Continuous Photometric Determination of Carbon Dioxide in Gas Streams W. D. MAXOhT A N D JIARVIX J. JOHNSON Department of Biochemistry. College of Agriculture, Cnirersity of Wisconsin, Madison, Wis,
I
For in\estigations in\ol\ ing carbon metabolisni in aerobic fermentations i t was found desirable to follow t h e carbon dioxide output of growing microThe method described organisms rather exactly was designed to fill the need for a n easilj fabricated system to determine carbon dioxide continuousl? in gas streams. I t makes use of the equilibriuni hetween gaseous carbon dioxide and aqueous bicarbonate ion a n d its photometric measurement using t h e indicator phenol red. I t has been iniestigated for determination of carbon dioxide concentrations in the range 0.06 to 129'0, and exhibits a standard deFiation of about 2 parts per hundred throughout. I t nia) be used continuously for periods of indefinite length, provided t h a t minor adjustments are made ever) 12 hours or more. idaptation of the method to many systems where i t is desired to determine carbon dioxide continuousl) in almost a n ) concentration should be possible.
1 PERCEXT
.
PERCENT
.
WAVELENGTH
T
HE reaction between gaseous carbon dioxide and water to
form bicarbonate ion and hydrogen ion has an equilibrium position which is dependent for a given aqueous solution of bicarbonate upon the partial pressure of carbon dioxide in the gas phase in contact with it. Furthermore, the position of the equilibrium is measurable by the hydrogen ion concentration. These facts form the basis of several previously described methods for the determination of carbon dioxide. Higgins and PIIarriott (2) used visual comparison of the color of the indicator phenol red to estimate t,he position of the equilibrium. The accuracj- of suvh a method is, of course, limited. A potentiometric determination forms the basis of the method of Vilson, Orcutt. and Peterson (3). Here the practical difficulty of obtaining precise enough measurements with a glass electrode limits results. The method of Winzler and Baumberger (4)involves the photometric determination of the color of the indicator methyl red. The range over xhich carbon dioxide partial pressures may he eatimated is someyhat limited in this case. Sone of the above workers attempted to adapt the method to continuous operation. Brown and Felger (1) have developed a continuous determination based on electroconductivity measurement. Their mrthod, Jvhile apparently dependable, involves rather comples apparatus :tnd has limited precision. In the present case the hydrogen ion caoncentration is determined by the photometric measurement of the red color of phenol red. The apparatus is easily and rather inespensively fabricated. I t is capable of continuous operation over long periods and the results may be automatically recorded. I t is easily adapted to estremely ivide limits of carbon dioxide concentr:ition. THEORETICAL BASIS
- millimicrons
Figure 1. .4bsorption Spectrum of Basic Phenol Red and Transniittance Spectrum for Evelyn Light Filter
KO. 565
(CO,)
=
2 L PCO?
22.4
where
(C02) = concentration of dissolved COS pCOl = partial pressure of CO, in atmospheres 2.
The Carbon Dioxide-Bicarbonate Equilibrium Reaction.
CO?
+ H2O
HCOi-
+ €€-
The yunntitative relation is expressed by the ionization constant:
where
(H-) = concentration of hydrogen ion (HCO,-) = concentration of bicarbonate ion 3. The Indicator Equilibrium Reaction. ionization constant is:
In thia caae the
where
( R ) = concentration of phenol red in the basic or red form ( T ) = total concentration of indicator Combination of the above three equations gives the following relationship for ideal behavior:
The reactions upon which the method depends are: 1. The Solution of Carbon Dioxide in Water. The quantity which expresses this numerically is a,the absorption coefficient (the volume of carbon dioxide, reduced to 0" C. and T60 mm. of mercury, which will dissolve in 1 volume of water when the partial pressure of carbon dioxide is 1 atmosphpre).
h con-tant, b, iq now defined which is proportional t o (HCOB-):
1541
ANALYTICAL CHEMISTRY
1542
This constant is, of course, variable with temperature and will include all deviations from ideal behavior in the solution. The Beer-Lambert law gives the following relationship for the transmittance of light by this system:
E = log Io/I
k(R)
(3)
where:
E = extinction coefficient
Io = per cent transmittance for infinite pCOz Z
=
per cent transmittance for other p C 0 ~
I; = a proportionality constant
If another constant, i , which is proportional to (2') and equal to the extinction coefficient a t zero pCOz, is introduced, Equations 2 and 3 may be combined to give:
E =
i pCOn/b
+1
(4)
Re arranging :
If p C 0 2 / b is now plotted against l/E, a straight line will result, whose slope and intercept both equal l/i. In Figure 1 the absorption spectrum for the red form of phenol red and the transmittance spectrum for the light filter used are shoan. The fact that their peaks do not coincide exactly m-ould lead one to expect deviation from the Beer-Lambert law in this system. Figure 2 shows such a deviation to occur to a signifiThis plot cant extent for transmittances lower than 30%. has been used as a correction to Equation 4, leading to the theoretically predicted relationship betxeen pCOz/b and per cent transmittance which is illustrated by Table I and the curve in Figure 3. An i value of 0.900 was found to be optimal and was used for construction of this line. APPARATUS
A schematic representation of the complete apparatus is shown in Figure 4. The heart of the system is the absorption cell in which the bicarbonate-phenol red solution is contained and through which the gas stream to be analyzed is bubbled. The arrangement of the components of this cell is diagrammed in Figure 5. The cell itself is simply a 6-ounce soft-glass prescription bottle of rectangular cross section (3.5 X 7 X 13 cm.). The sparger is constructed of glass tubing bent to face upward along the side of the bottle. The end of the tube is flared and over it is stretched a small piece of perforated rubber dam held n j t h a rubber band. Suitably sized holes in the dam are made previously by laying it upon a piece of linoleum or similar surface and puncturing it with a sharp pin. A thermometer is inserted into the solution through a hole drilled in one shoulder of the bottle. 811 components must be carefully arranged, so that there is space for a beam of light to pass through the bottle unobstructed by bubbles or objects whose position might change slightly. The absorption cell is firmly mounted by encasing it in a snugly fitted R-ooden sleeve. -4leaf spring is mounted in the sleeve to bear against one edge of the bottle, so that the bottle may be readily removed and returned to the same position. The photosensitive element, a General Electric photovoltaic cell, type PV-1, is mounted in one side of the sleeve so that it is held in the position relative to the absorption cell shown in Figure 5 . Directly opposite the photocell in the other wall of the wooden sleeve is mounted an Evelyn filter, No. 565, supplied by the Rubicon
co.
Rubicon Co. catalog KO.4625, 0.5 Ma. full scale, and a Leeds and Piorthrop Speedomax Type G recording potentiometer, 10 mv. full scale. Any instrument with a linear response and a suitable sensitivity could be used. In order to avoid the influence of ambient light upon the photocell, the entire apparatus m-ith the exception of the measuring instrument is housed in a box. A hinged lid to allow access to the absorption cell is provided. A small blower which directs an air stream on the lanip is valuable in decreasing the operating temperature. This results in a shorter XTarm-up time and a lower rate of evaporation of water from the solution in the absorption cell. OPERATIOU A Y D CALIBRATION
The steps involved in operation of the apparatus are enumerated : 1. A solution of sodium hydroxide of known concentration is prepared and together with formaldehyde to a concentration of 1%, is added to the bottle up to a carefully noted level. The sodium hydroxide is used for convenience and is converted to a carbon dioxide-sodium bicarbonate buffer v-hen carbon dioxide is added during the normal course of the determination. The formaldehyde n as found necessary n-hen the instrument was ubed to measure the carbon dioxide in the exit gas stream from 100 an aerated fermentor 80 over long periods, for without it bacteria 60 Tvould grow in the ab2 50 s o r p t i o n cell a n d c cloud the solution. 40 K i t h the absorp10 tion cell in place the 2 30 m coarse ( 5 0 0 0 - o h m ) and fine (300-ohm) + 2 20 sensitivity resistors V are adjusted to give a w full-scale reading on themeter. This condition (no indicator) 10 corresponds to in40 6o s' loo finite pC02, since the PERCENT OF MAXIMUM CONC'N. a b s o r p t i o n of t h e Figure 2. Deviation from Beeryellow (acid) form of Lambert Law for Basic Phenol Dhenol red is nenligible in the band-of Red and Evelyn Light Filter No. 565 the light filter used.
' ''
10
-
6
-
5
-
4
-
3
-
2
-
t
; i l i
I n the assembled apparatus, Figure 4, a light beam passes through the filter and the absorption cell and falls upon the photosensitive cell. The light source is a 6- to %volt auto lam , S o . 81. I t is powered by the 6-volt secondary of a 15-watt Sora constant voltage transformer, catalog S o . 30488. I n series with the lamp is a Kichrome wire resistor of 1.3 ohms to cut d o a n the voltage to the lamp to about 5 volts, thus increasing the operating lifetime of the lamp. The wiring of the measuring circuit to the photocell is designed for adaptability to a variety of measuring instruments. Used with success have been a direct current spotlight galvanometer,
-
8
1
Pf,,
0. 5
0. 0.43 20
30
40
50
80
70
80
90
PERCENT TRANSMITTANCE
Figure 3.
Calibration Curve for Apparatus
Solid l i n e represents theoretical relationship. Points are derived from CO2 determinations made by alkali absorption method 0 0.01 N bicarbonate apparent (HCOs-) = 0.00933 A 0.005 N bicarbonatd, apparent (HCOs-) = 0.005 x 0.001 N bicarbonate. apparent (HCOa-) = 0.001
V O L U M E 24, NO. 10, O C T O B E R 1 9 5 2 The per cent transmittance when the absorption cell is removed (“air blank”) is noted so that it may be reset to correct for photocell drift when necessary (see discussion). 2. Phenol red (0.02% aqueous) is added carefully until a per cent transmittance of 14.8 is reached. This condition corresponds to zero pCO2 and an i value of 0.900. All conditions must be made reproducibly to maintain a valid calibration. SAMPLE IN
4
’ \ -M PAL
1543 of a fairly constant concentration of carbon dioxide in air was obtained by using the exit gas from an aerated mixture of 10% glucose and 1% active dry baker’s yeast in water. The results of these determinations are presented in Figure 6. I t is evident that, as expected from theory, the ratio of b/(HC03-) is constant for the lower bicarbonate concentrations. However, a t the 0.01 N level the effect of increased ionic strength on the activity coefficients is noted and the effective (HCOs-) is lowered to 0.00933. Wilson, Orcutt, and Peterson (3) noticed the same effect to approximately the same degree. The decided increase of b with temperature is a reflection of the effect of temperature on 01, K l , K , (Bee Equation 1, theoretical basis), and the deviations from ideality. SAMPLE IN I
.
TRANSF‘ORMER
13
115
RMOMETER
volts
A. C.
- +
T O METER
Figure 4.
Schematic Diagram of Apparatus
3. A gas stream of known carbon dioxide concentration is passed continuously through the absorption cell a t a rate less than about 500 cc. per minute (see discussion). When equilibrium is reached the reading of per cent transmittance is referred to Figure 3 (which may be reconstructed from Table I if desired) t o obtain the value of pCOt/b. The value of b is then determined by dividing the known pCOz by the theoretical pC02/b. As b varies with temperature, the temperature of the solution should be noted. 4. The gas stream of unknown carbon dioxide concentration is then passed continuously through the cell. With the knowledge of b obtained by the single-point calibration described in step, 3 Figure 3 relates per cent transmittance to pCO2. If the temperature of the solution has changed from its value during calibration, a correction may be applied according t o the variation of b with temperature as illustrated in Figure 6. For example, if the temperature is 16” C. during the calibration and 26’ C. during the determination, b will change by a ratio of 0.7/0.5 according to Figure 6. The actual carbon dioxide concentration will then equal the apparent carbon dioxide concentration multiplied by this ratio. EXPERIMENTAL
The values of 6 have been determined for a variety of bicarbonate concentrations and temperatures, by dividing the gas stream to be analyzed, and passing part through the absorption cell and part through a suitable alkali absorption column. The volume of gas passing through the column n-as measured in a wettest meter. Titration of the excess alkali in the presence of barium chloride to a phenolphthalein end point permitted calculation of the concentration of carbon dioxide. A convenient source
Table I. Calculation of Theoretical pCOz/b According to Equation 4 for i = 0.900 Actual
% Transmittance 14.8 20 25 30 40 50 60 70 80 85 90 100 (I
Theoreticala
% Transmittance 12.6 18.8 24.2 29.8 40 50 60 70
8n ._ 85 90 100
pCOi/b 0 0.240 0.460 0.710 1.26 1.99 .. ~
3.05 4.80 8.27 11.67 18.55 0
Corrected for Beer-Lambert law deviation according t o Figure 4.
PHOTOCELL PERFORATED
Figure 5 .
Schematic Diagram of Absorption Cell
To check the validity of the method, point determinations of carbon dioxide pressure were made by the alkali absorption method on gas streams of various concentrations passing through the analyzer cell. The values obtained were divided by b as determined from Figure 6 according to the measured temperature and the bicarbonate concentration used in the cell. The resulting independent determinations have been plotted for comparison on Figure 3 against the observed per cent transmittance of the absorption cell. The standard deviation of 18 determined points from the theoretical curve is 2.1 parts per hundred. DISCUSSION
Consideration of Figures 3 and 6 shows immediately the wide range over which carbon dioxide may be determined by this method. Figure 3 shows that pCOp/b may be varied from 0.6 to 12 with approximately the same percentage accuracy throughout, since the calibration is nearly linear on a semilog plot in this range. Figure 6 shows that b may be varied from approximately 0.001 for 0.001 N bicarbonate to approximately 0.01 for 0.01 K bicarbonate. Within this range of concentrations pCOz may be determined a t levels from 0.06 to 12%. A single calibration point is sufficient to define this entire range for one solution temperature, and the variation Rith temperature is shown in Figure 6. If pressures of carbon dioxide higher than about 0.12 atmosphere are to be determined, it is only necessary to employ a bicarbonate solution of higher concentration in the absorption cell. Because the bicarbonate activity will be lower than its normality in this range, extrapolation of b from other normalities will not be possible and a further one-point calibration Kill be necessary. The use of concentrations of bicarbonate loxer than 0.001 LV a1lo-m extension of the method to
ANALYTICAL CHEMISTRY
1544
concentrations of carbon dioxide below 0.0670. Certain difficulties are encountered a t very 1011- concentrations, however. Experiments with 0.0001 II' bicarbonate solution indicate that the value of b increases markedly over periods of several hours presumably because of the dissolution of glass from the bottle or absorption of alkaline reacting gas from the atmosphere. The rate a t which the gas stream is passed through the absorption cell may be varied over a wide range. At rates above about 500 cc. per minute bubbles from the sparger begin to interfere slightly with the light transmittance and undue quantities of solution may be lost by entrainment and evaporation. The loner limit is determined by the rate of approach to equilibrium that is desired. For a gas rate of about 200 cc. per minute equilibration of the system is rapid, For an instantaneoils change in carbon dioxide pressure the approach to equilibrium behaves as a first order reaction mith a time constant of 0.5 min. -1 This indicates that essentially complete equilibrjuni is reached mithin 10 minutes of a sudden change, and that the usual gradual changes are folloaed very exactly.
!HCOji 1.0
.
0.9
-
0.8
-
0.i
-
0.6
-
0.5
-
0.41
IO
pCO2 refers to apparent pCOz b refers to the initial value This equation is derived directly from Equation 4. It is plotted in Figure 7 for more convenient use. The parameter was converted from pCOr/b as indicated in the equation to per cent transmittance by use of Figure 3. The correction is expressed both as 6,actual pCO?/apparent C 0 2 ,and as an addition to the observed per cent transmittance. The relation between these two is derived from the straight portion of the theoretical curve in Figure 3, and the latter is therefore valid only in the range of 30 to 8 0 7 transmittance. When it is desired to correct for continuous evaporation over a long period it is assumed for convenience that the evaporation occurred a t a constant rate between measurements. For eeveral reasons it is necessary that the instrument be checked at intervals during a continuous determination. The cell should be filled periodically to correct for evaporation. Furthermore, since operating temperature affects constant b, variations in ambient temperature during continuous operation will have an effect upon the calibration. For this reason the buffer temperature should be read periodically if room temperature fluctuates appreciably. Thirdly, although the drift of the photocell is inappreciable after a warm-up period of 1 or 2 hours, ambient temperature changes ill have an effect upon its reaction, thus causing a change in air blank, n-hich must be reset to its originally observed value. The attention required to correct for these variations, in liquid level, buffer temperature, and air blank, is needed after varying lengths of time dependent upon the accuracy desired and the operating conditions, but should not be necessary in any case before 12 or more hours of operation, provided interpolated corrections are applied for the interim period. With these adjustments periodically made, the apparatus should he operable in a continuous manner indefinitely.
1. E
1
'
:4
"
ia
"
22
'
'
1
26
30
34
-
11
P E R C E N T TRANSMITTANCE
10
*
'
38
T E M P E R A T U R E - ' Centigrade Figure 6. Effect of Temperature on Calibration 0 0.01 N bicarbonate, apparent (HCOr) = 0.00933
A 0.005 N bicarbonate, apparent (HCOr) = 0.005 X 0.001 .V bicarbonate, apparent (HCOI-) = 0.001
Because the temperature of the solution in the absorption cell during operation is slightly above room temperature, a certain amount of water ordinarily evaporates into the gas stream. For unusual conditions or for long periods of operation this may result in a considerable increase in the concentration of indicator and of bicarbonate. The resultant change in constants i and b of Equation 4 will cause a change in calibration. T o correct for this it is merely necessary to add water to the absorption cell up to the originally marked level. If i t is desired, however, to determine carbon dioxide during a period when evaporation has occurred-for example, when the instrument has been operating nith a recording meter for a long period without attentionit is possible to make correction n i t h the following relation:
where k = actual pCO?/apparent pCO? v = volume of solution in the absorption cell after evaporationhnitial volume
P E R C E N T OF ORIGINAL VOLUME IN ABSORPTION C E L L
Figure 7 .
Correction Curves for Evaporation of Water from Absorption Cell
The measured standard deviation in this method of 2.1 parts per hundred corresponds to a deviation of 10.5in per cent transmittance. The measurement of pH bl- this method and Kith this error mould be accurate to izO.01 pH unit. Since a potentiometric pH measurement with this degree of accuracy is difficult, photometric method appears to be more precise. The main sources of error appear to lie in the method used for calibration and in fluctuations in the light source. Improvements a t both points v-ould undoubtedly be possible if i t were desired. Accuracy to 0.1% transmittance would result in only 0.4% error in carbon dioxide. The adoption of this method by other laboratories should be
V O L U M E 24, N O . 10, O C T O B E R 1 9 5 2 easily made. The value of constant i is determined in the apparatus itself and is, therefore, easy to reproduce. The value of b is determined a8 described under "Operation and Calibration," m d depends for its reproducibility upon reproducible preparation of the absorption cell. Values for the desired buffer concentrations should therefore be determined by the individual analyst. This involves one calibration point far each buffer concentration. The degree of variation of b with temperature ehould not differ markedly from that found here, but if wide temperature fluctuations a ~ eencountered it u.ould be simple and advisable for the to determine at temperatures and construct 8. graph such 8 s Figure 6 for his oun use.
WJU
nf VI
1545 LITERATURE CITED
( 1 ) Brown, E. H., and Felger, M. M., IND.ENG.CXBX.,ANAL.ED..
17,283 (1945). L.r and w. M., J . Am. '' h e n . Sac., 39, 68 (1917). (3) Wilson, p, w,, orcutt,F. s,, s,,d peterson,w'. H., IND. ENG. CHEM.,ANAL.ED.,4,357 (1932). (4) winder, R. J., and Baumberger, J. P.,I b d . , 11, 371 (1939). (')
Higglns,
R E C E W ~for ~ review J~~~ 4. 1952. Accepted August 4. 1952 Pre.mSa i n Dart before the Division of Agrieult,ural and Food Cherniatry, Bioengineering SYrnPosiurn, &tthe lZlst Meeting of AhlERlCAN C A E M ~ C A S Lo n ~ ~ u , Milwaukee, Wis. Published with the approval of the direotor of the U'is. conrin Aerieultural Experiment Ststion. Suworted in part by s grant from the Red Star Yeast and Products Co., ?diiasnkee, Wis,
Micrometer Baly Cells with Beckman and Cary nmot nrc Ultraviolet Spect rnnhnt upllu~u,l~~L~~a 11111
I
ROBERT C. HIRT AND FRANK T. KING Str imford Research Laboratories, American Cyanamid Co., The practice of varying .~ cell length rather than concentration in the production of ul traviolet absorption spectra offerspreater meed. the convenience of fewer manipulations, the avoiding of Beer's law deviations on dilution, and extension rv snorter wave lengths. The choice of e l l lengths available to the ultraviolet sueotrosmpist is extended helow 5 mm. to a few hundredths of a millimeter by the use of Hilger miemmeter Raly cells in adapters built for their use with the Beckman and Cary spectrophotometers. The aeouracy of these cells is sufficient oal purposes except at very sh,mt lengths, and even these lengths may th due care. Attention is al so called to insert-type cells, and to a meter cell designed for use wiith infrared spectrometershut adaptable let instruments. ~~
-.
~ . ~ "_. " . ~..". " +ectrophotometry appears to 1hive become bound up with the I-em. length absorption cell. I
11
This impression is gained from articles appearing in the literature taday,where thevastmajorityof mtharsoitetheuseof 1-cm.cells, and indeed many do not mention the cell length used for their measurements, apparently assuming that the reader will know I-cm. cells were used. This tradition of using 1-em. cells has probably arisen because these are the cells furnished with the Beckman spectrophotometer when purchased, i t is expensive to
Figure 1. Hilger Micrometer Baly Cells in Adapter for Beelcman Speotmphotometer
purchase a variety of cells, and it is convenient to have the cell length in Beer's law calculations be unity (when the concentration is expressed in grams per liter). If the analyst intends to m,ork with very dilute solutions, he may obtain an accessory permitting the use of cells up to 10 cm. in length. For work with samples of very limited volume, specialized cells (9) and holders are available (from Pyrocell Mfg. Co., 207 East 84th St., New York 28, N. Y., and Microchemical Specialties, 1834 University Ave., Berkeley 3, Calif.). This paper points out certain advantages to the spectroscopist in varying the cell length rather than the concentration, Before advent of the Beckman instrument and of fused allquartz cells, many different styles of cells of various lengths were used. Most were of a demountable type, with tubes, gaskets, threaded rings, and clamps of many types. Vsiablble-length cells of the B d y plunger type and an assortment of demountable cells were very convenient for producing absorption spectra on photographic instruments, such as the Hilger Spekker photometer. A modification of the Baly cells which used a micrometer to determine the cell length was useful in preparing plates with stepped exposures and for obtaining densities in the optimum range for quantitative measurements. These cells have been briefly described and pictured by Lothian (8), Brode (S), and Harrison, Lord, and Loofbourow (7). They are manufactured by Hilger and Watts, Ltd., and sold through Jarrell-Ash Co., 165 Newbury St., Boston 16, Mass., as micrometer liquid cell H 436. The micrometer cells had proved 60 useful in these labaratories that, shortly after a Beckman Model DU was acquired in 1944, an adapter was built to permit the use of these cells. The cells, and this adapter, are shown in Figure 1. The housing resembles that supplied by Beckman for the accommodation of cells up to 10 cm., and the cells are moved back and forth, into and out of the beam from the manacbra-
