cult, and manual approiinxite ni,>thods are normally used (9). ACKNOWLEDGMENT
Deep appreciation is expressed to Fred Fritz, Robert H. Wood, and Harold C. Beachell for their hplpful discussion in connection nith the development of this instrument.
h 0
N
n
.9
REFERENCES
.3
(1) Avizonis, P. V., Kriston, J. C., Jr., unpublished work. (2) Hariharan, P., Bhalla, M. S Rev. Sci. Znstr. 27, 3 (1956). (3) Haupt, G. W., J . Research Natl. BUT. Standards 48, 414 (1952). ( 4 ) PutEam, F. W., “The Plasma Proteins, Vol. 1, p. 111, Academic Press, Kew York, 1960. (5) Rodda, S., “Photo-Electric Multipliers,” pp. 48, 69, PIIacDonald and Co., London, 1953. (6) Snowden, F. C., Page, H. T., Rev. Sci. Znst~.21, 179 (1950). (7) Sober, H. A., Gutter, F. J., Wyckoff, M. M., Peterson, E. A , , J . Am. C h m . SOC.78, 756 (1956). (8) Sober, H. A., Peterson, E. il., Federation Proc. 17, 1116 (1958). (9) Spackman, D. H., Stein, W. H., hfOOre, S., ANAL. CHEM. 30, 1190 (1958). (10) Sumner, FV., “Photosensitors,” p. 212, Macmillan, New York, 1958. ~
0
Tube n d m b e r
Figure 6.
DEAE cellulose chromatography of dialyzed guinea pig serum
Column 2.5 X 40 cm. Step 1. 0.005M,pH 7.0,phosphate buffer Grad. 1. Gradient to 0.02M,pH 6.0,phosphate buffer and 0.01 M NaCl Grad. 2. Gradient to 0.05M NaHSPOI and 0.1M NaCl Step 2. Step from last gradient to 0.1M NaH2P04 and 0.5M NaCl
These initial patterns were obtained with the modifications of the sodium phosphate-NaC1 gradients employed by Peterson and Sober (4, 7 , 8 ) . Further work has been done on guinea pig serum protein fractionation (1). This method of analysis of chromatographic column effluent has also been used here for nucleic acid fractionation a t 260 mp, and for amino acid and peptide fractionation in conjunction n-ith the methods developed by Spackman,
Stein, and Moore (9) a t 570 mp. Certain modifications had to be introduced in connection with the latter application ( 1 ) . I t is evidcnt that once a linear function is obtained on a recorder trace, it may easily be integrated for total amount of material under a peak by planimetry or commfrcial integrators. If, on the other hand, transmittance is plotted by use of an exponential function of concentration, integration is diffi-
RECEIVED for review July 6, 1961. 4ccepted October 3, 1961. Presented in part a t Science Symposium, Delaware Section, .4CS, February 1960. Work supported in part by the Elizabeth Storck Kraemer Memorial Foundation, Wilmington, Del.
Device for Measuring the Resistance of Solutions Linearly and Continuously PETRAS V. AVIZONIS, FRED FRITZ, and JOHN C. WRISTON, Jr. Deparfment o f Chemistry, University of Delaware, Newark, Del.
b Continuous linear a x . resistance monitors described previously need special compensation methods for linearization. This necessitates switching ranges to maintain linearity, and represents a serious lack of flexibility in situations where long-term operation is required over a wide range of resistances. A servomechanical monitor of high stability and linearity has been constructed here, for studying salt gradients used for the ion exchange fractionation of proteins, and for determining salt holdup in such columns, although the device may b e used in any system where the continuous measurement of resistances i s necessary. 58
ANALYTICAL CHEMISTRY
I
exchange fractionation of proteins on “modified cellulose” columns, as described by Sober et al. (6),requires gradient or stepwise changes in the pH and/or salt concentration of solutions applied to the column for elution purposes. It would be useful to be able to monitor the salt concentration and the pH of such solutions, either as they are applied to the top of the column, or as they emerge from the column. Recording pH meters are available commercially, but resistance monitoring presents several problems. Protein fractionation on cellulose columns may require up to 24 hours of continuous operation, depending upon ON
experimental conditions, and i n addition, n-ide ranges of salt concentration are used. In experiments performed in this laboratory and reported elsewhere ( I ) , concentrations ot SaCl frequently ranged from 0 to 0.5Jf and sometimes to 2 X , and phosphate buffer concentration ranged from 0.005M to 0.1M. For continuous column monitoring in such cases, it appeared that there was a need for an instrument having a very high degree of stability, and having in particular one continuous linear range of conductivity or resistance, since switching ranges would not be convenient, espeeislly for overnight operation.
1 00-mfd. 25-volt. d.c. electrolytic capacitor 5,000-ohm, 1 -watt resistors
20,000-ohm, 1 0-turn helipot ganged with R s
Figure 1.
Circuit of conductivity monitor
Several all-electronic a x . circuits have been investigated here for their range linearity, stability, and adaptability for continuous operation ( 3 4 , but none was suitable for the purposes described. A d.c. conductance circuit by Taylor and Furman ( 7 ) was also considered, but not tried because of the necessary physical dimensions of the cell. It was finally decided to design a circuit based upon electromechanical measurements, rather than to rely on an allelectronic system. The instrument as finally constructed uses a servoamplifier as a detector of the voltage imbalance which is caused by a change of resistance in the measurement cell. The servoamplifier drives a servomotor, which in turn drives a Helipot in series with the conductivity cell, thus compensating for the change in resistance of the measurement cell. This forms a servomechanical feedback loop, which acts to restore any imbalance sensed by the servoamplifier. Another Helipot ganged Kith the com-
30
2,500-ohm, 2-watt resistor 1 000-ohm, 1 0-turn Helipot 2000-ohm, 1 -watt resistor 50,000-ohm potentiometer 10,000-ohm, 1 0-turn Helipot ganged with R, 1 -megohm, 0.5-watt resistor 1 -megohm potentiometer Flow cell 1 000-ohm, 1 -watt resistors 1 N 5 4 A diodes 3-volt battery 1.5-volt battery Sola constant voltage transformer 12-volt a.c. filament transformer Leeds and Northrup servoamplifier Diehl servomotor, geared down to 600 r.p.m.
-
x------x
pensating Helipot taps off an appropriate voltage from a battery as a source of recorder signal. APPARATUS
The electronic circuit may be seen in Figure 1. The 100-volt a x . line was a convenient a x . source after stabilization by a Sola constant voltage transformer. This same transformer supplies regulated a s . t o the servoamplifier. The regulated 110-volt a x . is then reduced to 6 volts by a filament transformer, 2'2, which is connected across the cell, Rll, in series with R1, R2, and R3. The purpose of R1 and R2 is to provide enough impedance in the circuit so as not to load down the filament transformer when measuring highly concentrated solutions, thus causing fluctuation in the a x . voltage source for the cell. R3 is the servomotor-driven compensating Helipot completing the feedback loop. Voltage appearing across points a and b (across the cell and Helipot R3)is rectified by a diode bridge rectifier, and then filtered. This voltage is then balanced out by
Rb and R? to yield zero signal voltage t o the servoamplger across R6. The purpose of R, is to provide a large resistance across B1 so as to conserve the battery, and t o give a coarse initial balancing adjustment. When the resistance of cell Rn changes, the potential across a and b changes from that against which the servoamplifier was balanced. This potential then drives the servoamplifier, which runs the servomotor, which in turn drives Helipot R3 so as to compensate for the change of resistance in the cell, and provide a constant resistance across points a and b, thus restoring the previously balanced state. At the same time that the servomotor drives R3,it drives another ganged Helipot, Rs, which taps off an appropriate voltage from battery Bz as a signal source for a 10-mv. Varian recorder. The purpose of Re and Rlo is to provide 10 mv. across Rs. Since the Helipots used are rated as =!=0.25y0 linear, the recorder voltage follows the change in R3 linearly, depending solely upon Ohm's law. The flow cell was made from a piece of 6-mm. o.d. Pyrex (KO.7710) tubing
20.1 Ye Calibrating Resistors NaCl
Solutions
20 IO
14
Figure 2.
15
16
17
18
19
20
Calibration curve of instrument VOL. 34, NO. 1 , JANUARY 1962
59
.IO
0
.09
2
.O8
4
ti % .07;
\
5
6 ;s
-p
*to0
0
d .06 E
c
-05
IO
;
a .04
12
--
.03
14 Curve
4
Curve
b
curve
c
7-
.02
16
.o I
18
20
0 0
Figure 3.
Plot of recorder tracing A. 6. a.
b. c.
of resistance change in a flowing solution produced b y a Mariotte tube gradient device and of corresponding salt concentrations
O.OOSM, pH 7.0 phosphate buffer pumped through flow cell b y metering pump a t constant flow rate Mariotte tube NaCl gradient started. Chamber volume V,, 0.25 liter. Limit concentration, Co, 0.1M [see text) Recorder tracing of ax. resistances measured by instrument in flow cell Theoretical plot of Equation 4 Plot of concentration gradient from recorder tracing as described in text
with a pair of platinum wires sealed into the tube approximately 1 cm. apart. If the seals are rigidly made, there does not seem to be any vibration or instability in the cell with a solution flowing through it.
It is necessary that the R3 resistance value be equal to or greater than the cell resistance which would be obtained with a solution of the lowest concentration that one might encounter. Theoretically, then, it should be possible to measure cell resistances from Rll = Ra to RI1 = 0 with complete linearity, since Rii
+ aRa = k
where k is a RIU,~) and a =
constant equal
h(max)
- R11
Ra
(1)
to (2)
taking Rl1 to be the resistance of the cell a t any time and Rn(,.) to be the maximum resistance that the cell may have. EXPERIMENTAL
Since no solutions with a concentration lower than 0.005Af phosphate buffer, p H 7.0, were used in the chromatographic experiments, the instrument was constructed so that the maximum measurable resistance would be that of the 0.005M phosphate buffer solution. This resistance was deter-
60
o
ANALYTICAL CHEMISTRY
mined using a Serfass bridge, and it was found that the 0.005M phosphate buffer, pH 7.0, had a resistance inside the flow cell of 19.5 X IO3 ohms. This is close to 20.0 X lo3 ohms, and this value was chosen for Rs. The instrument was calibrated initially by using selected carbon resistors of .tO.1% tolerance in place of the flow cell. The calibration results may be seen in Figure 2. Next, a series of 0.005M phosphate buffer solutions, pH 7.0, was made up, containing in addition different amounts of NaCl. These solutions were passed through the flow cell, and their resistances measured with a Serfass bridge. Solutions passing to the cell, and the cell itself, were refrigerated by a circulating alcohol-water bath ( +0.lo (3.). The same solutions were passed through the flow cell again, and this time the recorder pen deflection corresponding to the resistances of these solutions was obtained with the instrument described The pen deflections for these NaCl solutions are also plotted against the resistances obtained with the Serfass bridge in Figure 2. The resistance values represent the average of six determinations for each solution. Flow rates through the flow cell ranging from 0 to 750 ml. per hour were used to determine whether flow rate has any effect upon instrument behavior; it was found to have no effect. Figure 3, a, shows the measured
resistances of the changing SaCl solutions obtained with a 3lariotte tube gradient-producing device ( 7 ) . .4 theoretical expression for this type of gradient was reported by Cherkin, RIatinex, and Dunn ( d ) , and may be rewritten in a more convenient form as
c/co
1
- ,-V/J’o
(3)
where C is the concentration of salt leaving the constant volume mixing chamber after a volume T’ has left, CO is the limit concentration being run into the mixing chamber, and V0 is the volume of the mixing chamber. Taking the logarithm of this expression, one obtains V/Vo = 2.3 log [c,Co __ CI
(4)
and a theoretical plot of 1’ e’s. C is also presented in Figure 3 as curve b. If one now takes the measured resistances of the effluent from the recorder tracing, determines the equivalent salt concentration from a calibration curve of salt concentration os. conductivity (reciprocal of resistance obtained on the Serfass bridge), and plots this in the same way as the theoretical equation, the results are those shoa-n in Figure 3 as curve c. DISCUSSION
Figure 2 clearly shows that a good linear relationship exists between re-
corder pen deflection and the calibrating resistors. A good linear relationship also exists between pen deflection and solution resistances. The two solutions of highest concentration seem to deviate somewhat from the calibration curve. The possibility that this deviation is due to insensitivity of the instrument a t very high concentrations is ruled out by the fact that standard precision resistors of low resistance fall on the curve. Also, if there were such a loss of sensitivity, an increase of a x . voltage across the cell should increase its sensitivity. Consequently, Tz was replaced with a 12-volt filament transformer] and the measurements were checked. There was a slight increase in sensitivity as the dead band decreased from 0.5% to 0.25%, and the points in question (the two uppermost points in Figure 2) were moved closer to the calibration line by that amount. This still leaves a deviation from the calibration curve of l to 2.5y0and further increase of cell voltage did not improve the results. The explanation for thi? deviation probably lies in two factors. The Serfass bridge used is somewhat inaccurate a t high concentrations, and the cell used is of poor design for measurements of low resistances. I t nould appear that the former is the major cause of error, since the a x . resistance readings made with the Serfass bridge using calibrated noninductirely wound resistors with +O.lYo tolerance gave low readings with a similar magnitude of error. Resistance ranges from 0 to 1000 and
from 0 to 100,000 ohms were tried (using appropriate changes in RB), with much the same results. Theoretically, this instrument should measure the resistances of solutions of high as well as of low concentrations. In practice, however, some sensitivity is lost a t high concentration (above 0.2.V). This may be overcome either by changing the dimensions of the cell, or, preferably] by replacing the 10turn dual Helipot (R3-R8 in Figure 1) with a 25- or 40-turn device (available from the Beckman Instrument Co.). The stability and reproducibility of the instrument were excellent. Over one period of 24 hours, no drift or deviation was observed with a 0.005X phosphate solution flowing through the cell a t various flow rates. The over-all response of the instrument could be improved considerably by reducing the over-all time constant. This would involve the reduction of the time constant (resistance times capacitance) of the rectifier and balance circuits, although the existing circuits represent nearly the optimum value possible with good filtering of the a x . It would be simpler to use a recorder having a better a x . rejection mode (a Varian recorder was used in this case), a shorter response time, and a smaller dead band. Since concentration is linear with conductance ( l / R ) , it would be convenient to have the recorder deflection linear with the reciprocal of resistance (linear with conductance). Since the resistance of the flowing solution is
being measured here, this means that one would need a hyperbolic function generator. Such a device may be constructed based upon Ohm’s law, since current is inversely proportional t o resistance, and if one keeps a constant source voltage, variation of resistance Rs will generate variable current nhich will be inversely proportional to the resistance. Unfortunately. because of the nature of the hyperbolic function, very small changes in the current output would be generated for large changes in the resistance along the horizontal asymptote of the hyperbola, thus giving very poor sensitivity in that area. For this reason no simple hyperbolic function generator was included in the instrument. LITERATURE CITED
(1) Avizonis, P. V., Triston, J. c:.) ANAL. CHEM.34, 54 (1962). (2) Cherkin, A., Matinex, F. E., Dunn, M. S., J . Am. Chem. SOC.75, 1244 (19531. (3) Creamer, R. hl., Chambers, D. H., ANAL.CHEM.26, 1098 (1954). ( 4 ) Daniels, F., Mathews, J. H., JT’illiams, J. R., staff, “Experimental Physical Chemistry,” 4th ed., pp. 454-6, McGraw-Hill, Kew York, 1949. ( 5 ) Fischer, R. B., Fisher, D. J., . ~ N A L . CHEM.24, 1459 (1952). (6) Sober, H. A., Gutter, F. J., \Tyckoff, SI. M., Peterson, E. A , . J . Am. C‘hem. SOC.78, 756 (1956). (7) Taylor, R. P., Furman, S. €I., AXAL. CHEM.24, 1931 (1952). RECEIVED for review July 6, 1961. rlccepted October 19, 1961. Work supported in part by the Elizabeth Storck Kraemer \ - - - - I
Memorial Foundation, Wilmington, Del.
Quantitative Gas-Solid Chromatog ra phic Determination of Carbonyl Sulfide as a Trace Impurity in Carbon Dioxide HARRY L. HALL The Coca-Cola Co., Atlanta 7 , Ga.
b Carbonyl sulfide at the parts per million level in carbon dioxide has been determined linearly over the investigated range of 0.5 to 2700 p.p.m. Minimum detectable corlcentration under these conditions is 0.3 p.p.m., with an estimated uncertainty at the 3-p.p.m. level of h0.07 p.p.m. The method is rapid and reliable and has been successfully applied to the determination of carbonyl sulfide in commercial carbon dioxide which produced carbonated water having offtaste and off-odor.
C
ARBONYL sulfide has been
found as a trace impurity in some commercial carbon dioxides in this country and abroad. In contact with water this compound slowly hydrolyzes to form hydrogen sulfide. A 1-p.p.m. carbonyl sufide content in carbon dioxide produces a discernible off-taste in some carbonated beverages. A faint odor of hydrogen sulfide will be present in most carbonated beverages if the carbon dioxide contains as much as 5 p.p.m. of carbonyl sulfide. A rapid, reliable, direct, and specific method of analysis
was required and provided the motivation for this work. Carbonyl sulfide has been determined in various gases by a number of methods, including spectrophotometric (9), potentiometric (3)’ colorimetric ( I I ) , selective solvent extractive (@, and gravimetric ( 1 ) . Xone of these methods satisfy all of the abovementioned requirements. A recent gasliquid chromatographic method ( l a ) was sensitive a t the 25-p.p.m. concentration level but with a +l5-p.p.m. uncertainty. Gas-solid chromatographic separation of carbon dioxide VOL. 34,
NO. 1,
JANUARY 1962
61
