appears that all of these may be considered as insignificant variations of the four basic arrangements, A, B, C, and D, shown in Figure 2 . Arrangements A and B are preferable over C and D since the flow-through (swept volume) design is more convenient for rapid changeover of mobile phase. Between A and B it was found that arrangement A is the more efficient per length of dampener, which accords with the analogy of an electrical capacitance circuit; but arrangement B was found to be preferable over A from a practical viewpoint because it is
simpler and requires barely 20 or 30% additional length to have the same effect as A. The dampener was wound as a 6-in. coil or helix and was not held rigidly in place. In another laboratory, the proposed dampener was coiled to a somewhat smaller diameter and held in place (attached) at three or more points with no discernible decrease in dampening effect (8). However, at operating pressures above 2200 psi, a slight movement or the coil was noticed (Bourdon tube effect), which means a smaller dampening effect might result if the coil is too small and is rigidly fixed in position. Operating Pressure Range. The dampener was tested at operating pressures between 500 and 2500 psi and was found to be equally (uniformly) effective a t all pressures in this range. It was also operated between 2500 and 5000 psi but a t such high pressures the dampener looses some of its effectiveness when returned to operation at lower pressures. (As the operating pressure increases above 100 psi, there is a slight but measurable increase in the outside small axis of the dampener; however, this bulging does not decrease the dampening effect until pressures of 2000-2500 psi are reached. At still higher operating pressures it was found that the small axis is doubled or tripled compared to its original dimension, and as the pressure is returned to normal, below 2000 psi, the dampening effect is somewhat diminished.) In view of the pressure limit and considering how often it is possible, even easy, to subject the dampener to a high pressure inadvertently, we recommend using a pressure relief valve set ca. 2000 psi. Pressure release devices are available commercially (11) or can be assembled locally from standard fittings at greatly reduced cost (12). Dampening Effect. The dampening effect was estimated from pressure gauge fluctuations or base-line oscillations of a recorder as it monitored either the UV detector or a pressure transducer (8). It was found that the peak to peak pressure fluctuations were 20 to 25 psi over the entire operating range of 500 to 2000 psi a t a mobile phase flow rate of 1 to 2 mL/min. Since the undampened pressure pulses (gauge only) are of the order of 400 psi, the dampening effect amounts to a 20-fold or 95% decrease in the pressure pulses. As shown in Figure 3, the recorder base line (10 mV, f.s.) is virtually indistinguishable from the HPLC system noise at UV detector sensitivity 0.01 AU and operating pressure 1500 psi.
ACKNOWLEDGMENT The authors gratefully acknowledge the technical assistance and materials provided by George I. Bartleson, Jr., of Handy & Harman Tube Company, Norristown, Pa. 19401. LITERATURE CITED (1) P.Achener, K. Judah, and D. Boehrne, Varian Instrument Division, Paper No. 36, Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 5 to 9, 1979.
1588
ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
(2) M. L. McConnell, Chromatix. Ref. 1, Paper No. 37. (3) Pulse Dampener Model 709, Laboratory Data Control Division, Milton Roy Company, Riviera Beach, Fia. 33404. (4) Pulse Dampener, Model 80-600Liquid Chromatograph, Gow-Mac Instrument Co., Bound Brook, N.J. 08805. (5) Gienco Scientific, Inc., Houston, Texas 77007, 1978 Liquid Chromatography Products Catalogue, p 9. (6) L. R. Snyder and J. J. Kirkland, "Introduction to Modern Liquid Chromatography", Wiley-Interscience, New York, 1974, pp 101-102. (7) J. N. Done, "Idealized Equipment Design for HPLC", in "Practical High PerformanceLiqua Chromatography", C. F. Simpson, Ed., Heyden & Son, New York, 1976, pp 71-72.
(8) E. Durrum, Eldex Laboratories, Inc., Menlo Park, Calif. 94025, private
communication. (9) D. A. Ventura and J. G. Nikelly, Anal. Chern., 50, 1017 (1978). (IO) N. Parris. duPont Company, Scientific 8 Process Instruments Division, private communication. (1 1) Model 7037 Pressure Relief Valve. Rheodyne. Inc., Berkeley. Calif. 94710. ( 1 2 ) Unpublished work.
RECEIVED for review February 5 , 1979. Accepted April 23, 1979.
Extraction of Semi- and Nonvolatile Chlorinated Organic Compounds from Water A. J. Burgasser"' and J. F. Colaruotolo" Hooker Chemical Company, Research Center, Grand Island Complex, M.P.O. Box 8, Niagara Falls, New York 14302
The category of semivolatile and nonvolatile extractable organic compounds comprises those compounds which are not capable of being analyzed by the purge and trap technique ( I ) but are preferentially soluble in nonaqueous solvents. Eighty-three (83) compounds on the EPA priority pollutant list fall into protocols in this general category ( 2 ) . The compounds fall into three subgroups-base/neutral, acid, and pesticide fractions. Analyses based upon these protocols call for p H adjustment, a triple extraction procedure with methylene chloride/hexane mixture, drying through sodium sulfate, and a concentration/solvent exchange step using a Kuderna-Danish evaporator. For chlorinated organic compound analyses by gas chromatography, the solvent exchange step is essential to remove all of the methylene chloride such that its detector response does not interfere. Once concentrated, the extracts are analyzed by gas chromatography using an electron capture or Hall detector, or concentrated further by dry nitrogen purge for GC/MS analysis ( 3 ) . The extraction procedure specified in the EPA protocols is widely used and considered to produce results with precision and accuracy acceptable to the requirements of the EPA. The primary drawback is that it is inherently slow, requiring long extraction and phase separation times. This effectively cancels out the benefits derived from the use of modern automated chromatographic instrumentation. Also, the procedure has a large number of manipulation steps, increasing the potential for poor performance as a result of technique related problems. We have developed a procedure to greatly increase the efficiency and speed of this extraction step using a Brinkmann Polytron Homogenizer to perform the extraction and a Sorval refrigerated centrifuge to speed up the phase separation process. The extraction of chlorinated organic compounds from water can be carried out in a single vessel in one step taking only 10 min to complete. Previous work on the extraction of pesticides from soils using the Polytron Homogenizer was reported by Johnson and Starr in which the soil was suspended in water and the compounds of interest were extracted from the soil particles into acetone ( 4 ) . Four compounds (hexachlorobutadiene, hexachlorocyclopentadiene, octachlorocyclopentene, and hexachlorobenzene) were evaluated for their extraction efficiencies and linearity of extraction from water by the Polytron Homogenizer 'Present address, Hewlett-Packard,102 8th Ave., King of Prussia, Pa. 19401.
~
~~
~~~
~~~~
Table I. Gas Chromatographic Conditions column: G.C. :
6 ft X 2 mm i.d. glass, 3% Dexsil 300 on 801100 Supelcoport Hewlett-Packard 5840A detector: electron capture ( 6 3 N i )300 'C injector: 220 C column temp: 150 " C carrier gas: 10% methane argon flow: 30 mL per min
__-
technique. The accuracy and precision obtained for the method is also reported.
EXPERIMENTAL Apparatus. The extractions were carried out in Pierce 125-mL hypovials using a Brinkmann Polytron Homogenizer, Model PT-1035, equipped with a model PT-1OST generator. Phase separation was carried out by centrifugation of the sample in the same vessel using a Sorval RC2-B centrifuge. Analysis of the extracts was carried out using a Hewlett-Packard 5840 gas chromatograph with an electron capture detector. The gas chromatographic conditions used are shown in Table I. Reagents. Water used for preparation of standards was triple distilled from potassium permanganate solution and nitrogen sparged to ensure that no organic residues were present. All organic solvents used (acetone, hexane, benzene) were pesticide grade Burdick and Jackson disbilled in glass. All glassware was cleaned by treatment with dichromate and subsequent rinsing with water, acetone, and hexane. The glassware was then oven dried at 250 "C. Procedure. One hundred (100) mL of the water to be analyzed were placed into a Pierce 125-mL hypovial and the pH was adjusted to suit the nature of the compounds being analyzed (for the chlorinated compounds, the pH was adjusted to 9-11). Then 10 mL of 15% benzene in hexane were added t o the vial. The sample was extracted for 30 s with the Polytron Homogenizer at 50% of full speed (approximately 11000 rpm). Following each extraction, the PT-1OST generator was cleaned by successive washings in acetone, hexane, acetone, and hexane. The hypovial containing the emulsified solution was then centrifuged for 5 min at 1500 rpm and 4 "C. This was sufficient time to completely separate the organic and aqueous layers. The organic layer was removed with a Pasteur pipet and sealed in an autosampler vial for analysis on the Hewlett-Packard gas chromatograph. RESULTS AND DISCUSSION The total emulsification of the organic and aqueous phases by the homogenizer had the effect of optimizing the partitioning of the extractable compounds. For compounds with
0003-2700/79/0351-1588$01.00/0 0 1979 American Chemical Society
