ANALYT

If. AI.

92,

BIOCHEMISTRY

A Sensitive

255-264

(1979)

Continuous and Discontinuous Photometric Determination of Oxygen, Carbon Dioxide, and Carbon Monoxide in Gases and Fluids

The

paper

describes

and discontinuous specific color

a sensitive,

rapid.

and

determination of OZ. reactions: O1 is determined

precise

COZ, by

photometric

and CO. its reaction

method

The

for the

method is with alkaline

continuous

based catechol

on

highly + Fe’&

yielding intensively colored products, CO, is determined by its color reaction with a solution of fuchsin + hydrazine: and CO is determined by its reaction with hemoglobin. The basic experimental equipment is that of the AutoAnalyzer (cf. Wolf, Zander. and Lang. 1976, A/)cr/.

Bir~c~/~cm. 74, 585).

vvith

the case of the discontinuous of I ml’min~ (STPD). the COL.

and

50 ppm

experimental 0.1 ~1 (STPD)

for

equipment for 0,.

CO.

an additional

analysis. lower limits The

chamber

discontinuous

plus an airtight 0.2 ~1 for CO,. and

analysis chamber. 0.1 ~1 for

Numerous gas analytical procedures are known for the determination of the concentration (or content) of 0,. CO,, and CO in gases and Ifluids. They are based on physior biological principles. cal, chemical, Among these, photometric methods (reviewed in Refs. l-4) are described, using the following indicator systems: Determination of 0, can be accomplished by the following methods: the chemically or photochemically reduced leuco-dyes such as indigo carmine, safranine red T. or methylene blue (5); the starch-iodine complex (6); copper (7); pyrogallol or catechol (8,9); Tris(dihydroxyphenanthroline)iron ( 10); ferrous hydroxide ( I I ); hydroquinone (12): nitric oxide ( 13); photometric modifications ofthe classical Winkler method (14); anthraquinone-2-suffonate (15); the Mn(IlI)-CyDTA complex (16,); N.N-bis-(2-hydroxypropyl)’ To

whom

correspondence

should

be sent.

for the

injection

Continuously analyzing of the sensitivities are of the The CO.

lower

of small

gas samples

in a standardized 50 ppm for 0,. three

gases limits

requires of

the

in

gas flow 100 ppm for the amounts

basic are

o-phenylenediamine (17). and fluorescence methods (18). Determination of CO, can be accomplished by the following: various pH-indicator systems (e.g., 19, 20, 21), fuchsine combined with hydrazine (22), turbidimetric methods (23). or other chemical indicators (24). However, most of these methods listed above show one or even more notable disadvantages, such as lack of specificity, high sample volumes. lack of applicability to gaseous and liquid samples, high operating costs, high costs of the apparatus, etc. Considering these numerous and somewhat serious disadvantages, the method of choice should meet the following requirements. (i) The method should be applicable to a great variety of different gases with a minimum of change in the equipment. (ii) The method should be sensitive and specific. i.e.. no cross sensitivity should occur between the different gases. 25s

0003.2697/79/020255-10$0’.00/0 C’op)nghf i 1979 by Acadrmx Prc\\. Inc All rtghtr of rrproductmn in aok form rrerved

256

LANG.

WOLF,

(iii) In order to minimize work. the calibration curve should be linear for each gas to be analyzed. (iv) The method should be applicable to gaseous and liquid samples. (v) The determination of extremely low amounts of gas (below 1 ~1) even in small sample volumes (5- 100 ~1) should be possible. Consequently, the method should be able to operate discontinuously-in the case of sample volumes too small for continuous measurement -and continuously, as well. (vi) The method should be reliable, i.e., the handling should be easy and feasible for untrained people. (vii) The readibility of the method should be high, preparation of the samples and measurement should be possible within a few minutes. (viii) The method should be cheap with respect to the costs of the equipment and operation. Recently, we have published a method for the continuous determination of 0, (25). In order to meet most of the requirements mentioned above, the original method was modified. In this modification, the sample is measured discontinuously by injecting it into a special airtight chamber (injection chamber), which is connected to a tube to the AutoAnalyzer system. The combined experimental equipment, including the injection chamber, provides the possibility to determine O,, COz, and CO continuously in gases or discontinuously in 5- to IOO-~1 samples of gases and liquids. MATERIALS

AND METHODS

Cherniccrls. Pyrogallol. catechol, NaOH, Na,CO,. Fe(NH,),(SO,),, fuchsin, and hydrazine were purchased from E. Merck, Darmstadt (reagent grade p. A.). The water was redistilled twice over quartz. Hemoglobin solutions were prepared from human blood. Standard solutions. Catechol: 25 mM cat-

AND

ZANDER

echo1 in 1.0 N NaOH; pyrogallol: 25 mM pyrogallol in 1.0 N NaOH; Fe(NH,),(SO,),: 5 mM Fe(NH,),(SO,), in water; fuchsin: 0.15 mM; hydrazine: 14.1 mM: the hemoglobin solution was prepared by a 1: 1000 dilution of human blood in distilled water; Na,CO, solution was prepared from Titrisol using distilled water free of carbon dioxide. The stock solution was stored under nitrogen. The NaOH and the water used for preparation of the standard solutions were thoroughly gassed by purified nitrogen, passed through an alkaline pyrogallol solution to remove traces of 0,. Tygon tubes for the proportioning pump were obtained from Ismatec, Ziirich. Stmdurd KLISCS.Standard gases with variable content of 0, and CO, were mixed from purified N,, O,, and CO,. They were analyzed by the method of Scholander (26) on the basis of 2-ml samples. Gases with known concentrations of CO in N, were purchased from Linde, Mainz. Equipment. As shown in Fig. 1, the experimental arrangement is that of the AutoAnalyzer, i.e., proportioning pump, mixing coil, flow cell, photometer, and recorder. These components are the basic device for continuous determination (cf. 25). Additionally, the apparatus contains an injection chamber (“injection equipment”) and an integrator for discontinuous determination. The different positions of the three-way valve in the cases of continuous and discontinuous determination are shown in the upper right of Fig. 1. In the case of the continuous determination, a stream of the gas to be analyzed is pumped into the mixing coil, in which it reacts with the color-developing reagents, The choice of the tube diameter depends on the concentration of the gas in the sample. In the case of the discontinuous determination, the gaseous or liquid sample is injected by means of a Hamilton syringe into the injection chamber. This chamber contains 4 to 5 ml of a fluid suitable for the elution of the gas to be analyzed (dis-

DETERMINATION

photometer

llnear recorder

OF 0,. CO,.

Integrator

i,, gas sample

AND

(cant

CO

1

257

4D

‘on,l”“o”s

CM.

valve

lnierttnn

“‘J--“-”

IL II

-

equipmen

uvnctn ,,I-

solution

2

FIG. I. Flsow diagram for the automated method for the continuous and discontinuous determination of 0,. CO?. and CO. The equipment, based on the AutoAnalyzer principle. consists of a special injection chamber. proportioning pump, debubbler, flow cell. photometer. recorder. and integrator. The special injection chamber and the integrator are used only in the case of discontinuous measurement. The different positions of the three-way valve for continuous and discontinuous analysis are shown in the upper right of the figure.

tilled water in the case of O.,, 0.01 N CH,.COO.H in the case of CO,). The gas is eluted by a stream of nitrogen which is finely distributed into small bubbles after passage of a glass filter, thus producing a large surface. A most rapid and complete elution is achieved when the chamber is small (total volume about 6 ml) and narrow. Generally, for transport of the nitrogen stream. a tube with a pumping capacity of 3.2 ml min--’ was used. In the experiments of the discontinuous determination, the peak area was integrated electronically. For 0, determination A = 490 nm, solution 1 is alk,aline catechol, and solution 2 is Fe(NH,),(SO,),: in the case of CO, A = 545 nm, solution 1 is hydrazine and solution 2 is fuchsin. and in the case of CO h = 420 nm, solutions I and 2 are hemoglobin from human blood diluted I: 1000 with distilled water. Alkaline catechol reacts in the presence of Fe”+ ions rapidly and quantitatively with 0, to yield deeply colored products. Fuchsin is decolored almost completely by hydrazine; the resulting faintly pink mixture

reacts with CO, yielding a red reaction product. RESULTS Corttinuom

AND DISCUSSION

Drtert,lincltion

The original recordings of the continuous determination of 0,. CO,, and CO are presented in Figs. 2A, B. and C. The baseline, produced by purified nitrogen (i.e.. in the absence of oxygen) is very stable. A fairly constant level of the absorbance is obtained within 1 to 2 min after the beginning of the addition of the sample. The original baseline is regained after a period of 2 to 3 min. The dependence of the absorbance on the concentration of oxygen and carbon dioxide is shown in Figs. 3A and B. The amount of the gas determined, given as microliter minute-’ , is calculated from the flow (milliliter.minute-‘) and the concentration of the gas in the sample (microliter.milliliter-I). In both cases, a strongly linear dependence is obtained by applying appropriate concentration ratios of the color-developing reagents. which especially in the case of CO,.

358

I>ANG.

1.0

WOLF.

AND

ZANDER

I\L

0.6

02

0 .

tL0 4

,

20

I

15 time

A


= 6.59.~ + 0.8 (n = 31, r = 0.994). The method described here provides a rapid and accurate tool for the continuous and discontinuous determination of O,, CO,. and CO in gases and liquids. Comparing other methods, which are generally used, several profound advantages become evident: the method is rapid, since a single analysis, independently. whether it is performed continuously or discontinuously, requires only 3 min. It is possible to measure at least three different gases(02, CO,, and CO) using the same experimental equipment. When different gaseshave to be measured, it is only necessary to change the gas-specific reaction solutions. This advantage is only met by some physical methods such as gas chro-

DETERMINATION

OF

0,.

CO,.

AND

261

CO

B 12

10

08

5 gas FIG.

3. Calibration

as a function of the trations of 0, in the with three (vertical),

curves

for

flow

x CO2

determination

concentration

and given

of CO,

concentration in Fig. 2B.

ml [ iiiii-

cone of O1 (A)

amount of 0, or CO,, i.e.. gas flow times three gases and the gas flow are given in

SD for absorbance (vertical), gases and the gas flow are and

continuous

15

10

and

x%iFq CO,

concentration. Fig. 2A. Mean

of 0, (horizontal). Mean values of five

(B).

representing

the

absorbance

Conditions: (A) The concenvalues of eight measurements

(B) The concentrations measurements with

SD

for

of CO, in the absorbance

(horizontal).

matography and mass spectrometry and chemical (manometric or volumetric) methods. such as the Van Slyke method. The method is highly sensitive and specific. Using a gas flow of 1 ml ‘rnin’ (STPD), the lower limits of the sensitivities are 50 ppm for O,., 100 ppm for CO,, and 50 ppm for CO in the case of the continuous analysis; the minimum amounts are approximately 0.1 ELI of O,, 0.2 ~1 of CO,, and 0.1 ~1 of CO (S’TPD) in the case of discontinuous analysis, based on an absorption difference of AE = 0.01 between sample and blank value. No cross reactivity occurs be-

tween 0, and CO,. which is especially important in the analysis of blood. The sample volume. which is sufficient for discontinuous analysis, is 5 to 10 ~1. In fact, this value is determined by the smallest volume of gas or liquid, which can be measured in a precision syringe, rather than by the sensitivity of the method. There are some other methods, which are suitable for measurement within the parts per million range (10,27-31), and even in the parts per billion range (32). However, with the exception of one method for the determination of dissolved O? in water (10) using a sample

LANG.

262

peak

absorbance

WOLF.

height [cm1

A

L

I

I

I

I

3

2 [mln]

1

0

time

ZANDER peak

absorbance IO

-

- 16

0.8

-

- 12

0.6 -

-0

0.4 -

-L

0.2 -

-0

-c

AND

height Ccml

0 2

I I B

I

I

I

3

2 [min]

1

time

1 0

FIG. 4. Typical original registration of discontinuous determinations of 0, (A) and of CO, (B). representing the absorbance as a function of time. The additional injection chamber used in these experiments is shown in Fig. I. Conditions: (A) Flows and reaction solutions as given in Fig. 2A. Peak I. IO ~1 of air; peak 2. 100 ~1 of distilled water. saturated with 0, at 37°C; peak 3. 5 ~1 of air. (B) Flows and reaction solutions as given in Fig. 2B. Peak 1. 8 ~1 of CO, (=7.22 ~1 of CO,. STPD): peak 2.30 ~1 of IO mM Na,CO,, (equivalent to 6.68 ~1 CO,. STPD).

of 1 ml, most of the methods require sample volumes between 60 ml and 20 liters (27-32, cf. 33). It is obvious that a linear correlation between the absorbance or the peak area and the gas concentration or the amount of gas is of great advantage, since a linear calibration curve leads to a minimum of work for calibration. The method described here shows a linear dependence of the absorbance both for O2 and CO, in contrast to other photometric methods (10,20,34,35).

In most of the other analytical procedures for the determination of O,, a major problem arises from the fact that atmospheric 0, migrates into the apparatus, thus markedly interfering with the experimental results. In the case of the method proposed here, any 0, constantly diffusing into the apparatus is eliminated from the results of the analyses, since the 0, blank value is automatically suppressed by the baseline. The discontinuous measurement of 0, and CO, is possible in gases and in liquids

FIG. 5. Calibration curves for the discontinuous determination of 0, (A) and CO, (B) using the injection technique, representing the peak area (arbitrary units) as a function of the amount of gas injected. Conditions: (A) Forty-four samples of 2 to 12 PI of air (0). and 31 samples of 10 to 100 PI of distilled water (Cl). which had been saturated with IOO’X 0, at 37°C (solubility coefficient = 0.0241 ml of O,.ml-‘.atm-‘). were injected into the chamber containing distilled water. Identical straight lines were obtained for both gaseous and dissolved 0,. All amounts of 0, shown are reduced to STPD. (B) Thirty-five samples of gaseous CO, (0). corrected to STPD conditions. and 38 samples of 10 mM Na*CO:, (0. 1.00 ~1 equivalent to 0.223 ~1 of CO,, STPD) were injected into 0.01 K acetic acid. N? flow = 3.2 ml.min-I (purple-orange tube), [fuchsin] = 0.118 mM (final concentration, purple-purple tube), [hydrazine] = 3 mM (upper curve). [hydrazine] = 4 mM (lower curve. in both cases final concentrations. white-white tube). The straight lines for gaseous CO, and N&O, are identical in both cases of different hydrazine concentrations.

DETERMINATION

A

OF 0,.

amount

CO,.

of 02

AND

Injected

CO

[,ul]

a0

20

0

2

B

6

L amount

of CO2

a Injected

[,uI]

IO

LANG.

264

WOLF.

without any variation ofthe apparatus, since both the gaseous and the liquid samples are injected into the same type of chamber. In the case of physical methods, a special extraction chamber is necessary for the analysis of liquid samples (e.g., 36). In comparison to other methods, especially gasometric methods, the handling of the experimental equipment is extremely easy. It can even be done by an untrained person. Furthermore, the simplicity of the equipment leads to a high reliability of the system. Since the complete apparatus consistswith the exception of the extraction chamber, which can be built easily by any glassblower-of components normally present in every laboratory, and since the operating costs (especially of the chemicals used) are low, the method may be run with a minimum of financial effort. The method is applicable to the determination of other gases. provided that sensitive color reactions are available. This work is under investigation. Until now, the method has been applied successfully to problems of blood gas analysis, e.g., the measurement of 0, and CO, concentrations in a lo-p.1 blood sample (37). These data will be presented in detail in a forthcoming article (Zander, Lang, and Wolf, submitted). REFERENCES 1. Lange. B. (1956) Kolorimetische Analyse. 5th ed.. Verlag Chemie. WeinheimiBergstrasse. 2. Marczenko. Z. (1976) Spectrophotometric Determination of Elements, Wiley. New York. 3. Charlot. Ci. (1964) Calorimetric Determination of Elements. Elsevier. Amsterdam/New York. 4. Snell. F. D.. and Snell. C. T. (1949) Calorimetric Methods of Analysis. Van Nostrand. Toronto. 5. Hamlin. P. A.. and Lambert. J. L. (1971) AIIN/. Chcrlr. 43, 618-620. 6. Pieters. H. A. J.. and Haussen. W. J. (1948) Am/. Chim.

Acfo

2, 712-726.

7. Brooks, F. R.. Dimbat, M., Treseder, R. S., and Lykken, L. (1952) Ancrl. Chcm. 24, 520-524. 8. Binder. K.. and Weinland, R. F. (1913) Brr. Derct. Chrm. Ges. 46, 255-259.

AND

ZANDER

9. Williams. D. D.. Blachly. C. H.. and Miller. R. R. (1922) Antrl. Clrc,n. 24, 1819-1821. IO. Poe. D. P.. and Diehl. H. (1974) Ttrltrtrrtr 21. lO651071. II. Exton, W. G.. Schattner. F.. Kormann, S.. and Rose. A. R. ( 194513. Ltrh. C/it?. Med. 30,84-95. I?. Winkler. L. W. ( I91 I) 2. Atr,qcw. Chc,m. 24, 34 I 343. 13. Sheaff. H. M. (1922) J. Biol. Chum. 52, 35-50. 14. Fadrus, H.. and Maly, J. (1971) Antrlyst 96, 59l597. Stafford. C.. Puckett. J. E.. Grimes. M. D.. and Heinrich. B. J. (1955) Aud. Clrc,r~r. 27, 20122014. 16. Sastry. G. S.. Hamm, R. E.. and Pool. K. H. (1969) Awl. Clwm. 41. 857-858. 17. Ostrowski. S.. Zasinska, Z.. and Zolendziowska. Z. (196512. P/r(lr/?(. Po/.\!+I 21, 743-746(Chrrrr. Ah.str. 65, 1959 a (1966)). 18. Longmuir, I. S.. and Knopp, J. A. (1973) irr Advances in Experimental Medicine and Biology (Bicher. H. I.. and Bruley. D. F.. eds.) Vol. 37A. pp. 55-57. Plenum. New York. 19. Hales. J. R. S.. Little. A.. and Webster, M. E. D. ( 1966) Ano/. Rioc~hc,/u. 16, 114-I IS. 20. Maxon. D. W.. and Johnson. M. J. (1952) Atrtrl. Ghc/~. 24, 1541-1545. ?I. Skeggs. L. T. (1960) Awr. J. Clir~. Pnthd. 33, 181-185. 22. Dubrisay. R.. and Gion. L. (1934) Proc. 14’ Congres de Chimie Industrielle, pp. 582-583. 23 Roller. P. S.. and Ervin, G. (1939) Irrdus/r. Enp. Chc~ur. A,rtr/. Ed. 11, 150-13. 24 Exton. W. G.. Schatter. F., and Rose. A. R. ( 1941) Amer. J. C‘li~. Patho/. 11, 632-642. 25. Wolf. H. U.. Zander. R., and Lang. W. (1976) Antrl. Bi~~hcrrr. 74, 585-591. 26. Scholander, P. F. (1947) J. Bide/. C‘hcm. 167, 23% 250. 27. Needleman. M. 11959) Atrcc/y.st 84, 720-735. 28. Sweetser. P. B. (1967) Autrl. Chem. 39, 979-982. 29. Pepkowitz, L. P.. and Shirley, E. L. I 1953) Antrl. c‘hem. 25, 1718- 1720. 30. Winslow. E. H., and Liebhafsky, H. A. (1946) f+ duvtr.

31. Silverman, 32. 33. 34. 35. 36. 37.

Eng.

Chrm.

L..

Antrl.

Ed.

18, 565-568.

and Bradshaw. W. (19561 A~cll. Chirtr. At fcr 14, 514-526. Buchhoff. L. S.. Ingber, N. M.. and Brady. J. H. (1955) Aucrl. C-hem. 27, 1401-1404. Potter, E. C. (19.57) J. Appl. Ckcru. 7, B-297. Spector. N. A.. and Dodge. B. F. (1947) Atrc~l. Chc~m. 19, 55-58. Brady. L. J. (1948) Autrl. Chrm. 20. 10331037. Albers. C.. and Fdrhi. L. E. (19651 Z. E.rp. .Mcd. 139,485-505. Zander, R.. Lang. W.. and Wolf. H. U. (1976) Pflii,gcr’s AK/I. 365, R 17.