Description of Instrumentation and Procedures - Advances in Biochemical Engineering Biotechnology - page 7

The temperature was measured at the top of the column with a thermistor
(10 k 6 mm, or equivalent) epoxied at the tip of a 2 mm (OD)

acid-proof steel tube. The temperature was measured as an unbalance signal of
a sensitive Wheatstone bridge. At the most sensitive setting, the recorder output
produced 100 mV at a temperature change of 0.01°C. Placing the temperature
probe at the very top of the column, rather than in the effluent outside the
column, reduced the turbulence around the thermistor and gave a more stable
temperature recording.
The solution was pumped through the system at a flow rate of 1 ml/min with
a peristaltic pump. The sample (0.1-1 ml) was introduced with a three-way
valve or a chromatographic sample loop valve. The height of the resulting tem-
perature peak was used as a measure of the substrate concentration and was
found to be linear with substrate concentration over a wide range. Typically it
was 0.01-100 mM, if not limited by the amount of enzyme or deficiency in any
of the reactants.
For example, this type of instrument was adequate for the determination of
urea in clinical samples. The sensitivity was high enough to permit a 10-fold
dilution of the samples, which eliminates problems of nonspecific heat. The
resolution was consequently about 0.1 mM, and up to 30 samples could be mea-
sured per hour.
In order to achieve more sensitive determinations, we developed a two-chan-
nel instrument,in which the water bath was replaced by a carefully thermostated
metal block. A specially designed Wheatstone bridge permits temperature
determinations with a sensitivity resolution of 100 mV/0.001°C. The calorime-
ter was placed in a container insulated by polyurethane foam. It consists of an
outer aluminum cylinder which can be thermostated at 25, 30 or 37 °C with a sta-
bility of at least

W at 25 °C, 1.5 ¥

0.01°C. Inside is a second aluminum cylinder with channels
for two columns and a pocket for a reference thermistor. The solution passes
through a thin-walled acid-proof steel tube (ID 0.8 mm) before entering the
column. Two-thirds of this tubing acts as a coarse heat exchanger in contact with
the outer cylinder, while the remaining third is in contact with the inner cylin-
der. This has a higher heat capacity and is separated from the thermostated
jacket by an airspace. Consequently, the column is held at a very constant tem-
perature, and the temperature fluctuations of the solution become exceedingly
small.
The columns were attached to the end of the plastic tubes by which they are
inserted into the calorimeter. Columns could therefore be readily changed with
a minimum disturbance of the temperature equilibrium. Inside the plastic tube
were the effluent tubing and the leads to the thermistor were fastened to a short
piece of gold capillary with heat-conducting, electrically insulating epoxy resin.
Veco Type A 395 thermistors (16 k

±

at 25°C, temperature coefficient 3.9%/°C)
were used. These are very small, dual-bead isotherm thermistors with 1% ac-
curacy; as such, they were interconvertible, comparatively well matched, and
follow the same temperature response curve (within 1%). An identical ther-
mistor was also mounted in the reference probe.
The Wheatstone bridge was built with precision resistors that have a low tem-
perature coefficient (0.1%; temperature coefficient 3 ppm) and was equipped

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