Butler, Patrick J.

Martin, Graham R.

Portugal, Steven J.

White, Craig R.

Respirometry is a standard technique in comparative physiology laboratories, with measurements of rates of O(2) consumption and CO(2) production available for hundreds of species. A common recommendation when carrying out respirometry is that water vapor be removed from the air stream before entering the analyzer. Often, this is accomplished with the use of chemical desiccants, such as Drierite. However, here we show that Drierite has an affinity for CO(2) when new and completely anhydrous, and therefore it has an adverse effect on the washout characteristics of this gas. Exposing the Drierite to room air reduces this CO(2) affinity, and a 2-min exposure at 20 degrees Celsius and 50% relative humidity is sufficient to reduce the 99% washout time by almost a factor of 4, at the cost of only a 5% reduction in water vapor capacity. When Drierite is exhausted and then recharged according to the manufacturer's instructions, the CO(2) affinity is further reduced, and washout times are less than 60% greater than when no desiccant is used

White, Craig

Portugal, Steven

Martin, Graham

Butler, Patrick

University of Birmingham Research Portal

Respirometry: Anhydrous Drierite equilibrates with carbon dioxide and increases washout times

University of Queensland eSpace

Division of Comparative Physiology and Biochemistry, Society for Integrative and
Comparative Biology
Respirometry: Anhydrous Drierite Equilibrates with Carbon Dioxide and Increases Washout
Times
Author(s): Craig R. White, Steven J. Portugal, Graham R. Martin, and Patrick J. Butler
Source: Physiological and Biochemical Zoology, Vol. 79, No. 5 (September/October 2006), pp.
977-980
Published by: The University of Chicago Press.  Sponsored by the Division of Comparative
Physiology and Biochemistry, Society for Integrative and Comparative Biology
Stable URL: http://www.jstor.org/stable/10.1086/505994 .
Accessed: 04/11/2015 22:30
Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .
http://www.jstor.org/page/info/about/policies/terms.jsp
 .
JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of
content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms
of scholarship. For more information about JSTOR, please contact support@jstor.org.
 .
The University of Chicago Press and Division of Comparative Physiology and Biochemistry, Society for
Integrative and Comparative Biology are collaborating with JSTOR to digitize, preserve and extend access to
Physiological and Biochemical Zoology.
http://www.jstor.org 
This content downloaded from 23.235.32.0 on Wed, 4 Nov 2015 22:30:59 PM
All use subject to JSTOR Terms and Conditions
977
T E C H N I C A L C O M M E N T
Respirometry: Anhydrous Drierite Equilibrates with Carbon Dioxide
and Increases Washout Times
Craig R. White*
Steven J. Portugal
Graham R. Martin
Patrick J. Butler
Centre for Ornithology, School of Biosciences, University of
Birmingham, Edgbaston, Birmingham B15 2TT, United
Kingdom
Accepted 3/2/2006; Electronically Published 8/16/2006
ABSTRACT
Respirometry is a standard technique in comparative physi-
ology laboratories, with measurements of rates of O2 con-
sumption and CO2 production available for hundreds of spe-
cies. A common recommendation when carrying out
respirometry is that water vapor be removed from the air stream
before entering the analyzer. Often, this is accomplished with
the use of chemical desiccants, such as Drierite. However, here
we show that Drierite has an affinity for CO2 when new and
completely anhydrous, and therefore it has an adverse effect
on the washout characteristics of this gas. Exposing the Drierite
to room air reduces this CO2 affinity, and a 2-min exposure at
20C and 50% relative humidity is sufficient to reduce the 99%
washout time by almost a factor of 4, at the cost of only a 5%
reduction in water vapor capacity. When Drierite is exhausted
and then recharged according to the manufacturer’s instruc-
tions, the CO2 affinity is further reduced, and washout times
are less than 60% greater than when no desiccant is used.
Introduction
Respirometry (the measurement of rates of oxygen consump-
tion, , and carbon dioxide production, ) has been a˙ ˙Vo Vco
2 2
standard technique in comparative physiology laboratories for
decades, with and measurements now available for˙ ˙Vo Vco
2 2
hundreds of species (e.g., White and Seymour 2005). Typically,
* Corresponding author; e-mail: c.r.white@bham.ac.uk.
Physiological and Biochemical Zoology 79(5):977–980. 2006.  2006 by The
University of Chicago. All rights reserved. 1522-2152/2006/7905-5079$15.00
the technique involves measurement of changes in partial pres-
sure of O2 and CO2 as air is either passed through a chamber
containing an animal or drawn through a mask into which an
animal breathes. Details of the procedures necessary for suc-
cessful respirometry measurements are available in a number
of articles that describe the specifics of measurement tech-
niques, chamber design, and the derivation of appropriate
equations for and calculation (Depocas and Hart˙ ˙Vo Vco
2 2
1957; Withers 1977, 2001; Fedak et al. 1981; Frappell et al.
1989; Koteja 1996). A common recommendation is that water
vapor be scrubbed from the excurrent air stream before it enters
the analyzer. This is necessary because water vapor dilutes O2
and CO2 and potentially interferes with CO2 measurement with
infrared analyzers, although technological advancements mean
that this interference is now less of a problem than it has
previously been.
A number of commercially available chemical desiccants can
be used to remove water vapor, including silica gel, phosphorus
pentoxide, fused potassium hydroxide, sulfuric acid, and fused
sodium hydroxide. Alternatively, water vapor can be removed
by condensation at low temperature; that is, liquid air would
give a dew point of about 190C and a dry ice/acetone mix
one of about 78C (Withers 2001). Possibly the most well-
known and commonly used chemical desiccant in comparative
physiology laboratories is Drierite (anhydrous calcium sulfate;
Hammond Drierite, Xenia, OH). When CO2 is measured,
Drierite is used in preference to silica gel, another common
laboratory desiccant, because silica gel is known to equilibrate
with CO2 (Withers 2001).
Drierite dries air by chemically binding water as the hemi-
hydrate of calcium sulfate, and it leaves a residual water content
of about 0.005 mg L1. It is often impregnated with a cobalt
chloride indicator (marketed as self-indicating Drierite) so that
a color change from blue to pink indicates when the Drierite
is exhausted. As part of a study of the energetics of birds, we
used new Drierite that had never been recharged to scrub water
vapor from the excurrent air stream of a positive-pressure open-
flow respirometry system and observed that the CO2 washout
time during automated baseline checks was poor compared
with that of O2 and that the situation improved when smaller
quantities of Drierite were used. Despite having chosen Drierite
in preference to silica gel to avoid interfering with CO2 mea-
surements, we nevertheless observed an affinity for CO2. Here
This content downloaded from 23.235.32.0 on Wed, 4 Nov 2015 22:30:59 PM
All use subject to JSTOR Terms and Conditions
978 C. R. White, S. J. Portugal, G. R. Martin, and P. J. Butler
Table 1: Calculated 99% equilibration times (SE) for
drying columns of varying Drierite capacity
Capacity
(g)
99% Equilibration Time (s)
Empty Full (New) Full (Recharged)
30 15.0  .0 188  4 22  1
50 16.8  .4 312  36 26  2
100 39  1 456  34 44.7  .6
Note. Columns are either empty or contain new, unused Drierite or
Drierite recharged according to the manufacturer’s instructions. The ef-
fects of column capacity (30, 50, or 100 g) and Drierite (empty, new,
recharged) on log(equilibration time) are significant (ANOVA: F p1,21
, and , , respectively); their inter-200 P ! 0.0001 F p 1,101 P ! 0.00012,21
action is not ( , ).F p 2.0 Pp 0.162,21
Figure 1. Model of the apparent changes in respiratory exchange ratio
(RER; A) arising from differences in the washout characteristics of O2
(B) and CO2 (C). The system was modeled with a 30-g tube of new,
anhydrous Drierite (Table 1); the O2 washout was modeled as equiv-
alent to that of CO2 in the absence of Drierite. Fractional O2 and CO2
concentrations ( and , respectively) were calculated assumingFo Fco
2 2
an actual RER of 0.8, with an automated 5-min baseline ambient
( , ) period every 20 min.Fo p 0.2095 Fco p 0.0
2 2
we report the nature of the affinity of Drierite for CO2 and
describe a simple procedure to minimize it.
Methods
Dry gases (100% N2 or 1% CO2 in N2) were drawn through a
drying column by the internal pump of an ML206 O2 and CO2
analyzer (ADInstruments) at 200 mL min1. The analog voltage
outputs of the analyzer were connected to a PowerLab ML750
analog-to-digital converter (ADInstruments) and recorded with
Chart software (ADInstruments) at 4 Hz. A solenoid valve
(SMC EVT307, Radio Spares, Corby, UK) under computer con-
trol (PowerLab ML750, ADInstruments) was used to automate
the transition between the gases, which were provided in excess
at atmospheric pressure.
The responses of the analyzer to changes in CO2 content
were compared when gases were passed through three different
lengths of drying column, each of which were tested both with
and without the presence of new, unused Drierite, as well as
with Drierite that had been exhausted during routine animal
respirometry studies and then recharged according to the man-
ufacturer’s instructions. Drierite capacities of the tubes were
30 g (small), 50 g (medium), and 100 g (large). The medium
and large columns were probably larger than those that would
be typically used for most measurements but were used in this
study to ensure that differences were noticeable. Three complete
CO2 loading/unloading cycles were completed for each of the
nine Drierite/column combinations.
Washout constants (k) for each of the columns were deter-
mined for the 0%–1% CO2 transition as the absolute value of
the slope of the relationship between andln (Fco  Fco ), eq2 2
time, where is the fractional CO2 content of excurrent airFco2
at a given time and is the equilibrium fractional CO2Fco , eq2
content (Bartholomew et al. 1981; Seymour et al. 1998). Wash-
out times (Dt) to a fraction of equilibrium (z; 0.99) were then
calculated as (Withers 2001).Dtp ln (1 z)/ k
To examine the effect of water vapor uptake on the CO2
washout characteristics of Drierite, the washout of the full me-
dium column was examined with new, anhydrous Drierite and
following three successive 1-min exposures of the Drierite to
room air (ca. 20C, 50% relative humidity [RH]). A quantity
of Drierite equal to or greater than the amount required to fill
the column was exposed to air in a layer one granule thick on
a paper towel and immediately was used to fill the drying
column. Any excess was allowed to remain on the paper towel
until pink, at which time it was considered used and was re-
charged according to the manufacturer’s instructions.
Results and Discussion
Drierite clearly has an affinity for CO2. The presence of new,
anhydrous Drierite in a column increases CO2 99% washout
times by more than an order of magnitude (Table 1), a tendency
that has been noted previously for a range of desiccants, in-
cluding Drierite (Elia et al. 1986). During steady state mea-
surements, the affinity of Drierite for CO2 is unlikely to have
an impact on measurements because the Drierite will be in
equilibrium with the gases passing through the desiccant col-
umn. However, whenever the CO2 content of the gas passing
This content downloaded from 23.235.32.0 on Wed, 4 Nov 2015 22:30:59 PM
All use subject to JSTOR Terms and Conditions
Technical Comment 979
Figure 2. Relationship between time of air exposure and time to 99%
equilibration (left axis, filled circles) and residual water vapor capacity
of Drierite (right axis, open circles).
through the column changes, the slow washout of CO2 is likely
to be detrimental. Such problems will be apparent during non–
steady state (instantaneous) measurements (Bartholomew et al.
1981; Seymour et al. 1998; Withers 2001) or when baseline
(ambient) conditions are checked frequently to account for
analyzer drift and barometric pressure changes. During instan-
taneous measurements, the slow washout of CO2 through the
column is likely to be a problem only when the washout of the
respirometry chamber is faster than the washout of CO2
through the column. When the washout of the chamber is
slower than that of the drying column, as is often the case, the
slow washout of CO2 through the column will be largely masked
by the slower washout of the chamber. However, when the
drying column contributes most or all of the total washout,
such as when air from a respirometry chamber is substituted
with atmospheric air to check for baseline drift, the slow CO2
washout will be a more serious problem.
Consider, for example, a positive-pressure open-flow system
where a subsample of excurrent air is passed through a 30-g
tube of new Drierite at a flow rate of 200 mL min1 before
entering the O2 and CO2 analyzer. Because of barometric pres-
sure fluctuations, the temperature sensitivity of the analyzer,
and electronic drift, a baseline check is required every 20 min,
and the excurrent subsample and ambient air streams are
switched by a solenoid valve located upstream of the drying
column so that both air streams are dried by the same column.
Such a system is in routine use in our laboratory, where we
currently measure and of great cormorants Phala-˙ ˙Vo Vco
2 2
crocorax carbo and barnacle geese Branta leucopsis. Following
a switch to ambient, the Drierite will require 3 min to equil-
ibrate with the altered CO2 level (Table 1), followed by 1 or 2
min of stable baseline measurement and a further 3 min to
equilibrate after the switch to excurrent air from the respirom-
etry chamber. Thus, 7 or 8 min are required for the complete
baseline measurement cycle, leaving only 12 or 13 min for
measurement of excurrent air from the chamber. During the
equilibration period following the switch from ambient air to
excurrent air from the respirometry chamber, will be un-V˙co
2
derestimated, and because CO2 washes out of the desiccant
column at a slower rate than O2, the respiratory exchange ratio
(ratio of to ) will also be underestimated (Fig. 1). This˙ ˙Vco Vo
2 2
observation illustrates the importance of examining raw data
traces to ensure that systems are performing as expected and
that the presence of chemical absorbents is not interfering with
measurements.
Articles on standard respirometry (Depocas and Hart 1957;
Withers 1977, 2001; Fedak et al. 1981; Frappell et al. 1989;
Koteja 1996) make no mention of this problem and therefore
provide no solution. Elia et al. (1986) reported that the affinity
of Drierite for CO2 decreased with increasing hydration, but
they do not indicate the extent of hydration that is necessary
to decrease affinity to an acceptable level. We found that ex-
posing Drierite to room air (ca. 20C, 50% RH) for only 2 min
decreased 99% washout times from 5 to 1.3 min, at a cost of
only a 5% reduction in water vapor capacity (Fig. 2). Further
air exposure provides diminishing improvements in washout
constant, with a linear decrease in water vapor capacity of ca.
2.6% min1. The CO2 washout characteristics of Drierite can
therefore be improved by exposing it to room air for a short
period, but the duration of the period will be influenced by
the temperature and humidity of the air to which it is exposed.
A 2-min exposure at 20C and 50% RH was sufficiently long
for the indicating Drierite to begin to undergo a slight color
change from blue to pink, so this change can be used as a rough
indicator of the appropriate exposure time when ambient con-
ditions differ substantially. Given that CO2 washout time is
related to Drierite quantity (Table 1), minimizing the size of
the drying column also reduces washout times, and a respi-
rometry system should be designed to incorporate the smallest
quantity of Drierite that is compatible with the experimental
duration required. Finally, Drierite that has been used and then
recharged according to the manufacturer’s instructions has a
lower affinity for CO2 and far shorter CO2 washout times than
does new Drierite (Table 1), so the use of recharged Drierite
will largely circumvent CO2 washout problems.
Acknowledgments
We thank Joan Hammond for suggesting a brief air exposure
to reduce the CO2 affinity of Drierite and two anonymous
reviewers for providing constructive comments that greatly im-
proved this technical comment. This work was supported by
grant NER/A/2003/00542 from the Natural Environmental Re-
search Council.
This content downloaded from 23.235.32.0 on Wed, 4 Nov 2015 22:30:59 PM
All use subject to JSTOR Terms and Conditions
980 C. R. White, S. J. Portugal, G. R. Martin, and P. J. Butler
Literature Cited
Bartholomew G.A., D. Vleck, and C.M. Vleck. 1981. Instan-
taneous measurements of oxygen consumption during pre-
flight warm-up and post-flight cooling in sphingid and sa-
turniid moths. J Exp Biol 90:17–32.
Depocas F. and S.J. Hart. 1957. Use of the Pauling oxygen
analyzer for measurement of oxygen consumption of animals
in open-circuit systems and in a short-lag, closed-circuit ap-
paratus. J Appl Physiol 10:388–392.
Elia M., T. McDonald, and A. Crisp. 1986. Errors in measure-
ments of CO2 with the use of drying agents. Clin Chim Acta
158:237–244.
Fedak M.A., L. Rome, and H.J. Seeherman. 1981. One-step N2-
dilution technique for calibrating open-circuit measur-V˙o
2
ing systems. J Appl Physiol 51:772–776.
Frappell P.B., H.A. Blevin, and R.V. Baudinette. 1989. Under-
standing respirometry chambers: what goes in must come
out. J Theor Biol 138:479–494.
Koteja P. 1996. Measuring energy metabolism with open-flow
respirometric systems: which design to choose? Funct Ecol
10:675–677.
Seymour R.S., P.C. Withers, and W.W. Weathers. 1998. Ener-
getics of burrowing, running, and free-living in the Namib
Desert golden mole (Eremitalpa namibensis). J Zool (Lond)
244:107–117.
White C.R. and R.S. Seymour. 2005. Allometric scaling of mam-
malian metabolism. J Exp Biol 208:1611–1619.
Withers P.C. 1977. Measurement of , , and evaporative˙ ˙Vo Vco
2 2
water loss with a flow-through mask. J Appl Physiol 42:120–
123.
———. 2001. Design, calibration and calculation for flow-
through respirometry systems. Aust J Zool 49:445–461.
This content downloaded from 23.235.32.0 on Wed, 4 Nov 2015 22:30:59 PM
All use subject to JSTOR Terms and Conditions


Respirometry: Anhydrous drierite equilibrates with carbon dioxide and increases washout times

http://espace.library.uq.edu.au/view/UQ:165190/UQ165190_OA.pdf

Respirometry: Anhydrous drierite equilibrates with carbon dioxide and increases washout times

Abstract

Similar works

Full text

Available Versions

University of Birmingham Research Portal

University of Queensland eSpace

University of Birmingham Research Portal