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An improved method for microrespirometry using gas chromatography
Soil Biol. Biochem. Vol. 5, pp. 271-274. Pergamon Press 1973. Printed in Great Britain
SHORT COMMUNICATION An improved method for microrespirometry using gas chromatography M. J. MITCHELL Department of Biology and ~nviro~ental Sciences Centre (KanaRaskis), The University of Calgary, Calgary, Aberta, Canada (Accepted 30 June 1972)
IN SOIL zoology much attention is being given to energetics studies since they give fresh
insight into the functioning of the soil community. However, problems have been encountered in the measurement of various energetic parameters, of which respiration is one of the most important. Up until now the only generally available method for the measurement of microrespiration of mesofauna has been the Cartesian diver. Zeuthen (1964) gave an account of this method, the main disadvantages of which are the confmement of the animal under experiment in a very small space, the necessity of a constant temperature environment, the absence of carbon dioxide in the atmosphere, and the great skill needed for operation. The main advantage of the diver is its sensitivity, detecting oxygen uptake as small as lob6 pi/h (Petrusewicz and Macfadyen, 1970). To overcome some of the problems of the diver, a new method has been developed for microrespirometry utilizing gas chromatography. Wood, Wood and Dickinson (1970) and Tadmor, Applebaum and Kafir (1971) have described methods in which gas chromatography has been used in respiration studies of small insects. Using a thermal conductivity detector they were able to detect COz production at the 10-l pi/h level. This sensitivity is not adequate for the soil mesofauna. To increase sensitivity a helium ionization detector was used. This detector is highly sensitive for the analysis of permanent gases; Hartmann and Dimick (1966) using a 10 ml sample of COz in He demonstrated a minimum detectability of 10m6~1 COz. A He ionization detector was incorporated into a Bendix 21 IO-S-(R):gas chromatograph (Bendix Corp., Ronceve~e, W. Va.). Matheson ultrapure He was used as the carrier gas at a flow rate of 60 ml~min. The CO, was separated from the other gaseous components with a 3 mm x 4 m stainless steel, 50/80 mesh, Porapak “Q” column. Separation took about 1 min using the following conditions: inlet temperature 8O”C, column temperature 55”C, and detector temperature 80°C. The detector response was recorded on a Honeywell 1.0 mV Lab/Test recorder. All gas samples were introduced into the gas chromatograph via the injection port using a Hamilton gas-tight syringe (Hamilton Company, Whittier, Cal.). A calibration curve for CO, was provided by injecting known concentrations (Primary Standard, Matheson Gas Products, Whitby, Ont.) of CO2 at different volumes and the responses were measured from recorded peak heights. The concentration of the COz at a fixed volume can then be plotted against the peak height. An example of such a curve is given in Fig. 1 which demonstrates the relationship between the log of peak height (detector response) and the log of concentration. Hartmann and Dimick (1966) have shown this relationship to be linear over a dynamic range of 10,000 fold. 271
272
SHORT COMMUNICATION
-z ” Attenuation
x 50 2
(Each
point
of least
represents 4
the mean
of
-
2.54
injections)
‘3 S! 3: w Il.
Correlation
Analysis:
r = 0.990
Regression
Analysis:
log (Peak
Height,
in.) =
log Gmcentro110n, x 1.04335logrmlc.)
0.1 L 100
I
t
I
r11111
=
parts /IO61 1.70297
I
1.04335
I
I
1,000 CONCENTRATION
-
log(PH.)+1.70297
I1111
10,000 ( parts
/ IO61
FIG. 1. Calibration curve for COZ (50 pl as sample).
Any suitable vessel could be used for respiration, with the size being dependent on the size of the animal. In the present study Kontes Micro-Flex Tubes K-74400 (Kontes Glass Company, Berkeley, Cal.) were employed with an approximate volume of O-7 ml. The volume was calibrated for each individual tube using mercury in a gravimetric procedure. A septum used for the removal of gas samples was cut from a cap of natural gum rubber (“Sani-Tab” Caps, Davol Rubber Company, Providence, R.I.), placed over the mouth of the tube, and was secured by means of the screw cap. This type of septum showed no detectable leakage of CO2 over a period of 3 days. The animal (or animals) was placed in the container together with a small piece of filter paper saturated with water to maintain a high relative humidity. The volume of the filter paper was subtracted from the calibrated volume of each vessel, assuming a density of 1 g/cm3 for filter paper of known dry weight. For determining respiration rates the animals were placed in the respiratory vessels and the time noted. Also a sample of atmospheric gas was sealed off in a 1 1. flask with a natural gum rubber cap. As many vessels can be set up as can be conveniently handled, assuming a measurement time of 2 min per vessel per sampling period. The respiratory vessels were then placed in the temperature regime (either constant or fluctuating as required by the experiment). At given intervals of time a sample of gas was removed from the vessel using a gastight syringe and injected into the gas chromatograph. The amount of gas removed should be small in relation to the vessel volume, and in these experiments was less than 7 per cent. A sample of equal volume of gas was returned to the vessel from the 1 1. flask to allow for the depletion of gases. It was assumed that the removal of gas resulted in negligible error in this procedure; however, knowing the concentration of gas removed and added to the vessel it would be possible to correct for this error. At the conclusion of the experiment the peak height measurements were converted to
273
SHORT COMMUNICATION
CO2 concentrations using the regression equation calculated from the calibration procedure (Fig. 1). It is felt that one of the main sources of error in the present method was in the measurement of peak height using a visual method, but this could be overcome by the use of a digital integrator. The concentration was converted to gas volume by multiplying by the appropriate vessel volume. The gas volume was plotted against time to give a graphical representation of the respiration, such as is given in Fig. 2.
-
Vessel No.5 5 indtviduais
-
No. 6
0.6
0.4
0
4
8 TIME
FIG. 2. Respiration
12
16
20
(h)
of Liucarus sp. (CO2 production).
To determine the actual respiration rate the portion of the curve which is linear is used in a regression analysis, the slope of the curve being the respiration rate. The results of one such experiment using Lirzcarus sp. (Atari, Oribatei) are given in Table 1. Since the determined values are in COZ output, the O2 consumption is also given assuming a respiratory quotient of 0.82, which is considered to be average (Petrusewicz and Macfadyen, 1970). Gas chromatography can also be used in the measurement of O2 uptake although a smaller percentage of change of gas must be detected (Tadmor et al., 1971), but because of measurement limitations in the present technique it has not been attempted for the mesofauna although preliminary testing with macrofauna has shown this to be feasible. This technique enables not only the respiration rate of the mesofauna to be determined, but also information can be gathered on the effect of fluctuating temperatures and various atmospheric constituents including carbon dioxide. ~eknu~Ze~g~menfs_Th~ author is deeply indebted to Dr D. PARKINSCIN and Dr G. PRITCHARDfor helpful discussions and for aid in the preparation of the manuscript. I am also grateful for the advice of Dr M. H. BENN and Dr J. B. CRAGG. The gas chromatograph was purchased under Negotiated Development Grant D-4 (N.R.C., Canada). During the course of this work the author was supported by an Izaak Walton Killam Memorial Scholarship.
274
SHORT COMMUNICATION TABLE1. RESPIRATION OF Liacarus sp. AT A CONSTANT TEMPERATURE OF 21°C
Vessel no. No. of animals/vessel Correlation coefficient (r) CO2 volume vs. time Respiration rate/vessel (6,.,)* (IO- 3 ~1 of CO,/h) S2Y.X S* Respiration rate/animal? (1O-J pl CO,/h) Respiration rate/animal: (1O-3 ~1 0,/h)
1 5
2 1
3 5
4 1
5 5
6 1
0.976
0.975
0.996
0.996
0.999
0.989
1.05 5.12
8.6 0.05 1.13
61.6 0.39 3.10
7.7 0.01 0.41
47.8 0.01 0.47
10.4 0.03 0.89
7.9
8.6
12.3
7.7
9.6
10.4
9.6
10.4
15.0
9.4
11.7
12.7
39.4
* Computations are after Sokal and Rohlf (1969). Regression coefficient SZ,.& standard error of regression coefficient = &. t P = 9.4; s = 1.76; S.E. = 0.72. $ Assuming an R.Q. of 0.82.
= b,.,; unexplained
variance
=
REFERENCES HARTMANNC. H. and DIMICKK. P. (1966) Helium detector for permanent gases. J. Gas Chrom. 4,163-167. PETRUSEWICZK. and MACFADYENA. (1970) Productivity of Terrestrial Animals, Principles and Methods. IBP Handbook No. 13. Blackwell Scientific Publications, Oxford. SOKALR. R. and ROHLFF. J. (1969) Biometry: The Principles andpractice of Statistics in BiologicaI Research. Freeman, San Francisco. TADMORU., APPLEBAUMS. W. and KAFIR R. (1971) A gas-chromatographic micromethod for respiration studies on insects. J. exp. BioI. 54,437-441. WOOD G. W., WOOD F. A. and DICKINSONR. A. (1970) Carbon dioxide output of individual small insects measured with a gas chromatograph. Can. J. Zool. 48,902-903. ZEUTHENE. (1964) Microgasometric Methods: Cartesian Divers. In Second International Congress of Histoand C;vto-Chemistry (T. H. Schiebler, A. G. E. Pearse and H. H. Wolff, Eds), pp. 70-80. Springer, Berlin.
SHORT COMMUNICATION An improved method for microrespirometry using gas chromatography M. J. MITCHELL Department of Biology and ~nviro~ental Sciences Centre (KanaRaskis), The University of Calgary, Calgary, Aberta, Canada (Accepted 30 June 1972)
IN SOIL zoology much attention is being given to energetics studies since they give fresh
insight into the functioning of the soil community. However, problems have been encountered in the measurement of various energetic parameters, of which respiration is one of the most important. Up until now the only generally available method for the measurement of microrespiration of mesofauna has been the Cartesian diver. Zeuthen (1964) gave an account of this method, the main disadvantages of which are the confmement of the animal under experiment in a very small space, the necessity of a constant temperature environment, the absence of carbon dioxide in the atmosphere, and the great skill needed for operation. The main advantage of the diver is its sensitivity, detecting oxygen uptake as small as lob6 pi/h (Petrusewicz and Macfadyen, 1970). To overcome some of the problems of the diver, a new method has been developed for microrespirometry utilizing gas chromatography. Wood, Wood and Dickinson (1970) and Tadmor, Applebaum and Kafir (1971) have described methods in which gas chromatography has been used in respiration studies of small insects. Using a thermal conductivity detector they were able to detect COz production at the 10-l pi/h level. This sensitivity is not adequate for the soil mesofauna. To increase sensitivity a helium ionization detector was used. This detector is highly sensitive for the analysis of permanent gases; Hartmann and Dimick (1966) using a 10 ml sample of COz in He demonstrated a minimum detectability of 10m6~1 COz. A He ionization detector was incorporated into a Bendix 21 IO-S-(R):gas chromatograph (Bendix Corp., Ronceve~e, W. Va.). Matheson ultrapure He was used as the carrier gas at a flow rate of 60 ml~min. The CO, was separated from the other gaseous components with a 3 mm x 4 m stainless steel, 50/80 mesh, Porapak “Q” column. Separation took about 1 min using the following conditions: inlet temperature 8O”C, column temperature 55”C, and detector temperature 80°C. The detector response was recorded on a Honeywell 1.0 mV Lab/Test recorder. All gas samples were introduced into the gas chromatograph via the injection port using a Hamilton gas-tight syringe (Hamilton Company, Whittier, Cal.). A calibration curve for CO, was provided by injecting known concentrations (Primary Standard, Matheson Gas Products, Whitby, Ont.) of CO2 at different volumes and the responses were measured from recorded peak heights. The concentration of the COz at a fixed volume can then be plotted against the peak height. An example of such a curve is given in Fig. 1 which demonstrates the relationship between the log of peak height (detector response) and the log of concentration. Hartmann and Dimick (1966) have shown this relationship to be linear over a dynamic range of 10,000 fold. 271
272
SHORT COMMUNICATION
-z ” Attenuation
x 50 2
(Each
point
of least
represents 4
the mean
of
-
2.54
injections)
‘3 S! 3: w Il.
Correlation
Analysis:
r = 0.990
Regression
Analysis:
log (Peak
Height,
in.) =
log Gmcentro110n, x 1.04335logrmlc.)
0.1 L 100
I
t
I
r11111
=
parts /IO61 1.70297
I
1.04335
I
I
1,000 CONCENTRATION
-
log(PH.)+1.70297
I1111
10,000 ( parts
/ IO61
FIG. 1. Calibration curve for COZ (50 pl as sample).
Any suitable vessel could be used for respiration, with the size being dependent on the size of the animal. In the present study Kontes Micro-Flex Tubes K-74400 (Kontes Glass Company, Berkeley, Cal.) were employed with an approximate volume of O-7 ml. The volume was calibrated for each individual tube using mercury in a gravimetric procedure. A septum used for the removal of gas samples was cut from a cap of natural gum rubber (“Sani-Tab” Caps, Davol Rubber Company, Providence, R.I.), placed over the mouth of the tube, and was secured by means of the screw cap. This type of septum showed no detectable leakage of CO2 over a period of 3 days. The animal (or animals) was placed in the container together with a small piece of filter paper saturated with water to maintain a high relative humidity. The volume of the filter paper was subtracted from the calibrated volume of each vessel, assuming a density of 1 g/cm3 for filter paper of known dry weight. For determining respiration rates the animals were placed in the respiratory vessels and the time noted. Also a sample of atmospheric gas was sealed off in a 1 1. flask with a natural gum rubber cap. As many vessels can be set up as can be conveniently handled, assuming a measurement time of 2 min per vessel per sampling period. The respiratory vessels were then placed in the temperature regime (either constant or fluctuating as required by the experiment). At given intervals of time a sample of gas was removed from the vessel using a gastight syringe and injected into the gas chromatograph. The amount of gas removed should be small in relation to the vessel volume, and in these experiments was less than 7 per cent. A sample of equal volume of gas was returned to the vessel from the 1 1. flask to allow for the depletion of gases. It was assumed that the removal of gas resulted in negligible error in this procedure; however, knowing the concentration of gas removed and added to the vessel it would be possible to correct for this error. At the conclusion of the experiment the peak height measurements were converted to
273
SHORT COMMUNICATION
CO2 concentrations using the regression equation calculated from the calibration procedure (Fig. 1). It is felt that one of the main sources of error in the present method was in the measurement of peak height using a visual method, but this could be overcome by the use of a digital integrator. The concentration was converted to gas volume by multiplying by the appropriate vessel volume. The gas volume was plotted against time to give a graphical representation of the respiration, such as is given in Fig. 2.
-
Vessel No.5 5 indtviduais
-
No. 6
0.6
0.4
0
4
8 TIME
FIG. 2. Respiration
12
16
20
(h)
of Liucarus sp. (CO2 production).
To determine the actual respiration rate the portion of the curve which is linear is used in a regression analysis, the slope of the curve being the respiration rate. The results of one such experiment using Lirzcarus sp. (Atari, Oribatei) are given in Table 1. Since the determined values are in COZ output, the O2 consumption is also given assuming a respiratory quotient of 0.82, which is considered to be average (Petrusewicz and Macfadyen, 1970). Gas chromatography can also be used in the measurement of O2 uptake although a smaller percentage of change of gas must be detected (Tadmor et al., 1971), but because of measurement limitations in the present technique it has not been attempted for the mesofauna although preliminary testing with macrofauna has shown this to be feasible. This technique enables not only the respiration rate of the mesofauna to be determined, but also information can be gathered on the effect of fluctuating temperatures and various atmospheric constituents including carbon dioxide. ~eknu~Ze~g~menfs_Th~ author is deeply indebted to Dr D. PARKINSCIN and Dr G. PRITCHARDfor helpful discussions and for aid in the preparation of the manuscript. I am also grateful for the advice of Dr M. H. BENN and Dr J. B. CRAGG. The gas chromatograph was purchased under Negotiated Development Grant D-4 (N.R.C., Canada). During the course of this work the author was supported by an Izaak Walton Killam Memorial Scholarship.
274
SHORT COMMUNICATION TABLE1. RESPIRATION OF Liacarus sp. AT A CONSTANT TEMPERATURE OF 21°C
Vessel no. No. of animals/vessel Correlation coefficient (r) CO2 volume vs. time Respiration rate/vessel (6,.,)* (IO- 3 ~1 of CO,/h) S2Y.X S* Respiration rate/animal? (1O-J pl CO,/h) Respiration rate/animal: (1O-3 ~1 0,/h)
1 5
2 1
3 5
4 1
5 5
6 1
0.976
0.975
0.996
0.996
0.999
0.989
1.05 5.12
8.6 0.05 1.13
61.6 0.39 3.10
7.7 0.01 0.41
47.8 0.01 0.47
10.4 0.03 0.89
7.9
8.6
12.3
7.7
9.6
10.4
9.6
10.4
15.0
9.4
11.7
12.7
39.4
* Computations are after Sokal and Rohlf (1969). Regression coefficient SZ,.& standard error of regression coefficient = &. t P = 9.4; s = 1.76; S.E. = 0.72. $ Assuming an R.Q. of 0.82.
= b,.,; unexplained
variance
=
REFERENCES HARTMANNC. H. and DIMICKK. P. (1966) Helium detector for permanent gases. J. Gas Chrom. 4,163-167. PETRUSEWICZK. and MACFADYENA. (1970) Productivity of Terrestrial Animals, Principles and Methods. IBP Handbook No. 13. Blackwell Scientific Publications, Oxford. SOKALR. R. and ROHLFF. J. (1969) Biometry: The Principles andpractice of Statistics in BiologicaI Research. Freeman, San Francisco. TADMORU., APPLEBAUMS. W. and KAFIR R. (1971) A gas-chromatographic micromethod for respiration studies on insects. J. exp. BioI. 54,437-441. WOOD G. W., WOOD F. A. and DICKINSONR. A. (1970) Carbon dioxide output of individual small insects measured with a gas chromatograph. Can. J. Zool. 48,902-903. ZEUTHENE. (1964) Microgasometric Methods: Cartesian Divers. In Second International Congress of Histoand C;vto-Chemistry (T. H. Schiebler, A. G. E. Pearse and H. H. Wolff, Eds), pp. 70-80. Springer, Berlin.