NBSIR 73-180

Testing of the NBS Clinical

Microcalorimeter

NAT'L INST. OF STAND & TECH REFERENCE

E. J. Prosen and R. N. Goldberg

Thermochemistry Section

Physical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

April 1973

Interim Report

_ ared for \i(C^ mal Institute of General Medical Sciences

/^O inal institutes of Health

CL^^ !sda, Maryland 20014

NBSIR 73-180

TESTING OF THE NBS CLINICAL

MICROCALORIMETER

E. J. Prosen and R. N. Goldberg

Thermochemistry Section

Physical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

April 1973

Interim Report

This report is to be superseded by a future publication which will receive general distribution and should be cited as a reference. Please consult the NBS Office of Technical Information and Publications

to obtain the proper citation.

Prepared for National Institute of General Medical Sciences

National Institutes of Health

Bethesda, Maryland 20014

* 1 7 *

U. S. DEPARTMENT OF COMMERCE, Frederick B. Dent. Secretary

IMATIOIMAL BUREAU OF STANDARDS, Richard W. Roberts, Director

Foreword

The use of clinical laboratory tests of physiological

fluids for specific components has been rapidly increasing in

the past few years. One aspect of this has been an increase

in the number of tests for well-known particular substances

for which satisfactory tests are available. Another equally

important aspect is the increasing need of the physician and

of the research biochemist for ready methods for determining

other substances present in varying amounts in normal and

pathological states. For this reason^ the development of new techniques applicable to problems for which optimum solutions

have not been found are of considerable interest in clinical

and biological chemistry.

The universal occurrence of energy changes in chemical

and physiological processes has caused the calorimeter to be

considered as a potential tool for clinical chemical analysis

and for observing biological processes for some years. The

actual applicability^ however^ has been limited previously by

lack or insufficiency of one or more necessary factors. Such

factors include: instrument sensitivity and operational noise

levels^ instruments adapted to use of small samples, simplicity

of instrumental operation and data reduction, and suitable

mechanisms for sorting out specific processes from a melange

of potential interferences. It was the judgment of the authors

and of the National Institute of General Medical Sciences (NIGMS)

that the understanding of these factors had developed to a point

where a vjell coordinated attack on the application of microcalorimetry would now lead to successful results.

A program was established in the Thermochemistry Section

(Physical Chemistry Division, Institute of l-laterials Research)

at the National Bureau of Standards, and after some months was augmented with support from the NIGMS to demonstrate the

feasibility of microcalorimetry in some specific areas useful

in the Clinical Chemistry Laboratory. Dr. Robert S. Melville

is the program officer for NIOIS. Collaborative support has

been provided by the Clinical Chemistry Service (Dr. Donald

Young, Chief), and by the Laboratory for Technical Development

(Dr. Robert L. Bowman, Chief, and Dr. Robert L. Berger)

This is a report provided at the end of the first year of

the NIGMS support of this work and summarizes a portion of the

work done. This report is one of four covering different aspects

of the NBS Program. For the information of the reader, the titles

and NBS Report numbers of the four reports are as follows:

NBS Report 73-178

73-179

73-180

73-181

Microcalorimetric Assay for Glucose in

Human Serum and Plasma

Design and Construction of the NBS Clinical

MLcroca 1 or ime te r

Testing of the NBS Clinical Microcalorimeter

Fine Structure in Thermal Growth Patterns

of Bacteria by Microcalorimetry

George T. Armstrong ,/

Principal Investigator

Edward J. Pr(^en

Project Leader

Testing of the NBS Clinical Microalorimeter

by

E. J. Prosen and R. N. Goldberg

ABSTEACT

The NBS Clinical Microcalorimeter has been tested for stability,

sensitivity^ ease of operation, and accuracy. The accuracy was tested

by means of electrical calibration and the determination of the heat

of neutralization of HCl(aq) V7ith NaOH(aq). The heat of this reaction

agrees with the best literature value within the precision of the

calorimeter. The precision is about 0.6 percent when measuring about

50 mJ of heat of chemical reaction. The accuracy is estimated as 1 percent. The precision of electrical energy determination is about

0.4 percent for energies of from 1 to 1400 mJ.

Table of Contents

Page

1. Introduction

2. Measurement of Thermopile Voltage 2

3. Calculation of Areas 4

4. Temperature of the Calorimeter 6

5. Time Constant and Speed of Operation of

Calorimeter 7

6. Quality of Baseline 8

7. Electrical Calibrations 9

8. Procedures Used in Calorimetric Experiments 11

9. Blank Heat Effects " 14

10. Acid-Base Test Reaction 15

11. Ease and Reliability of Operation 18

12. References 19

13. Acknowledgements 21

Tables

1. Results of Electrical Calibrations (Series I)

Temperature is 29.85 'C 22

2. Results of Electrical Calibrations (Series II) 23

Temperature is 30.80 "C 23

3. Blank Heats of Mixing 24

4. Results of Acid-Base Calorimetric Experiments .... 26

1. Introduction

This report describes tests of the performance of the NBS Clinical

Microcalorimeter . This calorimeter was designed and constructed [1]

in order to be able to perform microcalorimetric measurements as part

of a program, sponsored by the National Institutes of Health, investigating the applicability of microca 1 crime try to clinical chemistry and

to biochemistry.

The reader is referred to several books and articles [2, 3, 4] to

obtain further background on the subject of microcalorimetry

1

2 . Measurement of Thermopile Voltage

The thermopile voltage of the NBS Clinical Microca lorime ter vas

1 amplified using a linear D.C. amplifier (Keithley Model 140)^ the gain

setting of which was generally set at 10^ and only rarely set at lO'^. The rise

time of the amplifier was set at 5 seconds and it was not found necessary to use either the amplifier's zero suppression or input filter. Electrical

ground for the thermopile signal was established at the input to the

amplifier.

Calibration of the amplifier was periodically performed in the

following manner: a stable current from a D.C. power supply (Sorensen

Model QHS 40-. 5) was passed thru four standard resistors (1-Q, 10-Q,

100-Q, and 10-k Q) in series with each other. The input voltage to the

amplifier was then selected to be across one of the lower valued

resistors. By measuring the voltage across both the entire voltage divider

net^jork and the amplifier's output terminal and by application of a

small correction for the amplifier zero^ the gain of the amplifier was

calculated. The amplifier's gain, as measured in this manner, was found

to be within 0.02% of its nominal value on the 10^ scale and stable to

within ± 0.006% over a period of one year.

Certain commercial products and instruments are identified in this paper

in order to specify adequately the experimental procedure. In no case

does such identification imply recommendation or endorsement by the National

Bureau of Standards, nor does it imply that the products or equipment

identified are necessarily the best available for the purpose.

2

The amplifier output was recorded both in analog and in digital

fom. The purpose of the analog record (obtained either with a Leeds and

Northrup Speedomax XL-600 or Varian F80 recorder) was to give the

calorimeter operator a visual picture of the course of an experiment,

while the digital record was used to obtain all numerical results. The

analog to digital conversion was accomplished using an autoranging

digital voltmeter (Hewlett-Packard Model 3450A, ±1 |av or ± 10 \iv

sensitivity, depending on range). The stability and accuracy of this

voltmeter were checked periodically against saturated standard

cells calibrated by the National Bureau of Standards. Using this procedure,

the accuracy of the digital voltmeter was found to be within 0.0025%

at a reading of 1.018 v, and to have a stability of ±0.0017o over a period

of one year. During an experiment, a digital clock, having the line

frequency as its time base, sent "read" commands to the digital voltmeter

every 3 seconds. The time and voltage information was then processed thru a

digital coupler (Hewlett-Packard 2547A) and recorded in typewritten form

and on punched paper tape.

3

3. Calculation of Areas

In a properly designed heat conduction calorimeter, the thermopile

voltage is a measure of the rate of heat transfer between calorimeter

vessel and heat sink. A calorimetric experiments thus consists of

measuring the thermopile voltage as a function of time continuously

(or at 3 second intervals). These readings may be divided into three

periods: a fore period, a main period, and an after period. During

the fore period, no process heat is being liberated in the reaction

vessel. Ideally, the mean of the measured voltages would be zero volts

and flucuations about the mean would be due only to Johnson noise. The

main period begins with the initiation of the reaction or electrical

heating and the accompanying heat effect, which is detectable as a heat

flow across the thermopile and a corresponding change in the thermopile

voltage. After completion of the reaction or electrical heating, there

will be an exponential decay of the voltage to the after period baseline

which ideally should be the same as the fore period baseline.

4

The total heat effect, Q, to be associated with the reaction is proportional

to the area. A, of the plot of voltage readings, E, versus time, t.

Q = k.A

This area. A, in millivolt-seconds (mvs) is the area of the curve

above the extrapolated baseline from time t^ (at the end of the fore

period) to time t^ (at the beginning of the after period).

To obtain the extrapolated baseline during the reaction period,

a straight line was assumed from E(t^) j the voltage at the end of the

fore period, to E(t^) , the voltage at the beginning of the after period.

E(tj^) and E(t2) were obtained by a least-squares straight line fit of the

fore and after period readings, respectively. The area was obtained by

the use of the trapezoidal rule.

All of the above calculations were done on a digital computer

utilizing the digital voltage and time readings (taken at 3-second

intervals throughout the experiment)

The electrical calibration experiments gave the value of k:

k = Q/A

where, in this case, Q is the electrical energy added to the calorimeter

heater.

5

4. Temperature of the Calorimeter

The temperature of the microcalorimeter was controlled by controlling the current through a heater wound on the inner jacket [1].

A modified Quartz Thermometer [6] was used to check the temperature

stability of the inner jacket. In performing this test, the quartz

thermometer probe was taped to the inside of the calorimeter's inner

jacket, and a temperature record (sensitivity ±10 H^K) was obtained over

a 15 hour period. This record indicated a steady drift of 0.00094 K

for this entire period^ while short term variations in temperature were

with ±0.0002 K.

The absolute temperature of the inner jacket, as measured with

an NBS calibrated mercury- in- glass thermometer and a copper-constantan

thermocouple junctioned between the inner jacket of the microcalorimeter

and the mercury-in-glass thermometer, was determined initially to be

29.85°C. Several weeks later an abrupt change in temperature to 30.80°C

was noted. It is believed that this jump was due to a break in a wire

that connected a 20-kfi resistor, which had been put in parallel with

one of the manganin arms of the wheatstone bridge of the temperature

control circuit on the inner jacket. More recently, the temperature

of the calorimeter has been observed to be drifting gradually higher

to 31.00°C. This may be attributable to relaxation effects in the

resistors comprising the regulation network.

6

5 . Time Constant and Speed of Operation of Calorimeter

The half -response time of the microcalorimeter, as determined by

observation of the exponentially decaying portion of reaction peaks, was

observed to be about 30 to 40 seconds. This corresponds to a time constant of

about 50 seconds. This time constant has important implications as regards the

speed of operation of a microcalorimeter . For example, it has been

empirically found that a wait of twenty half-response times, or 10 minutes,

is generally adequate to insure proper thermal equilibration of a calorimetric cell with the calorimeter block and thermopiles when an external

heatsink, kept to within a degree of the calorimeter's temperature is

employed to pre-equilibrate the reaction cell and its contents before

inserting the cell into the calorimeter (pre-equilibration time

approximately 5 minutes). The amount of time required to complete a

run, following the thermal equilibration period in the calorimeter,

is about 15 minutes. This allows sufficient time for both a mix and

remix operation with the necessary recording of the fore, main, and

after period voltage readings for each. Thus, total time elapsed

from the inserting a cell into the calorimeter to end of a run is

about 25 to 35 minutes. Using manual methods it is possible to do

about 15 separate experiments during the course of an 8 hour day.

7

6 . Quality of Baseline

The quality of the baseline or thermopile voltage readings^ is

an important characteristic of a heat conduction microcalorimeter, and

is determined by the absence of electrial or thermal interferences

and is ultimately limited by Johnson noise. For measurement of the

heat effects accompanying rapid processes^ as a consequence of the

calorimeter's rapid response time, it is necessary to be concerned

about the quality of the baseline over only a 15 to 20 minute interval.

Observations of the baseline have indicated that peak-to-peak noise is

generally within 30 nv over this sort of time interval. Since the

-1 -1

sensitivity of the instrument is 16.7 J'v (see Section 7), or

60 nvM-W this 30 nv peak-to-peak corresponds to 0.5 |i.W of power

peak-to-peak over a 30 second interval. The long term drift of the baseline has been found to be less than 100 nv over a 24 hour period.

It is worthwhile to note that the measured value of the thermopile

voltage was generally within 0.5 [xv of electrical zero. Occasional severe

deviations from electrical zero were attributable to the following

causes: (1) water leaks from the reaction cells; (2) a severe thermal

disturbance of the calorimeter, such as unscrewing the lids, and (3) an

effect believed to be due to a pressure disturbance of the thermopiles

whenever a reaction cell was fit too tightly into the silver box of the

microca lorimeter . This latter effect was observed, for example, when

excess wax, used in sealing earlier cells, was left on the side of

that cell.

8

7. Electrical Calibrations

Electrical calibrations of the NBS clinical Microcalorimeter were

performed using the permanent resistance heater installed in the silver

box which is between the calorimeter thermopiles [1]. Current from a

well-regulated D.C. power supply (Sorensen Model QHS 40-. 5, stability

0.0005% + 50 uv), operated in the constant voltage mode, was passed thru

this calibrating resistor and thru a 1000-fi standard resistor in series

with it. Measurements of the voltages across the heater and across the

standard resistor were made using a digital voltmeter (Hewlett-Packard

Model 3450A, +1 yv or + 10 yv sensitivity, depending on range). The

interval of heating was determined [5] by means of an electronic counter

(Hewlett-Packard Model 5325B) set to read to the nearest millisecond.

Grounding of this calibration circuit was done at the heater connection

to the power supply. The saturated standard cells, against which the

digital voltmeter was checked, and the standard resistor were intercompared with other standards in this laboratory that had been calibrated

by the National Bureau of Standards. The internal oscillator of the

counter was checked periodically against the NBS 100 kHz frequency

(stability of 1 part in 10''"'^) and found to be stable to within 5 parts in

10^ over a period of one year. Power input to the calorimeter was calculated as the product of the potential across the heater and the current

thru it, with a small correction (0.005%) applied for power generated in

the heater leads. In applying this correction, it was assumed that onehalf the power generated in the heater leads could be apportioned to the

jacket (i.e. the aluminum block) and the other half to the calorimeter

vessel. Total heat input to the calorimeter was the product of power

9

and time interval of heating.

The results of electrical calibrations are given in Tables 1 and 2.

The results, shown in Table 1 were obtained when the calorimeter was at

a temperature of 29.85''C and were performed (with the exception of run

no. 52) with no sample cell contained in the silver box of the microcalorimeter. In Table 2, the results reported were taken at an assigned

calorimeter temperature of SCSO^C and a sample cell, containing approximately 300 y5- of water, was contained in the calorimeter's silver box.

The calibration constant (units of W'v ^ or J*v ^'s ^) was calculated as

the ratio of the heat input (mJ) to the calorimeter to the measured area

(units of mvs). A second way of obtaining this calibration constant

was by application of a steady state power for a long time (13 to 16

half-response times of the calorimeter). Using this procedure [2], the

calibration constant is calculated as the ratio of the steady state

power to the steady state deflection of the thermopile voltage from its

baseline. The values of the calibration constant, obtained in this manner, are given in parenthesis in Tables 1 and 2.

It will be noted that the calibration constant is the same, within

the precision of the measurements, whether we use the integral energies

or the steady power-deflection method (values in parentheses) . The

integral energy results are used since the areas were determined in the

same way as in chemical reaction experiments. The calibration constant

is thus taken as 16.67 W'v ''" at either temperature. Measurements have

not yet been made at widely different temperatures. Inspection of the

data in Tables 1 and 2 shows no obvious trend with power level from 0.01

to 2.00 mW, time of heating from 10 to 800 seconds, or energy from 1 to

10

1400 mJ. Below 1 mJ, the area or electrical energy was perhaps not measured accurately enough and runs number 21,24,27, and 209 were omitted

in averaging.

The precision of the determinations of the calibration constant with

electrical energy can thus be taken as approximately 0.4 percent for

energies from 1 to 1400 mJ.

8 . Procedures Used in Calorlmetric Experiments

A calorlmetric experiment consists of several distinct operations,

the details of which are described in this section.

A. Preparation of Reaction Cell

Since the Kel-F reaction cells are not yet disposable, it is first

necessary to clean a previously used cell by appropriate means. For

acid-base experiments, the cleaning procedure employed was to rinse the

cell several times with distilled water. After the last rinsing, the

water was sucked out using a vacuum line in such a way as to not completely dry the inside of the cell. This procedure, identified as the

"wet cell" procedure, was used to facilitate the mixing of the solutions.

However, as will be shown, this procedure introduces a heat of dilution

which can have serious effects on the accuracy of the results. More

recently, it has been found that a "dry cell" procedure, i.e. one where

the inside of the cell is completely dry, can be made to mix reliably if

the solutions being used contain a trace amount ( i part in 10^) of an

inert wetting agent such as alkylphenoxy polyethoxy ethanol (trade name,

Triton X-100) . New cells, which have a hydrophillic surface treatment,

do not require the use of the wetting agent nor the "wet cell" procedure.

The "wet cell" procedure was used in all experiments summarized in this

report.

B. Cell Loading and Sealing

Solutions were introduced into the reaction cells with syringes.

Following the loading of one side of a cell with that solution containing

an excess quantity of one of the reactants (solution A)^ that side of the

cell was tilted 90 degrees to the side so that the solution would wet

that side of the cell and thus would flow when mixing was desired. The other

side of the cell was then loaded carefully with the solution containing

the limiting amount of reactant (solution B) . Quantities of solutions

introduced into a reaction cell were determined by weighing the empty

cell_, the cell containing solution A alone, and the cell containing

both solutions A and B. Care was taken not to wet the "0"-ring seat

at the top of a cell.

Cell sealing was done in one of two ways: (1) use of small Kel-F

or Teflon plugs that fit into the cell entrance portals and that were

sealed over with melted wax and (2) use of no. 2-1 Buna-N "0"-rings that

were compressed by means of small Teflon plugs. The sealing process is

extremely important, for if a cell is not properly sealed, the heat of

vaporization of water, as indicated by a large endothermic voltage

signal from the microcalorimeter, will generally dwarf the heat effect

being measured and make its determination uncertain. Initial

difficulties with the "0"-ring technique necessitated use of method (1)

above. However, the use of wax was neither convenient nor desirable in

that excess wax left on the side of a cell could adversely affect the

thermal sensors (see Section 6). Recent experience with slightly

modified cells and "0"-ring plugs has given highly reliable and

convenient sealing.

12

A second possible point of leakage of water from the cell was at

the epoxy seal of the top of the cell to the base. If this point of

leakage could not be repaired by regluing as was found to be generally

the case^ the cell was worthless as far as meaningful measurements were

concerned. Even more serious, was the case when water from a leaky cell

would remain in the silver box of the microcalorimeter . Until evaporation of that water was complete, reliable measurements could not be

performed

C. Pre-equilibration and Actual Run

Following the sealing of a cell, it was then put into a pre-equilibrator or aluminum block maintained at a temperature within one Kelvin

of that of the microcalorimeter. Following a five to ten minute wait,

the cell was the transferred to the microcalorimeter for final equilibration.

This generally took about 15 minutes (see Section 5). The actual run

consisted of obtaining a digital and graphical record of the thermopile

output as a function of time for the fore period, main period, and after

period. The main period was initiated by a 360-degree rotation of the

microcalorimeter block and 360-degrees back. After an adequate number of

data points had been accumulated in the after period, a remix was

performed to ascertain the completeness of reaction. The cell was then

removed from the calorimeter and prepared for the next run.

13

9. Blank Heat Effects

A factor which effects the precision and accuracy of the microca lor imeter is reproducibility of the heat effect accompanying the necessary

experimental operation of reaction initiation by mixing. The results

of measurements of the magnitude of this heat effect are given in Table 3.

In performing these runs^ the "v?et cell procedure"^ as described in

Section 8^ was used. The results show that one simple operation of

mechanical rotation of the calorimeter block yields an apparent endothermic

heat effect of -40 ± 18 1-lJ (average deviation). The cause of this effect

is not fully understood and may be due to movement of wires during

rotation.

The results obtained when solutions are present in the Kel-F

reaction cell indicate that the heat effect accompanying the first

rotation is always larger than that accompanying the second rotation or

remix. The reason for this^ in the case of the mixing of water with

water, may possibly be attributable to a heat of wetting of Kel-F or

to some other surface interaction. However^ in the case when hydrochloric

acid and sodium hydroxide are mixed with themselves^ the effect is due

to the "wet cell" procedure used and was the results of the heat of

dilution of acid or of base by the pure water on the walls above the

solutions in the cell, A series of mixing experiments, employing the dry

cell procedure is planned.

14

10. Acid-Base Test Reaction

The heat of neutralization of hydrochloric acid with sodium

hydroxide has been used as a test of the accuracy of the

microcalorimeter . The hydrochloric acid used in these experiments was

obtained from George Marinenko of the Analytical Chemistry Division (NBS)

and had been analyzed by him using a coulometric procedure [6, 7].

This stock acid, as received, had a concentration

-4 -1

of 1.99242 X 10 equivalents g and vas believed to be accurate to within

± 0.02%. This stock solution was used in the preparation by weight of

more dilute acid solutions. A saturated stock solution of sodium

hydroxide was prepared using Baker analyzed sodium hydroxide pellets

and water distilled at the National Bureau of Standards. After allowing

this solution to stand for several weeks to permit precipitation of

sodium carbonate, a portion of the clear solution was withdrawn from the

saturated sodium hydroxide solution and diluted using freshly boiled

distilled water. The concentration of this sodium hydroxide solution

was determined by titration against potassium acid phthalate (NBS

Standard Reference Material No, 84 g) . In all calorimetric runs, the

sodium hydroxide was used in substantial excess of stoichiometric.

The cell loading procedure used was the "wet cell procedure".

15
)
The results of ca lorlinetric measurements of the heat of neutralization of hydrochloric acid v/ith sodium hydroxide are given in Table 4.

In analyzing these results^ a calibration constant of 16.67 J*v ^'S

was used (see Section 7). The measured heat (Q) , given in Table 4^

includes a correction^ -1147 M-J^ for the blank heat of mixing as determined when 0.08 N sodium hydroxide was mixed with itself. This correction

is not very satisfactory but until dry-cell experiments are available^

this effect is our best estimate of the correction to be applied. It

amounts to about 2 percent of the heat of the reaction measured. Corrections

for heats of dilution to the standard state [12] were applied using tabulated

data [8] for the relative apparent molal enthalpy and heat capacity

of the reactants and products. Also a correction was applied [8] to

convert the data to 25°C. The final value, AU°, is the internal

energy change of ionization of water at 25 °C.

No correction was applied for the change in vapor pressure of water

above the solutions in the reaction of this space upon reaction. The

total estimated effect was '^.02%. The vapor pressure before reaction

was taken as the average of that of the NaOH and of the HCl and after

reaction was that of NaCl and NaOH in water. The change in volume of

the liquid phase was taken from Harned and Owen [9]_, the enthalpy of

vaporization of water from Wagman et al [10], and vapor pressures

calculated from osmotic coefficients of Robinson and Stokes [11]. Since

(Z^V)° is small is taken as equal to AH°, the enthalpy of ionization

of water.

16

The resulting values of given in Table 4 were averaged (with

the exceptions of runs no. 56^ 151, 156, and 166) to yield a value of

= -55.74 kJ«mol with an average deviation of 0.33 kJ'inol The

four runs discarded, out of a total of 22 measurements, all differed

from the average of the remaining results by at least a factor of seven

times the average deviation. It is believed that these four runs yielded

results much lower than the rest due to poor handling of the reaction

cell and some attendant pre-reaction of the hydrochloric acid. The

result of -55.74 kJ«mol ^ agrees, within the experimental precision,

with the "best" literature value of -55.835 ± 0.104 kJ'mol"''" [8]. At

present, new lots of hydrochloric acid sodium hydroxide are being

prepared and analyzed by G, Marinenko. Another series of calorimetric

acid-base measurements using these materials and the "dry cell" technique

is anticipated.

The precision of measurement of the heat of a chemical reaction is

thus about 0.6 percent when the heat is of the magnitude of 50 mJ or

more. In the present experiments it was necessary to make a 1 mJ correction

for the blank mixing effect caused by the "wet cell" procedure. It is

estimated that this correction may introduce a systematic error of about

one percent in the final result. Thus, at present, we can estimate

the accuracy of the calorimeter to be about 1 percent.

17

11 . Ease and Reliability of Operation

Various aspects of the ease and reliability of operation of the

microcalorimeter have been discussed at various points throughout this

report. Difficulties were encountered with sealing the cells and

obtaining reliable mixing of the solutions at the appropriate time. Both

of these problems now appear to be under reasonable control. As pointed

out in Section 5, the speed of operation of the calorimeter itself is

such that, at present, a run can be performed once every thirty to forty

minutes. The use of automated cell loading and running techniques,

automated computation of results, disposable cells, and other devices

could in principle greatly improve the reliability, speed of operation,

and number of measurements possible in a given period of time.

18

12. References

[1] E. J. Prosen^ NBS Report on the design and construction of microcalorimeter^ in preparation. NBS Report No. 73-179 (1973).

[2] E. Calve t and H. Prat, "Recent Progress in Microcalorimetry", The

MacMillan Company, New York, 1963.

[3] T. H. Benzinger in "A Laboratory Method of Analytical Methods of

Protein Chemistry", P. Alexander and H. P. Lundgren, editors,

Pergammon Press, Oxford and New York, 1969.

[4] "Biochemical Microcalorimetry", H. D. Brown, editor. Academic

Press, New York, 1969.

[5] A. P. Brunetti, E. J. Prosen, and R. N. Goldberg, "The Enthalpy of

Reaction of Tris(hydrox3nnethyl)aminome thane with Hydrochloric Acid",

manuscript in review.

[6] G. K. Marinenko and J. K. Taylor, Analytical Chemistry, 40, 1645

(1968).

[7] G. Marinenko and C. E. Champion, Jour. Res. Natl. Bur. Stds., 75A,

421 (1971).

[8] V. B. Parker, "Thermal Properties of Aqueous Uni-univalent

Electrolytes", Report NSRDS-NBS 2, U.S. National Bureau of

Standards, U.S. Government Printing Office, Washington, D.C., 1965.

[9] H. S. Earned and B. B. Owen, "The Physical Chemistry of Electrolytic

Solutions", Reinhold Book Corporation, New York, 1958.

[10] D. D. Wagman, W. H. Evans, V. B. Parker, I. Halow, S. M. Bailey,

and R. H. Schumm, NBS Technical Note 270-1, U.S. Government

Printing Office, Washington, . D.C. , 1965.

19

[11] R. A. Robinson and R. H. Stokes^ "Electrolyte Solutions", 2nd ed.,

Butterworth and Co., Ltd., London, 1959.

[12] C. E. Vanderzee and J. A. Swanson, J. Phys . Chem. 67, 2608 (1963).

20

13. Acknowledgements

We acknowledge the assistance of Ms. Ruby N. Boyd with some of the

calorimetric measurements. We thank George Marinenko for providing the

coulometrically analyzed hydrochloric acid.

21

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Table 3. Blank Heats of Mixing

Simple Mechanical Rotation

Run No. Area (mv-s)

65 -0.0020

67 -0.0038

68 -0.0006

69 -0.0021

75 -0.0015

76 -0.0045

78 -0.0020 •

Average Area = -0.0024 = 0.0011 mv s

Q - -40 ± 18 KiJ

Water vs Water

Area (mvs)

Run No. 1st Rotation 2nd Rotation

188 +0.0114 +0.0045

189 +0.0056 +0.0035

190 +0.0150 +0.0000

191 +0.0075 +0.0009

192A +0.0104 -0.0013

1st Rotation: Average Area = +0.0100 ± 0.002S mvs

Q = +167 ± 47

2nd Rotation: Average Area = +0.0015 = 0.0020 mv-s

Q = +25 ± 33 ^iJ

24

Table 3. (cont.)

0.08 N NaOH vs 0.08 N NaOH (Wet- cell ) Area (mvs)

Run No. 1st Rotation 2nd Rotation

192B +0.0658 -0.0006

193 +0.0710 +0.0009

194 +0.0782 -0.0004

195 +0.0719 -0.0004

196 +0.0568 +0.0020

1st Rotation: Average Area = +0,0687 ± 0.0060 mvs

Q = +1147 ± 100 [iJ

2nd Rotation: Average Area = +0.0003 ± 0.0010 mvs

Q = +5 ± 17 I^J

0.01 N HCl vs 0.01 N HCl (Wet-cell) Area (mvs)

Run No. 1st Rotation 2nd Rotation

198 +0.0010 -0.0004

200 +0.0046 -0.0025

202 +0.0183 -0.0039

203 +0.0227 -0.0094

1st Rotation: Average Area =+0.0117 ± 0.0089 mvs

Q = +195 ± 148 [xJ

2nd Rotation: Average Area = -0.0041 ± 0.0027 mvs

Q = -68 ± 45 ^LJ

25

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OKM NBS-1 14A (1-71)

L'.S. DEPT. OF COMM. BIBLIOGRAPHIC DATA

SHEET

1. PriMTC A l ION OR RKI^OR l NO. NBSIR 73-180

2. Gov't Accession

No.

3. Recipient's Accession No.

4. riTLi; Axn >ubtitle

Testing of the NBS Clinical Microcalorimeter

5. Publication Date 4-73

6. Performing Organization Code

7. AUTHOR^S)

E. Jo Prosen and R. Goldberg

8. Perfornung Organization

NBSIR 7 3-180

9. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

UASHINGTON, D.C. 20234

10. Project/ Task/'^Sork Unit No. jiDU'tii and jioUiiC

11. Contract./ Grant .No.

12. Sponsoring Organization Name and Address National Institutes of Health

Bethesda^ ^^aryland 20014

13. Type of Report & Period Covered

Interim

14. Sponsoring -Agency Code

15. SUPPLEMENT.ARY NOTES

16. .ABSTR.ACT 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here.)

The NBS Clinical Microcalorimeter has been tested for stability^ sensitivity^, ease of operation^ and accuracy. The accuracy was tested by means of electrical

calibration and the determination of the heat of neutralization of HCl(aq) with

NaOH(aq). The heat of this reaction agrees with the best literature value within

the precision of the calorimeter. The precision is about 0.6 percent when measuring

about 50 mJ of heat of chemical reaction. The accuracy is estimated as 1 percent.

The precision of electrical energy determination is about 0,4 percent for energies

of from 1 to 1400 mJo

17. KEY WORDS (-Alphabetical order, separ ated by semicolons; Clinica 1 chemistryj clinical microca lorimetry

;

microcalorimeter: NBS microca lorime try ; testing of microcalorimeter.

18. .AVAILABILITY STATEMENT

^fuNLMTED.

I ; FOR OFFICIAL DISTRIBUTION. DO NOT RELEASE

TO NTIS.

19. SECURITY CLASS

(THIS REPORT)

UNCLASSIFIED

20. SECLT^m" CLASS

(THIS PAGE)

UNCLASSIFIED

21. NO. OF P.AGES

22. Price

USCOMM-DC 66244-P71