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A study of chemical tests for the vitamins

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A study of chemical tests for the vitamins

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Content A STUDY OF CHEMICAL TESTS FOR THE VITAMIN'S
A Thesis
Presented to
the Faculty of the Department of Chemistry
University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Master of Arts
by
William Zenter Bock
June 1941
UM! Number: EP41526
All rights reserved
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UMI
P u t t i i h i r t f
UMI EP41526
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This thesis, written by
.........W I L L I M . . . ^ N T K m . M C K.........
under the direction of h%&. Faculty Committee,
and a p p ro v e d by a ll its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fu lfill
m ent of the requirem ents f o r the degree of
.23AS.TER...QJL.ARTS.
Dean
Secretary
D a te..
Faculty Committee
TABLE OF CONTENTS
PAGE
I- INTRODUCTION . . . . . . . . . . . . 1
Introductory statement . . . . . . . 1
A short summary of the vitamins.......... 3
Review of chemical tests . . . . . . 7
II. EXPERIMENTAL WORK . . . . . . . . . . 10
Proposed experimental work . . . . . . 10
1. Vitamin A ................................ 11
Description of adsorption methods . . 13
Method of procedure for colorimetric
test................................ 16
Standardization of colorimeter
for vitamin A ................... 17
Vitamin A tests . . . . . . . . 19
1. Bills-Wallenmeyer photometer . 19
2. Photo-electric colorimeter . . 20
2. Vitamin .................................21
M e t h o d s ...................... 21
Vitamin t e s t s .......................23
Fluorophotometer for vitamin 26
3. Vitamin C .................................27
Methods ............................. 27
Vitamin. C tests.......................... 29
iii
1. Iodine number .................. 29
2. Indophenol dye titration . . . . 30
3. Photoelectric indophenol method . 31
4. Comparison of methods . . . . 33
III. CONCLUSIONS . ' ................ 35
BIBLIOGRAPHY .........................36
LIST OF FIGURES
FIGURE PAGE
1. Sketch of Klett-Summerson Photo-electric
Colorimeter ............... 11
2. Thiamine Chloride Calibration Curve for
Photo-electric Colorimeter . 25
3. Ascorbic Acid Calibration Curve for
Photo-electric Colorimeter . . . . . . . . 32
I. INTRODUCTION
INTRODUCTORY STATEMENT
Vitamins• have bean known to scientists since 1897
ana have been the objects of research in biology, biochem
istry, and chemistry. The present extreme popularity of
vitamins is due primarily to a concentrated advertising and
publicity campaign which has suddenly caused vitamin products
to be the foremost items of sale at drug stores.
In order to deter misrepresentation and fraud,
vitamin products have been placed under the Pure Pood and
Drug Laws, and labels must contain an accurate statement of
vitamin content. This involves the use of tests for the
vitamins which can be sufficiently rapid and accurate for
use in industrial control and government inspection.
Standard vitamin assays until recently have been
made entirely by biological methods. Such assays have been:
rat growth rate, pigeon growth rate, calcification of bones,
and other effects of the addition of the vitamin in question
to'a diet devoid of that vitamin. The results have been
valid, and quantitative standards are largely based on them.
However, the time factor, a matter of weeks or months, has
made biological assays unfeasible for control work.
Research in vitamin chemistry in recent years has
been directed principally toward the development of chemical
tests. This research has involved first the perfection of
chemical techniques, and second the search for reagents
which are specific for the vitamins, which give results in
the shortest time, and which can be used for quantitative
purposes. Methods of analysis are being developed which de
pend more on colorimetry and spectroscopy than on the older
techniques of gravimetric and volumetric analysis. More
rapid methods of isolation of the vitamins are being used,
but in order to obviate the necessity of isolation in some
cases, attempts are being made to find reagents which are
specific enough to give valid results regardless of inter
fering substances. Chemical tests in most cases may be used
to supplement or to replace the slower methods of biological
assay in industrial work or for inspection purposes.
It is the purpose of this research to investigate
the various methods of chemical tests and to ascertain their
practicability in routine test work.
A SHORT SUMMARY OF THE VITAMINS
At this point it may be well to review very; brief
ly .some of the important points pertinent to the principal
vitamins now known. Hie physiological properties are by now
well known and. this thesis is not concerned with these bio
logical aspects; therefore only chemical points will be con
sidered.
Vitamin A, anti-xeropthalmic, has had no definite
name given to its structure, though the following have been
suggested; "beta-ionone-diisoprenol, semi-beta-earotinol,
beta-ionol, or beta-ionone. “1 Essentially, it is an alcohol
made of a beta-ionone ring having as a side chain a group of
isoprene units;
H3 V CHjH » H H C ^ 3 H
H o/ c — c - c — c = c— c-c — c. — c—
<
Two. molecules of vitamin A could, by losing water, form to
gether a molecule of beta-earotene, but it is more likely
that the vitamin is formed by hydrolysis of the latter.
The beta-ionone ring seems to play some part in the vitamin's
activity, as only those carotenoids containing this structure
can be converted in vivo to the vitamin. Paul Karrer and
1l .S. Palmer, "The Chemistry of Vitamin A and Sub
stances Having a Vitamin A Effect," The Vitamins (A Sympo
sium, American Medical Association, 1939), p. 16.
his associates were instrumental in the determination of the
chemical structure of vitamin A.
Vitamin Bi, the anti-neuritic, or thiamine chloride,
is composed of a thi&zole nucleus attached to a pyrimidine
nucleus:
cl
1 1 w J.
|4C-C C— CL— N C
J 1/ II H H H r r\U
'» i ! n r C---c — C — OH
N C I - / H H
Whether the activity is due to one or both of these groups
is not known, but the vitamin is relatively heat labile and
may be split into two products, analysis of which shows the
existence of tne above nuclei. Thiamine chloride has been
synthesized and appears on the market generally as the syn
thetic product.
Vitamin B2, lactoflavin, or riboflavin, as it is
variously known, owes its now accepted chemical name of
riboflavin to the fact that it is made of a molecule of
d-ribose substituted as a side chain on the center nitrogen
of iso-alloxazine, a compound found in many of the flavins:
\A
— C— OH
H
Riboflavin crystallizes from absolute alcohol as yellow-
orange crystal needles, melting with decomposition at be
tween 240° and 280°C. The synthesis of riboflavin is not
difficult but is hampered considerably by the scarcity of
ribose.
Other members of the vitamin B complex, vitamin
Bg, the filtrate factor, the pellagra-preventive factor, and
nicotinic acid have not been analyzed as thoroughly as have
the foregoing. It is believed that perhaps the total B com
plex is essential im prevention and cure of pellagra.
Vitamin C, known also as cevitamic acid, more pro
perly is ascorbic acid. It is a six-carbon-atom carbohydrate
containing a lactone ring between atoms 1 and 4 and a double
bond between 2 and 3 (2,3-enediol-4-lactone) and is thus re
lated structurally to the hexose sugars:*
H
C — c.— c
I ------- I n
I r C . _- c C . H , OH
o=c— c = c— Y v
o ' h o h h 0H
1-xylose has been used in the synthesis of the vitamin, as
has d-glucose. Ascorbic acid exists in two optically active
states, the laevo having vitamin activity and the dextro none.
Anti-rachitic vitamin D exists in several forms,
all sterol derivatives. The most important are activated
ergosterol and activated 7-dehydro-cholesterols
H . C - C
iC
H H H H
-C— C— C- C— CM
H H H I
CH,
Vitamin E, the anti-sterility vitamin, is alpha-
tocopherol, a chromane nucleus with a substituted isoprene
side chain:
CHj ^
if
H
~c-
ch3 h
H
-C
H
CW
H t •
. c— c,—
H
ch3 cW*
\chJ c ~ cHi
3
(c^Jr c-
W H
The above include the more common vitamins. Others
such as factor H, the growth factors, vitamin P, and the re
cently discovered blood-clotting vitamin K, are of interest
but will not be considered in the work of this thesis.
REVIEW OF CHEMICAL TESTS
The principal chemical tests for the vitamins fall
under three headings. Two of these general type tests are
specific and rapid enough for commercial work, "but the third
is of laboratory interest only and will not be covered exten
sively in this discussion.
The first general-method of vitamin testing in
volves the use of substances which produce a color with the
vitamin. Colors so produced may be measured by any accurate
colorimeter, and the intensity of color in some cases gives
a quantitative estimate of the vitamin content. The photo
electric colorimeter has been found to be of particular
service for color testing, as it is quite sensitive and gives
results accurate enough to be used for standardization pur
poses. As it is also a rapid method, this instrument is of
use in the case of rapidly fading colors such as the brilli
ant blue produced by the reaction of vitamin A with antimony
tri-chloride. In the case of vitamin C, which decolorizes
indophenol dye, it has been found that direct titration with
the reagent is of sufficient accuracy to yield good results.
The recent literature in the field shows six possible color
reactions with vitamin A, only one of which is now used.
Five reactions have been found for vitamin B^, two of which
give results satisfactory for commercial work. Of six pos-
sible reactions for vitamin C, two are of commercial use.
Color tests have been advanced for vitamins D and E, but
none have yet been adopted as being sufficiently specific*
The second general method of vitamin assay is by
speetrographic methods. Absorption spectra data have been
gathered for most of the vitamins and are available in the
literature for research workers. The method used is simples
a solution of the vitamin is irradiated by light of a fre
quency including in its range the point of maximum absorption
of the vitamin, and the amount of absorption measured is a
quantitative estimate. As most of the vitamins have their
maximum absorption within the range of the near ultra-violet,
the obvious difficulties are sufficiently monochromatic
light sources and the question of solution cells which will
allow the passage of the ultra-violet radiation. Quartz is
generally used for the solution cell, but is quite expensive.
Good speetrographic equipment is also very expensive.
A third method for vitamin tests involves the for
mation of precipitates with certain reagent.. However, this
method is unsatisfactory, as no quantitative determinations
may be made due to the fact that the precipitates are gen
erally of an unstable nature and cannot be heated and weigh
ed as in ordinary gravimetric analysis. This method is then
confined to the laboratory, and although it will undoubtedly
be of use in identifying the vitamins as they are synthesized,
it can be of little practical use in assay work.
Thus it may be seen that of the many methods of
identification of the vitamins by chemical means, only a
few are of commercial importance. As only those in that
category will be considered in further work in this paper,
this limits the field considerably.
II. EXPERIMENTAL WORK
PROPOSED EXPERIMENTAL WORK
Search or 't/he literature showed that the following
are the chief chemical tests that have been proposed for
vitamin assays
Vitamin A - the Carr-Price Test with antimony tri
chloride , absorption spectrum and various absorption
methods using a monochromatic light source.
Vitamin - diazotized p-aminoac etophenone and
thiochrome fluorescence methods.
Vitamin C - iodine number, indophenol dye titra
tion, and colorimetric indophenol dye tests.
Vitamin Bo - fluorometric methods.
Vitamin D - absorption spectrum.
It was proposed x-o duplicate tests by these meth
ods in the following cases for which equipment was available.
Vitamin ' Test
A 1. Carr-Price Test
2. Bills-Wallenmeyer absorption test
B1
1. Diazotized p-aminoacetophenone
C 1. Iodine number
2 • Indophenol dye titration
3. Indophenol dye by photo-electric colorimeter
1. VTTAMII'T A
nEBCRIPTIOM OF COLORIMETRIC APPARATUS
The photo-electric colorimeter is the first in
strument to be used in this research and has been used in
connection with vitamin A as well as with vitamin and C.
The particular instrument used was the Klett-Summerson, de
signed by W,H. Summerson and manufactured by the-Klett Manu
facturing Company. Figure 1 shows a diagram of the instru
ment:
---------------- I p
□
c
0S
Figure 1
Klett-Summerson Photo-Electric Colorimeter
Light originates in the standard light source, S,
a small 100 watt lamp, and passes through a lens, L, which
makes a parallel beam. The light is then passed through a
filter, F, chosen according to the color of the solution to
be measured. Part of the filtered light passes through the
solution cell, C, and falls on the working photo-electric
cell, Pg. The rest of the beam strikes the standard photo
cell, P-^. The two photocells are connected in opposition, and
the net current generated is passed through a galvanometer, G.
When the two photocells are in balance, that is, when they
are equally illuminated, the galvanometer pointer is not de
flected and is said to be in its zero position. It is con
venient to set the zero reading of the instrument according
to the solvent to be used. The solvent is placed in a spe
cial test tube in the solution cell, and the position of the
standard photocell in reference to the light source is
changed until balance is again restored.
When a colored solution is placed in the solution
cell, the intensity of the beam falling on the working, cell,
P2, is decreased in proportion to the intensity of color of
the solution, thus producing an unbalanced condition and de
flecting the pointer of the galvanometer. Since the potential
of this cell has been decreased it becomes necessary to mea
sure the new potential in order that the galvanometer may again
read zero. In the wiring set-up, the reference photocell acts
as the potential source for a potentiometer. As the potential
of the working cell is decreased, there is a point along the
resistance of the reference potentiometer which will corres
pond to the new potential of the working cell. This point
is determined by a sliding contact operated by a knob.
13
The knob also operates a dial, D, from which readings may be
made. The dial is graduated in logarithmic spacings, thus
eliminating the necessity of taking logarithms of readings
or of plotting curves on semi-logarithmic paper.
Calibration may be made by plotting a curve for a
series of known vitamin concentrations, using to determine
the zero position a blank of distilled water or the solvent
used, reading concentrations of unknowns from the curve thus
established. The older technique of running a standard con
centration with each unknown may also be used with success.
Unknown concentration may then be found by proper proportion
ality calculations.
DESCRIPTION OF ABSORPTION METHODS
A satisfactory absorption instrument in industrial
use is the Hilger Vitameter2. The construction and use of
this instrument depend primarily on the fact that vitamin A
has a maximum absorption at 3280 Angstrom units. The ab
sorption coefficient, E, is measured on the basis of a 1%
solution 1 cm. in depth and is usually expressed as cm.
This coefficient is read directly on the vitameter, but ac
tually represents an optical constant determined as follows:
^Machine through the courtesy of Dr. Sven Lassen,
Van Camp Cannery.
E = Log I0 (intensity or incident light)
• Log I .(intensity of emergent light)
Production of monochromatic light of the proper
wavelength (3280 Angstrom units) is made possible by the use
of a copper arc whose light is passed through a filter made
of a thin film of silver and a thin plate of Woods glass..
The filtered light is then passed through two slits, behind
one of which is a quartz solution cell containing the vita
min material. Thus ¥/e have produced two beams, one of which
is absorbed, the other uriabsorbed. By an optical system, the
images of the two slits are thrown on a fluorescent screen
in such a manner as to be in juxtaposition. The intensities
of the two images thus produced will be different in propor
tion to the amount of light absorbed by the vitamin. It is
then possible, by varying the area of the slit through which
it passes, to reduce the intensity of the unabsorbed beam
until it approximates that of the absorbed beam. This so-
called match point is used as a measure of vitamin potency
on an already calibrated scale.
It has been found in practice that considerable
eye strain is experienced in the reading of the vitameter
and that readings may vary due to differences in vision of
different operators. For practical industrial use, the in
strument must be kept in a dark room and conditions so stan
dardized that one operator is solely responsible for all
readings. Only .then may valid results be obtained.
15
Another instrument used industrially is the Bills-
Wallenmeyer Electronic Photometer3. Essentially this is a
spectro-photometer with absorption measured photo-electrical-
ly instead of visually. The light source used is an argon
glow lamp provided with a suitable filter, producing nearly
monochromatic light at 3280 Angstroms. The amount of light
is controlled by a variable aperture, operated by the dial
on which readings are taken. To standardize the instrument,
the solution cell is filled with 99% isopropyl alcohol, and
the dial set at zero, the minimum aperture condition. By
proper manipulation, the photo-electric system can be balan
ced to this condition and the galvanometer set at zero. Ob
viously, this is a condition of no absorption. As the sol
vent is replaced by a vitamin A-containing oil, the absorp
tion will reduce the intensity of transmitted light, throw
ing the photo-electric system out of balance. This condition
may be remedied by opening the aperture sufficiently so that
the light transmitted is equal to the original transmitted
light. Since aperture increments are made by adjustment of
the dial, the vitamin potency may be read directly on a pro
perly calibrated instrument. Standard for calibration is
reference cod-liver oil. The material used in the deter
mination was shark-liver oil.
*%se of instrument through the courtesy of Ur. M.
A. Joffe, Culver City Vitamin Refining Company.
16
The technique of operation for a high potency oil,
such as shark-liver oil, is to make a dilution of one to two
thousand of the oil in isopropyl alcohol, reading this dilu
tion, and making proper dilution calculations in obtaining
potency of the oil in question. .Amount of dilution is, of
course, in proportion to potency of the oil. Low potency
oils would not be diluted to such an extent.
With either of the two instruments described above,
their
results would seem to justify/use in quantitative measure
ments of vitamin A.
METHOD OF PROCEDURE FOR COLORIMETRIC TEST
Vitamin A reacts with antimony tri-chloride to
give a brilliant aquamarine blue coloration, in chloroform
solution. The color thus produced must be read within thir
ty seconds of production, as it fades rapidly to a wine-red.
This Is known as the Carr-Price test, from its originators,
and was one of- the original tests for vitamin A. However,
the purity of sample must be great, as carotene and vitamin
D both give similar reactions with the reagent.
Originally, cod-liver oil was used as the vitamin
source for the Carr-Price test, but it was soon found that
vitamin D was present in too high a concentration to give
valid results. A change was then made to shark-liver oil,
which is extraordinarily high in A and contains almost no S.
Better results were observed, particularly in the purity of*
9
color produced and in the stability. Fading did not take
place until thirty seconds, and colors lasted sometimes as
long as a minute.
For qualitative work, the Carr-Price test was
found to be of great value, but from a quantitative stand
point it was not so successful. Results on oils of differ
ent potency showed that colorimetric readings were not linear
functions of concentration and gave only indications of rela
tive potency rather than valid potency measurements.
The technique employed in the Carr-Price test is
as follows: the oil is made up in a 20% solution with chloro
form as the solvent. A second solution is made of antimony
tri-chloride in chloroform to saturation. The ratio of oil
to tri-chloride is ten to one, with about 0.2 ml. of the oil
to 2 ml. of the tri-chloride solution being used as optimum
values for readings.
STANDARDIZATION OF COLORIMETER FOR VITAMIN A
Three samples of oil were used for this purpose.
Two were taken from an industrial concern which furnished
also the potency figures. One sample was of extremely high
potency, and the other was relatively low for industry, but
will be referred to as the medium sample in all calculations.
The third sample, potency up to now unknown, had been allow-
IS
ed to stand for several years and had lost considerable of
its original strength.
The oils were first run on a Bills-Wallenmeyer
electronic photometer which had been previously calibrated
by the use of reference eod-liver oil by another worker.
The industrial high potency oil, since it checked well with
the Bills-Wallenmeyer and the potency assigned it, was used
as the standard for calibration of the photo-electric color
imeter, on which the oils were next run. The Carr-Price
antimony tri-chloride test was used.
Because of the ¥/ide variation in potencies, it
was necessary to use different dilutions for the different
oils in order to keep readings on the scales of the instru
ments used.
Data are shown in Table I, page 19,
19
TABLE I
DATA FOR BILLS-WALLEKMEYER PHOTOMETER
Sample Dilution Reading Potency
High , , 1 in 10,000 71 74
73 72
72 70
av. 72
138,000 IU/ml.
f
Medium 1 in 2,000 69 71
70 70
70 70
av. 70
27,000 IU/ml.
Low
*
1 in 1,000 19 17
17 18
18 19
2,520 IU/ml.
av, 18
CALCULATIONS
Solvent factor - 6 scale readings
Instrument factor - .21 (Units per scale division)
Calculation method:
Reading - solvent factor --corrected reading
Corrected reading x instrument factor
x dilution factor = potency
Sample calculations:
High: (72 - 6) x .21 x 10,000 = 138,000
Medium: (70 - 6) x .21 x 2,000 r 27,000
Low: (18 - 6) x .21 x 1,000 - 2,520
TABLE II
2 0
Sample
High
(Standard)
Medium
Low
DATA FOR PHOTO-ELECTRIC COLORIMETER
Dilution
1 in 110
1 in 20
1 in 10
Reading
510
500
510
av. 507
525
630
535
av. 530
107
109
105
av. 107
Potency
138,000 lU/ml.
26,500 lU/ml,
2,675 lU/ml,
Using high potency oil as standards
507 scale divisions * * » 138.QOQ IU
110 (dilution factor)
Therefore:
1 scale division • 2.5 IU
Calculation method;
Reading x 2.5 x dilution factor * potency
Sample calculations;
High: 507 x 2.5 x 110 » 138,000 IU
Medium: 530 x 2.5 x 20 - 26,500
Low: 107 x 2.5 x 10 = 2,675
2. VITAMIN Bi
Of several tests for vitamin B^, the one most near
ly suitable for use ?/ith the photo-electric colorimeter is
one suggested by Melnick and Field4, based on a reagent pre
pared by Frebluda and McCollum^. Diazotized p-aminoaceto-
phenone is the reagent used and is prepared according to the
following directions:
Solution A - 3.18 g. of p-aminoacetophenone, dis
solved in 46 ml. of concentrated hydrochloric acid and dilu
ted to final volume of 600 ml. with water. This solution
may be kept for months away from strong light.
Solution B - 22.5 g. of MaNQg in 600 ml. of solu
tion. Solution must be kept at a low temperature and must
be made fresh every few days.
Solution C - 20 g. of NaOH dissolved in 600 ml. of
water, 28.6 g. of NaBCOs added and made up to final volume
of 1 liter.
Diazotization is carried out in an ice bath. At
first, equal parts of A and B are mixed, then four parts of
solution B are added and the mixture stirred for ten minutes
at a temperature between 0° and 5°C.
4J. Melnick and H. Field, ' ‘ Thiamine in Urine j'"
Journal of Biological Chemistry. 127:606-531,1939.
5H.J. Prebluda and E.V. McCollum, "A Chemical Re
agent for Thiamine," Journal of Biological Chemistry. 127:
496,1939.
2 2
The final reagent is made by mixing 20 ml. of the
diazotized material to 275 ml. of solution C and adjusting
the pH to between 5.0 and 6.0. The final reagent should be
stirred for five minutes or longer to remove a purple color
ation produced. It is then ready for use with the thiamine
chloride. Thiamine chloride test solution is made to the
desired dilution and is added to the prepared solution gen
erally 10 ml. of the thiaminecehloride solution to 20 of the
prepared solution. A reddish color is produced which devel
ops best when left overnight. The reddish color thus pro
duced forms a precipitate in water but is soluble in xylene.
Therefore the material is extracted with about 5 ml. of
xylene and the color evaluated with the photo-electric color
imeter. Standards used were vitamin Bq tablets manufactured
by the Merck Chemical Company.
This technique was used, as well as a simpler tech
nique using toluene as the solvent with the same reagent.
Here the standard used was pure thiamine hydrochloride madeo
by Merck. Tests were made on a standard vitamin syrup,
sold in drug stores. It was found that excellent color
could be observed for dilutions corresponding to as low as
5 micrograms of thiamine chloride. However, readings could
be made with accuracy only with (solutions labbve 20 micrograms,
dilutions between 20 and 100 micrograms yielding excellent
results by the colorimeter.
TABLE III
23
BATA FOR VITAMIN
Standard used for calibration of the instrument
was 3 isg. tablets which assay 900 IU of vitamin %. One
tablet was dissolved in 100 ml. of water giving a solution
which contained 30 micrograms per ml. (about 9 IU per ml.).
Duplicate samples were taken containing varying amounts of
thiamine, treated with twice the volume of freshly prepared
diazotized solution, and extracted with 10 ml. of xylene
after standing overnight.
Results;
Dilution Reading
9IU 11
16
27IU 35
29
45 IU 43
43
90 IU 96
98
135 IU 145
150
Blank
0
24
TABLE III (Cont.)
Standardization runs were made using the simpli
fied p-aiafnoacetophenone method before mentioned. In this
case the diazotizatiom. was carried out during the course of
the preparation of reagents instead of as a separate step,
thus saving considerable time. By using a 2.5 N sodium hy
droxide solution, the reaction was made to proceed more
rapidly, and tests could be made immediately, obviating the
necessity for waiting over night for colors to develop.
A few results are here listed, showing their close
agreement with the curve established (Figure 2, page 25).
A vitamin syrup was used as well as pure thiamine chloride.
Calculated
potency
Reading Potency by
curve
4.5 IU 6 ' 7 IU
4.5 IU 5
5 IU
25.5 IU . . 30 27 IU
25.5 IU 27
25 IU
51.0 IU • 59 52 IU
63.0 IU 77
69 IU
125.5 IU 142
129 IU
Color*'
CALIBRA'
PHOTO-ELECTRIC : COLORIMETER}
: 1 L__
»70
120
iio
So
‘ r
XO
to
zmiR-*
St 63 7 1 Ir/ 1o 1i
V i t a m i * "Potency ( l . U . )
m
FLUOROPHOTOMETER FOR VITAMIN \
Thiamine gives on oxidation a product known as
thiochrome which exhibits a strong bluish fluorescence. The
test is usually carried out in a methyl alcohol solution by
the use of potassium ferricyanide and sodium hydroxide. This
gives an excellent qualitative test for the vitamin which
can be made quantitative by the use of a fluorophotometer.
The test is finding some industrial application but is not
widely used due to the cost of the apparatus and its limited
use.
In the fluorophotometer, an exciting beam from a
standard light source strikes the solution in a cell, caus
ing the solution to fluoresce. The fluorescent light strikes
a photo-cell, causing an electric current which is measured
with a sensitive galvanometer. As fluorescence depends on
intensity of exciting light and concentration of solution,
it can be seen that if a fixed exciting intensity is used,
the concentration may be found from the amount of fluores
cence. Thus the galvanometer readings may be rather easily
calibrated in terms of vitamin potency. ’
Determinations were not made by such an instru
ment as none was available for use.
3. VITAMIN C
The first, method used for vitamin C was the direct
titration of the material with indophenol dye. The dye was
made up to a strength of 50 mg. per 100 ml. of hot water
and had to be restandardized often, as it is unstable in
water solution. The ascorbic acid source, lemon juice in
this case, was made up fresh each time in a 3% solution of
meta-phosphoric acid. The dye substance reacts rapidly with
ascorbic acid in a reduction process which removes the color.
The photo-electric colorimeter was also used in
the determination of ascorbic acid by indophenol dye reduc
tion. The decrease in concentration of the dye was in this
case measured, using an amount of ascorbic acid insufficient
to reduce completely the amount of indicator used.
Another method6, the iodine number, was also used.
An approximately 0.1 N solution of NagSgOg was prepared and
standardized against KMnG4 . In use, this solution was di
luted with boile d carbon dioxide-free water to approximately
0.01 N. An approximately 0.01 N iodine solution, containing
20-25 g. of KI per liter was prepared and standardized, just
before use, against the HagSg03 solution, using starch as
the indicator. KIO3 also be used in this determination.
^Method used by the California Bruit Growers
Association.
28
Approximately 20 g. of natural strength juice was
placed in a 250 ml. erlenmeyer flask and to this was added
20 ml. of 10% meta-phosphoric acid. An excess of the 0.01 N,
iodine solution was added from a burette and the flask allow
ed to stand for thirty seconds, after which an excess of
freshly diluted 0.01 N NagSgOg was added, and then 2 or 3
ml. of sensitive starch indicator. Then more iodine solution
was added until the color of the juice just began to darken.
The total amount of iodine added, less the iodine equivalent’
of the NagSgOg solution, equals the iodine used in the titra
tion. It was- found that approximately 1 ml. of 0.01 N iodine
solution is equivalent to 0.88 mg. of ascorbic acid. The
standard used at present is natural lemon juice, 0.1 ml. of
which is approximately equal to 1 I.U. of vitamin C.
Due to the fact that other reducing substances ex
ist which would have the same effect as the ascorbic acid on
the indophenol dye as well as on the iodine, it is necessary
to carry out the titrations rather rapidly. For this reason,
it is perhaps more accurate to use the photo-electric color
imeter, as the rate of fading due to other interfering sub
stances can be plotted against time.
The iodine number method ?,rould not be reliable in
the presence of unsaturated oils because these also absorb
iodine. Therefore it is used only with such sources as the
pure vitamin C or fruit extracts. See Table IV, page 29.
TABLE IV
29
IODISE MJMBER BATA
Standard used - commercial ascorbic acid, made up to strength
of 25 mg. per 100 ml. or 5 IU per ml. of solution.
BagSr£>s solution, 0.093 ¥ (standardized against KMnQ,^) ,
diluted to 0.0093 H for use in titration.
Iodine in KI solution, 0.012 H (standardized against KagSgOg)
Amt. as- 511. Ig Ml. BagSgOg lo equiv. Ig used Mg. acid
corbic or ¥agS^>3 for as- per ml.
acid____________________________________________ corbic Ip soln.
2.5 mg. 2.9 2.0 1.5 2.55 0.98
1.15
4.05
2.5 mg. 3.0 3.0 2.3 2.20 1.13
2.5
5.5
2.5 mg. 3.0 2.0 1.5 2.40 1.04
0.9
3.9
5.0 mg. 6.0 3.0 2.3 4.8 1.04
1.1
7.1
5.0 mg. . 5.5 2.0 1.5 5.0 1.00
1.0
6.5
5.0 mg. 6.3 3.0 2.3 4.9 1.02
0.9
'7.2
av. 1.035
Since the iodine used in titration was 0.012 I f f , the ascorbic
acid equivalent of 1 ml. of 0.01 Iff iodine= 1.035/1.2 = 0.86
Hie literature records 0.88 as the approximate quantity.
30
TABLE ¥
INDOPHENOL BYE TITRATION
Strength of dye solution: 50 mg. per 100 ml.
Strength of ascorbic acid solution: 25 mg. per ml.
Amount of ascorbic acid Amount of dye Mg. ascorbic
per ml. dye
0.25 mg. 0.9 ml. 0.278
0.95 ml. 0.263
0.85 ml. 0.294
av. 0.90 ml. 0.278
0.50 mg. 1.S5 ml. 0.270
1.80 ml. 0.278
1.80 ml. 0.278
1.75 ml. 0.285
av. 1.80 ml. 0.278
0.75 mg. 2,70 ml/ 0.278
2.73 ml. 0.274
2.68 ml. 0.280
2.72 ml. 0.275
2.70 ml. 0.278
av. 2.71 ml.
0.276
Each ml. of dye corresponds, on the average, to 0.278 mg. of
acid, or since 1 mg. of ascorbic acid equals 20 IU,
20 x 0.278 = 5.560 IU. 1 ml. dye = 5.560 IU.
TABUS VI
3 1
PHOTOELECTRIC METHOD
Strength of ascorbic acid, solution: 25 mg. per 100 ml.
Strength of dye: 5 mg. per 100 ml. of sodium acetate solution.
Technique: The original strength indophenol dye (50mg./100ml.)
was diluted to 1 in 10 with a dilute solution of sodium ace
tate brought to a pH of 7 with a few drops of acetic acid.
10 ml. of the dye solution were placed in a colorimeter tube
and read on the photo-electric colorimeter. To the tube was
then added, in 0.1 ml. portions, sufficient standard ascorbic
acid solution to reduce the dye. Readings were taken after
the addition of each portion until a constant value was ob
tained, which could be assumed as the end point.
Zero setting - distilled water.
Filter - blue.
/
Amt of C Colorimeter Reading
Sample I Sample II Sample III Sample IV Ave
0.000
240 240 240 240 240
0.025
230 226 224 228 227
0.050
208 206 202 206 205
0.075
185 182 182 190 184
0.100 166 163 168 161 164
0.125
147 148 143 145 146
0.150
127 132 124 128 128
0.175
108 109 107 110 108
0.200
. 92 90 93 88 91
0.225
72 71 74 66 71
0.250 58 49 59 47 53
0.275
33 30 37 32 33
0.300
21 26 23 24 24
Celorime?**' tf5
25*
(ASCORBIC AC ID. C .TION
COLORIMETER
PHOTO-
iff
■ ■ ■ T f - X j i r - J *
RSniiS
0.35-0
N O . 6 J M . J E S S E RAY M i L L E R , L O S A N G E L E S
ftilli ffafcis ofr ftscorbi'c- A g!A
TABLE VII
33
!
COMPARISON OF METHODS FOR VITAMIN C USING UNKNOWN
LEMON JUICE SAMPLES
I. Iodine number method: 20 ml. of natural juice used.
(1 ml. 0.012 N iodine solution * 1.035 mg. ascorbic
acid. The iodine titration value represents the average of
4 - 5 determinations.)
Sample Ml. Iodine used
mg.
Potency
mg./ml. IU/ml
I 8.90 9.20 0.460 9.20
II 8.50 8.80 0.440 8.80
I U
9.60 9.85 0.492 9.85
II. Indophenol dye titration: 1:1 in 3% m-phosphoric acid.
(1 ml. of dye * 0.278 mg* ascorbic acid.)
Sample Eye used per lye used per Potency of juice
ml. dil. juice ml. pure juice mg./ml. IU/ml.
I 0.80 1.60 0.44-5 8.90
II 0.70 1.40 0.390 7.80
III 0.85 1.70 0.472 9.44
34
TABLE VII (Continued)
III. Photoelectric indophenol methods
(For value of reading, see graph, pg. 32)
Sample Dilution Beading Potency of juice
mg. /ml. jLU/ml.
I 1*1. 80 Q;440 8.80
II 1:1 102 0.380 7.60
I I I . 1:3 155 0.480 9.60
Sample
Iodine
Number
Method I
COMPARISON
Direct
Titration
Method II
Photometer
Measurement
Method III Average
I 9.20 8.90 8.80 8.97
II .8.80 7.80 7.60 8.07
III- 9.85 9.44 9.60 9.63
It will he noticed that the iodine number shows a
slightly higher value for the samples chosen than the other
two methods. The indophenol titration method and the indo
phenol photo-electric method check reasonably well with each
other, but end point difficulties in the titration method
would seem to indicate that the photo-electric method is the
more desirable. Its accuracy is probably higher than that
of the iodine number method because of the difficulties and
chances for error in the standardization of solutions for
the latter.
III. CONCLUSIONS
In view of the success of* absorption spectro-pho-
tometers in the assay of vitamin A, it seems probable that
some similar devices may be used for the other vitamins in
commercial analysis. Much is known in regard to absorption
spectra of the other vitamins, but as yet the cost of spec-
trographie materials is prohibitive for commercial purposes
unless real mass-production methods are to be used in which
the cost is justified.
From the experimental work done, there are a few
general conclusions which may be drawn as to colorimetric
processes. Since even the titration processes used for
vitamin C seem to be slightly inaccurate due to end-point
difficulties, it would appear that some instrument such as
the colorimeter is necessary in vitamin testing for accurate
determinations of color for quantitative readings. Yet true
colorimetric tests seem to be quantitatively accurate only
in the cases of Bi and C and have as yet not yielded valid
results for the other vitamins. It would seem that other
methods should be investigated.
More research must be carried out on absorption
by the vitamins and less expensive devices be developed.
BIBLIOGRAPHY
Bessey, Otto A., “A Method for the Determination of Small
Quantities of Ascorbic Acid and Dehydroascorbic Acid
in Turbid and Colored Solutions in the 'Presence of
Other Reducing Substances," Journal of Biological
Chemistry, 126, December, 1938.
Chemical Abstracts, 1930 through |94X.
Melnick, J. and Field, H., "Thiamine in Urine," Journal of
Biological Chemistry. 127:605-531, 1939.
Merck and Company, Annotated Bibliographies of the Vitamins,
1940.
Mindlin, R.L. and Butler, A.M., "Ihe Determination of
Ascorbic Acid in Plasma: a Macro and Micro Method, "
Journal of Biological Chemistry,122:673, February, 1938.
Prebluda, H*J. and McCollum, E.V., MA Chemical Reagent for
Thiamine,1 1 Journal of Biological Chemistry, 127:495,
1.939.
Report of California Citrus (lowers Association (Unpublished)
Summerson, W. H., "A Simplified Test-tube Photo-electric
Colorimeter, and the'Use of the Photo-electric Color
imeter in Colorimetric Analysis," Journal of Biological
Chemistry. 130:149, 1939.
The Vitamins - A Symposium. Chicago: American Medical
Association, 1939. 613 pp.
"Vitamin Symposium, "Journal of Industrial and Engineering
Chemistry. Analytical Edition, 13:209-231, April, 1941.
(Published subsequent to the experimental work of this
thesis.)

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Asset Metadata

Creator Bock, W. Z (author)

Core Title A study of chemical tests for the vitamins

Degree Master of Arts

Degree Program Chemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, organic,OAI-PMH Harvest

Language English

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Advisor [illegible] (committee chair), Copeland, C.S. (committee member), Vollrath, Richard E. (committee member)

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