U.S. patent number 3,645,691 [Application Number 04/802,421] was granted by the patent office on 1972-02-29 for method of quantitatively determining iodine and thyroid hormones.
Invention is credited to Guenter Knapp, Hans Spitzy.
United States Patent |
3,645,691 |
Knapp , et al. |
February 29, 1972 |
METHOD OF QUANTITATIVELY DETERMINING IODINE AND THYROID
HORMONES
Abstract
Improved method of quantitatively determining iodine or thyroid
hormones according to the method of Sandell and Kolthoff by
replacing sulfuric acid by nitric acid. A preferred measuring
apparatus consists of a thermostat, a photometer, and an evaluating
and plotting instrument.
Inventors: |
Knapp; Guenter (Graz,
OE), Spitzy; Hans (Graz, OE) |
Family
ID: |
3523201 |
Appl.
No.: |
04/802,421 |
Filed: |
February 26, 1969 |
Foreign Application Priority Data
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Feb 27, 1968 [OE] |
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A 1889/68 |
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Current U.S.
Class: |
436/124; 436/87;
436/34 |
Current CPC
Class: |
G01N
33/78 (20130101); Y10T 436/19 (20150115) |
Current International
Class: |
G01N
33/74 (20060101); G01N 33/78 (20060101); G01m
021/24 (); G01m 031/22 (); G01m 033/16 () |
Field of
Search: |
;23/230,253,23B
;252/408 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Wolk; Morris O.
Assistant Examiner: Katz; Elliott A.
Claims
We claim:
1. In a method for quantitatively determining iodine and thyroid
hormones, the steps which comprise
a. admixing arsenous acid, a cerium (IV) compound, and nitric acid
to the test solution containing iodine or a thyroid hormone, the
amount of nitric acid added being sufficient to produce a reaction
mixture containing at least 6 percent, by weight, of nitric acid,
and
b. determining photometrically the cerium (IV) ion concentration in
said reaction mixture at a constant temperature between about
20.degree. C. and about 60.degree. C.
2. The method according to claim 1, wherein the reactants are
admixed in step (a) to the test solution in the order arsenous
acid, nitric acid, and cerium (IV) compound solution.
3. The method according to claim 1, wherein the test solution is
first mixed with arsenous acid, nitric acid, and cerium (IV)
compound solution whereafter the reaction mixture is adjusted to
the constant reaction temperature.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an improved method of determining
iodine and thyroid hormones and more particularly to a method of
determining small amounts of iodine, especially of protein bound
iodine, and of thyroid hormones and to an apparatus for carrying
out such a method.
2. Description of the Prior Art
A number of methods for the analysis and determination of smallest,
submicrogram amounts of iodine or thyroid hormones are known. These
methods are based on the reaction found by Sandell and Kolthoff.
Said reaction proceeds according to the following equation I:
2 Ce.sup.4.sup.+ +As.sup.3.sup.+ .fwdarw.2 Ce.sup.3.sup.+
+As.sup.5.sup.+ I.
Iodine or, respectively, thyroid hormones have a catalytic effect
upon the course of reaction I, i.e., the more iodine or thyroid
hormones are present in the preparation to be analyzed, the more
rapidly proceeds said reaction I. The speed of reaction is
proportional to the iodine concentration or, respectively, to the
thyroid hormone concentration. In this manner it is possible to
determine iodine or, respectively, thyroid hormones even in the
nanogram range. This known analytical method is carried out in the
following manner:
A predetermined amount of a solution of arsenous oxide As.sub.2
O.sub.3 in concentrated sulfuric acid is added to the test solution
and the temperature of the mixture is adjusted to reaction
temperature, i.e., usually to a temperature between 20.degree. C.
and 60.degree. C. A predetermined amount of a cerium (IV) sulfate
solution in sulfuric acid is added thereto. Thereupon, the mixture
is allowed to react at the predetermined temperature for a definite
period of time. Said reaction time is selected in accordance with
the order of magnitude of the amount of iodine to be determined and
with the respective selected reaction temperature. The reaction
time is usually between 10 minutes and 40 minutes. Thereafter, the
content of the test solution of cerium (IV) ions is determined
photometrically. The lower the photometrically determined cerium
(IV) ion concentration is, the higher is the speed of reaction and,
consequently, the amount of catalytic agent, i.e., of iodine or,
respectively, of thyroid hormones. In this manner the iodine or,
respectively, thyroid hormone concentration of the test solution
can directly and quantitatively be determined. However, these known
processes require very long measuring times and are quite
complicated in their execution.
SUMMARY OF THE INVENTION
It is one object of the present invention to provide a simple and
effective method of determining small amounts of iodine or thyroid
hormones which method is free of the disadvantages of the known
processes and can be carried out automatically.
Another object of the present invention is to provide a simple and
effective apparatus for carrying out said novel method.
Other objects of the present invention and advantageous features
thereof will become apparent as the description proceeds.
In principle the method according to the present invention is
characterized by using nitric acid, in place of sulfuric acid as
used heretofore, for acidifying the reaction mixture, i.e., the
test solution, the arsenous acid solution, and the cerium (IV)
compound solution. It is of importance to add nitric acid in an
amount sufficient to produce a reaction mixture containing at least
6 percent, by weight, of nitric acid and preferably between about
12 percent and about 30 percent, by weight, of nitric acid in order
to achieve satisfactory and accurate results. This change in
composition of the reaction mixture increases the sensitivity of
the reaction about twentyfold. As a result thereof test solutions
of the same iodine content which require, according to the
conventional catalytic reaction method using sulfuric acid, about
20 minutes reaction time to demonstrate an observable decrease in
cerium (IV) ion concentration, need a reaction time of only 1
minute, when carrying out the catalytic reaction with nitric acid,
in order to produce the same decrease in cerium (IV) ion
concentration. These results, of course, are achieved while
operating at the same reaction temperature.
The novel process according to the present invention is based upon
these and the following fundamentally changed reaction conditions.
While heretofore the iodine-containing test solution was mixed with
the arsenous acid and the mixture was brought to reaction
temperature in a thermostat, whereafter the reaction was initiated
by the addition of the cerium (IV) compound solution, all the
components of the reaction mixture, i.e., iodine-containing test
solution, arsenous acid, nitric acid, and cerium (IV) compound
solution, are mixed and all together are brought to reaction
temperature when proceeding according to the present invention.
The measuring technique used in the method according to this
invention also differs essentially from the measuring technique of
the known methods. Heretofore, the content of cerium (IV) ions and
thus the iodine content of the test solution was determined only
after termination of the reaction. In contrast thereto the speed of
reaction I is directly determined when proceeding according to the
present invention. This result is achieved by continuously
measuring the decrease in cerium (IV) ion concentration in the
reaction mixture. Reaction I proceeds normally as a reaction of the
first order as expressed by the following equation II:
-(d[Ce.sup.4.sup.+ ]t/dt)=k. [Ce.sup.4.sup.+ ].sub.t II.
Transformation yields equation III:
log[Ce.sup.4.sup.+ ].sub.t =-kt+log[Ce.sup. 4.sup.+ ].sub.t .sub.o
III.
Since the cerium (IV) ion concentration is proportional to the
extinction, the following equation IV finally results:
log Ext.sub.t =log Ext.sub.t .sub.o -k.t. IV
When plotting in a diagram the logarithmus of the extinction
against the reaction time, a straight line with the gradient
results. Said gradient corresponds to the speed of reaction and is
directly proportional to the iodine concentration.
The advantages of this method of determining the iodine content of
a solution over the heretofore known methods are, among others,
that
a. the speed of reaction can be measured at any desired moment
during the reaction and that
b. the resulting curve illustrating the course of the reaction
permits to instantly spot any changes and disturbances in the
course of reaction if it does not proceed as a reaction of the
first order.
Quantitative determination of iodine or, respectively, of thyroid
hormones by the method of this invention is preferably carried out
in a measuring apparatus which takes into account the specific
requirements of the novel method.
Such a measuring apparatus comprises a thermostat, a photometer,
and an evaluation and plotting instrument. The feedpipe supplying
the reaction mixture to the cuvette of the photometer passes
through the thermostat preferably in coiled form. The evaluation
and plotting instrument attached to the photometer transforms the
transmission values observed in the photometer into the logarithmus
of extinction and records the reaction curve log Ext-time. Said
double logarithmic transformation of the transmission values into
the logarithmus of extinction can be carried out
electromechanically by means of a cam disk or fully
electronically.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel method of determining iodine or, respectively, thyroid
hormones according to the present invention and a preferred
measuring apparatus for carrying out such determinations will be
understood more fully be reference to the accompanying drawings of
which
FIG. 1 illustrates schematically the manner in which the method
according to this invention is carried out in such a preferred
apparatus.
FIG. 2 illustrates schematically a preferred evaluation and
plotting instrument.
FIG. 3, which includes FIGS. 3a to 3d, illustrates calibration or
standardization curves at different temperatures and increasing
nitric acid content as they are used for comparison with the
samples to be tested.
FIG. 4 shows a curve illustrating the increase in sensitivity
depending upon the nitric acid concentration.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As shown in FIG. 1, the test solution of which the iodine content
or, respectively, the content of thyroid hormones is to be
determined is placed in flask 1. Arsenous acid which preferably is
prepared by dissolving arsenous oxide As.sub.2 O.sub.3 in sodium
hydroxide solution and weakly acidifying said solution with
sulfuric acid, nitric acid, and a cerium (IV) compound solution are
added thereto and are intimately mixed therewith as indicated in
FIG. 1. The amount of nitric acid to be added must be sufficient to
yield a test mixture containing at least 6.0 percent of nitric
acid. Any cerium (IV) compound may be used in this test. Especially
useful have proved ammonium cerium (IV) nitrate, ammonium cerium
(IV) sulfate, and cerium (IV) sulfate. The resulting reaction
mixture is introduced by suction by means of pump 7 (not shown)
through pipe line 2 into flow-through thermostat 3 whereby it
attains the desired reaction temperature within a few seconds.
Thereafter, the mixture passes into flowthrough cuvette 6 of
photometer 5 which is also kept at reaction temperature. As soon as
the cuvette 6 is completely filled with the reaction mixture, pump
7 is disconnected and the measuring operation starts. Due to the
reaction according to reaction equation I given hereinabove, the
yellow cerium (IV) ions disappear because they are converted into
the colorless cerium (III) ions. As a result thereof the reaction
mixture in cuvette 6 becomes more and more light-transmissive.
Thereby, photometer 5 yields the potential T which is proportional
to the light-transmission. Said photometer potential T is subjected
in evaluation and plotting instrument 10 to a double logarithmic
transformation to yield log E=log(2-log T), E being the extinction
value and T being the photometer potential which is proportional to
the transmission. The resulting line representing the course of the
reaction is recorded by said instrument 10. The gradient (tg
.alpha.) of said line directly measures the iodine concentration
or, respectively, the concentration of the thyroid hormones. After
testing is completed, pump 7 is again operated to draw another test
mixture into cuvette 6 while discharging the preceding test mixture
therefrom. The diagram shown in FIG. 1 below photometer 5 is
obtained when recording the reaction with a linear compensating
recording apparatus. The diagram at the left side of evaluation and
plotting instrument 10 represents the reaction in the form of a
straight line after correction of the nonlinear decolorization
curve.
It may be mentioned that most favorable results would be obtained
by using cerium (IV) nitrate solutions in nitric acid, because the
speed of reaction decreases with increasing sulfuric acid
concentration. However, nitric acid solutions are usually not as
stable as sulfuric acid solutions. Therefore, weakly sulfuric acid
solutions of the reactants to which nitric acid is added have
proved to be especially useful.
Best results are achieved when mixing the test solution with the
reactants in the order arsenous acid, nitric acid, and cerium (IV)
compound solution. When mixing the reactants in another order, it
was found that the reaction in its first part does not proceed
exactly as a reaction of the first order. As a result thereof
curved lines are obtained on evaluation and plotting. The test and
reactant solutions may be mixed at the desired reaction
temperature. The preferred procedure, however, is to first mix said
solutions and then to adjust the temperature of the reaction
mixture in the thermostat to the desired reaction temperature.
FIG. 2 shows schematically a preferred compensating recording
apparatus, the compensating potentiometer 11 of which is driven by
a servomotor 14 by means of cam disk 13 and rack 12 cooperating
with the pinion of the compensating potentiometer 11. The cam disk
is shaped according to the function x=log (2-log T). If such an
electromechanical converter with its recording apparatus is
attached to a photometer and if the curve of the iodine catalysis
reaction is plotted therewith, straight lines representing the
course of the reaction as illustrated in FIGS. 3a to 3d are
obtained. In order to eliminate cumbersome calculation of the
gradient of said straight lines, the recording instrument is
provided with a trailing or drag pen which is dragged or pulled
along from the zero point at the start of the measurement
independently from the light transmission value of the reaction
solution. In other words, the recorder is firmly connected with the
photometer 5 and the pen can be engaged at any given time during
the reaction. From the moment of its engagement the pen records the
straight line representing the course of the reaction for exactly 1
minute. It is then disengaged and is returned to the zero point.
The recorded height of the peak, i.e., the greatest distance of the
pen from the zero line is proportional to the gradient of the
straight line representing the course of the reaction and thus is
proportional to the iodine concentration or, respectively, the
thyroid hormone concentration.
As stated above, the testing and measuring system according to the
present invention has the great advantage that measurement may be
started at any time in the course of the reaction because the
gradient of the straight line representing the course of the
reaction is always of the same inclination independently from the
start of the reaction and at any period of time within the
measuring range.
Thermostat 3, photometer 5, and electronic evaluation and plotting
instrument 10 (FIG. 1) as well as the compensating recording
apparatus may be conventional instruments.
FIGS. 3a to 3d show standardization curves recorded with standard
solutions of known iodine content. It is evident therefrom that the
sensitivity of the reaction which is expressed by the gradient or
rate of increase of the standard curve or straight line, increases
with increasing temperature and increasing nitric acid content. The
peaks of said curves or straight lines are proportional to the
iodine concentration.
A suitable photometer 5 which has proved of value is the
spectrophotometer of the firm Carl Zeiss. It is provided with a
cuvette holder and a 1-cm. flowthrough cuvette 5. The thermostat
coil 3 is arranged within the inflow of said cuvette 5. The outlet
of the cuvette is connected via a shutoff valve to a water jet
pump. As stated above, however, any type of spectrophotometer may
be used for carrying out the method according to the present
invention.
FIG. 4 represents a diagram which shows that the sensitivity of the
reaction is dependent on the nitric acid concentration of the
reaction mixture. The curve illustrates the coefficient of
sensitivity increase. It is evident from the curve that at least a
normal nitric acid concentration is required to produce
satisfactory results and that best results are achieved starting
with 2N concentration.
For determining the iodine content of the solution to be tested it
is passed through the photometer 5 and its straight line of the
course of reaction is recorded. The resulting diagram is compared
with the respective standard diagram of FIGS. 3a to 3d or similar
diagrams recorded at different temperatures and different iodine
content. Thus it is possible readily and in a simple manner to
determine the iodine content of an iodine solution containing even
such small amounts thereof as nanograms, i.e., one billionth of a
gram. When determining the catalytic activity of thyroid hormones,
the iodine value found must be divided by 2, because said hormones
have only 50 percent of the activity of the iodine.
Variations in the amount of arsenous acid have no effect upon the
results. The amount of cerium (IV) compound, however, should be
sufficient to produce a reaction mixture the light transmissivity
of which is at the starting point of the photometer scale, i.e., at
about 5 percent to 10 percent transmissivity, at the start of the
reaction.
* * * * *