AN APPARATUS AND METHOD FOR THE MEASUREMENT OF
THE RESPIRATION OF COTTONSEED
A. M. ALTSCHUL, M. L. KARON, AND P. J. FYNN
(WITH TWO FIGURES)
Introduction
In the course of an investigation to determine the relationship of the
respiration of cottonseed to the other processes which take place during storage, studies were made of the effect of moisture content, maturity, and
inhibitors on respiration, rate of free fatty acid formation, and pigment
changes in the oil. For these studies, a series of measurements w-;as required
on about fifty different samples of cottonseed over a period of one year.
In view of the large number of analyses needed, an apparatus designed to
obtain accuracy and speed was built to follow the changes of the oxygen and
carbon dioxide concentrations in the atmosphere surrounding the seeds.
This apparatus and the methods used to make and record the measurements
are described below.
Methods
A sample of seeds for which a measurement of the respiration rate is
desired is stored in a stoppered flask. The size of the sample and the length
of the storage interval are determined by the estimated respiration rate of
the seed sample. Since an accumulation of carbon dioxide in the atmosphere surrounding the seeds inhibits respiration (2), a period of storage is
selected that will not permit the development of more than a 3-per cent.
concentration of carbon dioxide in the flask at the time of analysis.
At the conclusion of the storage period, a sample of the atmosphere in
the flask is withdrawn and analyzed for both oxygen and carbon dioxide
content. This is accomplished by the successive removal of carbon dioxide
and oxygen accompanied by measurements of the resulting changes in pressure at a constant volume. The gas sample is first dried, then drawn into
a measuring chamber of fixed volume kept at constant temperature, and its
pressure measured. The percentage of carbon dioxide is then determined
by passing the gas sample several times through a tube of Ascarite,' after
which it is pumped back into the measuring chamber. The percerntage drop
in pressure represents the carbon dioxide content of the sample. Oxygen
is determined on the same sample by passing the carbon dioxide-free gas
several times over a heated copper screen before pumping it back into the
measuring chamber. The difference between the percentage of oxygen
in a sample of air and in the sample which was analyzed is eaual to the
percentage of oxygen consumed by the respiring seed. After each oxygen
analysis, the copper is regenerated by passing hydrogen gas over the heated
oxidized copper screen.
I
A commercial preparation of asbestos coated with sodium hydroxide.
410
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ALTSCIIUL ET AL.: RESPIRATION OF COTTONSEED
411
Apparatus
Figure 1 is a schematic drawing to scale of the apparatus u-sed for the
gas analysis, and figure 2 is a photograph of the apparatus. All of the
FIG. 1. Diagram of respirometer.
connections in this apparatus were made with standard tapered ground
glass joints. Vacuum-tight seals were obtained by cementing the ground
glass joints with a phthallic anhydride resin. It will be noted that this
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412
~ ~.
PLANT PHYSIOLOGY
apparatus is similar in many respects to the one described by BAMFORD and
BALDWIN (1). It was found desirable, however, to sacrifice some of the
Al 4~~~~~4 rgt..
'
FIG. 2.
X
..
.N' .j ::....
Respirometer assembly.
accuracy obtained by BAMFORD and BALDWIN in favor of simplification of
operations to obtain the speed necessitated by the number of analyses to be
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ALTSCHUL ET AL.: RESPIRATION OF COTTONSEED
413
conducted. Nevertheless, almost negligible difference existed between the
oxygen content of air found with the apparatus used in this investigation
(20.93 + 0.05%c) and that found by BAMFORD and BALDWIN (20.94 ±
0.04%v).
Method of analysis
of
the
Description
procedure followed with a typical gas specimen will
serve to illustrate the method of analysis. The symbols used refer to those
indicated in figure 1.
MEASUREMENT OF THE INITIAL PRESSURE
The respiration flask, A, is connected by paraffin-covered rubber tubing
to the tube B which contains phosphoric anhydride layered on glass wool.
This tube serves to remove water and volatile bases. With stopcock I closed,
the entire apparatus is evacuated to approximately 0.05 mm. pressure by
means of a "Megavac" pump. The gas sample is admitted slowly into the
measuring chamber, FG, through B and the system of stopcocks I, VI, X.
Stopcocks- X and XI are closed; mercury is forced up through bulb, F, to the
mark in the capillary tubing; and the pressure on the manometer, K, is
read to the nearest half millimeter. The quantity of gas admitted is such
that the initial pressure is slightly less than atmospheric pressure. The
chamber, G, is maintained at 260 C. by circulating water from a constant
temperature water bath through the surrounding jacket; and the vacuum,
in the manometer, K, is renewed daily by forcing the mercury up through
the stopcock XIII, before closing it.
MEASUREMENT OF PERCENTAGE OF CARBON DIOXIDE
After the iniitial pressure has been determined, the sample of gas is
passed through the tube containing Asearite, I, into the Toepler pump, D,
through the system of stopcocks IX, VI, and V. Both ends of the tube I
contain small amounts of magnesium perchlorate to absorb any moisture
produced during the removal of carbon dioxide by Ascarite. Compressed
air and vacuum (5-mm. laboratory vacuum line) are used to regulate the
level of mercury in the chamber, G, and in the Toepler pump. The air,
as obtained from the laboratory source, was under 80 pounds' pressure.
This pressure was reduced by the use of the bleeding stopcock XIV, to that
required as measured by the manometer, L. The float value, H, prevents
mercury from rising into the capillary lines. A similar valve, E, above the
Toepler pump affords protection to the capillary lines above the pump.
When all of the gas has been forced out of chamber G into the Toepler
pump, the flow is reversed and the gas is pushed back again through the
Ascarite absorption tube. The process is repeated twice more, so that, in
all, the sample of gas is passed over the Asearite six times. At the end of
the sixth operation, all of the gas will have been forced out of the Toepler
pump, and the level of the mercury in the measuring chamber is set at a
point below bulb F. The gas remaining in the absorption tube, I, and in
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414
PLANT PHYSIOLOGY
the capillary lines is pumped back into the system FG by the use of the
Toepler pump. Four pumpings are sufficient to remove all the residual gas
from the lines. With stopcocks X and XI closed, the level of the mercury
in the measuring chamber is again brought to the mark in the capillary
tube below G and the pressure is measured. The fraction of carbon dioxide
in the gas sample is equal to the drop in pressure divided by the initial
pressure.
MEASUREMENT OF PERCENTAGE OF OXYGEN
Oxygen is removed from the carbon dioxide-free gas by passing the
gas over the heated copper screen in tube C into the Toepler pump through
the system of stopcocks X, VII, and V. Tube C is electrically heated by
means of 30 feet of size 22 Chromel-A wire (0.999 ohm per foot) Wound
around the outside of the tube. Fifty volts A.C., taken from a variable
transformer, are sufficient to give adequate heat for both the removal of
oxygen during the analysis and for the subsequent reduction of the copper
oxide by hydrogen. The operations used for the removal of oxygen in the
sample and for the return of all the residual gas to the measuring chamber
are similar to those carried out for the determination of carbon dioxide.
When the analysis of the sample has been completed, the copper surface on
the screen is renewed by passing hydrogen gas into the heated tube, C,
through the system of stopcocks II, VI, V, and VIII.
The drop in pressure resulting from the removal of oxygen in the sample
divided by the initial pressure is equal to the fraction of oxygen in the
sample. The change in oxygen pressure due to the consumption of oxygen
by the respiring seeds is obtained by subtracting the percentage of oxygen
obtained by analysis from the oxygen content of the air (20.93%7o).
Typical results
A set of typical recordings of experimental data and calculations of the
results of the gas analysis are presented in table I. In the case of analysis
No. 1, the oxygen was first removed, followed by carbon dioxide; in the
second analysis the order of removal of the gases was reversed. It will be
noted that the order of removal of the gases has no significant effect upon
the acculracy of the results.
TABLE I
MEASUREMENTS AND CALCULATIONS OF
THE RESULTS OF A TYPICAL GAS ANALYSIS
PRESSURE
ANALYSIS
NUMBER
1
2
INITIAL
mm. Hg.
766
739
AFTER
AFTER
CARBON
OXYGEN
REMOVAL
REMOVAL
mm. Hg.
610.5
584.0
m.
DIOXIDE
EVOLVED
CARBON
OXYGEN
CONTENT
%
%
0.65
0.61
20.30
20.36
0.63
0.57
g.
605.5
734.5
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OXYGEN
CONSUMED
415"a
ALTSCHUL ET AL.: RESPIRATION OF COTTONSEED
Starting with data of this type, it is possible to place the results of all
respiration experiments on a comparable basis by reducing the analytical
values for carbon dioxide and oxygen to the number of cubic centimeters
of gas at standard temperature and pressure that is evolved or consumed
per day by one gram of seed (moisture-free basis). To effect this transformation, it was necessary to measure the volume of the free air space in the
respiration flasks, so chosen that the volume of free space was constant for
each size of seed sample. For a 10-gram sample of seed the air space was
1108 cc.; for a 25-gram sample, 1079 cc.; and for a 50-gram sample, 1032 cc.
Corrected to standard conditions, the volume of gas in the above flasks was
1022 cc., 995 cc., and 953 cc. for 10-, 25-, and 50-gram samples, respectively.
The complete calculation of the respiration rate of the sample illustrated
in table I is shown in table II.
TABLE II
CALCULATION OF RESPIRATION RATE
CARBON OXYGEN
RESPI-
WEIGHT
INTER-
OF
VAL SAMPLE
VAL;
days
39
gm.
50
Mois -
CON-
WEIGHT CARBON OXYGEN CARBON OXYGEN
OF DRY DIOXIDE
CON-
DIOXIDE
CON-
DIOXIDE
PVE
PER
SEED EVOLVED SUMED EVOLVED SUMED GRAM
~~TENTGRM
PER DAY
%
10.1
gm.
45.0
%
0.63
%
0.60
cc.
6.0
cc.
5.7
CON-
cc.
0.0034
GRAM
RA
PER DAY
cc.
0.0032
Summary
1. An apparatus, developed for the determination of the respiration rate
of cottonseed, is described. Carbon dioxide and oxygen are successively
removed from the gas sample, and the changes in pressure of a fixed volume
of the sample, occasioned by removal of the gases, are used to calculate the
percentage of carbon dioxide and oxygen in the sample.
2. The results of the respiration rate measurements are expressed in
terms of the number of cubic centimeters of carbon dioxide or oxygen, at
standard temperature and pressure, evolved or consumed per gram (moisture-free basis) of seed per day.
SOUTHERN REGIONAL RESEARCH LABORATORY
NEW ORLEANS, LOUISIANA
LITERATURE CITED
1. BAMFORD, C. H., and BALDWIN, R. R. A method for the accurate analysis of gaseous mixtures. Jour. Chem. Soc. 26-29. 1942.
2. RAMSTAD, P. E., and GEDDES, W. F. The respiration and storage behavior
of soybeans. Minnesota Agr. Exp. Sta. Tech. Bull. 156. 1942.
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