THE OSAZONE METHOD FOR
DETERMINING ASCORBIC ACID
IN FRUITS AND VEGETABLES

Thank for tho Degree of M. S.
MICHIGAN STATE COLLEGE

Vivian Harris

1944

 

Date

This is to certify that the

thesis entitled
presented by

has been accepted towards fulfihnent
of the requirements for

Major professor

V/YV

THE OSAZONE METHOD
FOR DETERMINING ASCOEBIC ACID IN

FRUITS AND VEGETABLES

By

Vivian Harris

A THESIS
Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfilmnt of the
requiranents for the degree of

MASTER OF SCIENCE

Department of Bacteriology
1944

Acknowledgement

Grateful acknowledgement is hereby
given to Dr. P. W. Fabian whose never-
i’ailing interest and guidance have made
this work possible.

Acknoeledgement is given also to
Mr. c. K. Wadsworth and Mrs. L. R. Guile
whose friendly encouragement and assist-

anee have been invaluable.

x" t \ '54 i ‘ H

1 ‘-I ‘5
-3:3\jd\}&)

me.

table of Contents

Introduction................................................l
Literature Review.........................................."8
Preliminary Experimental York

she oeaaone nethed................................6

Pure ascorbic acid solutions......................9
asp-21.1mm

Preparation of eample............................16

rhe dye titration nethed.........................l$

rhe dye phetelceeter method......................l7

rm seasone method...............................28
Baeults....................................................25
General Discussion".......................................38

mneeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaeeeeeeeeeeu

Bihliognm000000000.00.0.0....00...OOOOOOOOOOOOOOOOOOOOOO“

Wan

For three hundred years medical science has been aware of a substance
in foodstuffs which protects against scurvy. It was in 1747 that Lind
round that the feeding of oranges and lemons to British sailors would
prevent or cure the disease. Progress in the study of this vitamin'was
slow until its isolation from lemon Juice by King in 1932.

Chemically, ascorbic acid is a six-carbon compound closely related
in structure to the simple sugars. Since it is a potent reducing sub-
stance, early chemical methods of measuring it were based on this preperty.
such as the one by Tillmans in 1927 using dichlore-phenol-indcphenol dye.
At the present time there are a great many methods and modifications for
detennining ascorbic acid.using various dyes and newer equipment such as
phctelcmeters based on the reducing ability of ascorbic acid.

Further study of this vitandn. however, revealed two fonns'besides
the active one, both.with.antiscorbutic preperties (9) yet no reducing
ability. One was called dehydro ascorbic acid, an cxixided fem” the
other ascorbigen, a combined form.of ascorbic acid with a protein. Only
in recent years have chemical methods of measurement been made available
for these two forms. Roe and his collaborators (29,30) have now deve10ped
a highly specific chemical method for determining the active and oxidized
forms of vitamin C by means of a csazcne reaction. Thisxnethcd was origin-
ally“'0fk9d out for body fluids but was recently'mcdified (31) so as to be
applicable to plant tissues.

who present work; as originally planned, was to be a study or the
applicability of the first Roe method for determining the active and

oxidised forms of vitanin o in fruits and vegetables. For carparative

-2...

purposes the ascorbic acid content of the same substances was determined
by dye titration and by the photelaneter methods. Upon publication of the

modified method by Bee gt, 3], for plant tissues, it was also included.

.3-

LITERATURE REVEW

The occurrence of fully developed cases of scurvy is, fortunately.
relatively rare in civilised cointries at the present time. Gannon even
in the days of the Greeks. the plague took a heavy toll of hmnan lives for
hundreds of years. Even Vascc do Gama lost a hundred men free his original
crew of a hundred and sixty fran the disease. No tonic or drug soared to
prevent the palpitations, the swelling and tenderness of the legs. the
bleeding gums, the vomiting, diarrhea, - all the cannon symptans of frank
scurvy. Finally in 1720 Kramer (11) found that citrus fruits both pre-
vented and cured the disease and thirty years later Lind, a British naval
surgeon, recumnended that all British sailors be required by law to have
fresh citrus fruits. Progress was slow, however, because of the lack of
quantitative methods of determining the antisoorbut ic properties in various
food. Impetus to the systematic study of the occurrence of the vitamin in
food material and its stability was given by the tremendous problan of
civilian and army rationing during the first World War. Thus the begin-
ning of our modern knowledge of the quantitative determinations are found
in the work of Holst and Frolich, (14) in 1907. Using the guinea pig
method they made conparisons of the ant iscorbut ic value of foods and gave
specific directions as to the preparation. Check and Hume (5). two
Englishnen, continued the studies of Holst and Frolich.

The real progress in the systematic study of vitamin C began with its
isolation from lemon Juice by King (18) in 1932. Then its structure as a
six carbon canpound related to the simple sugars and having a empirical
formula of 063506 was established. Fran the lmofledge of the chemical pro-

perties of ascorbic acid, chemical methods of detennination were devised

which today have almost entirely rcplaced the biological guinea pig method
of assay. Tillmns (37) relying on the reducing ability of vitamin e de-
veloped the first chenical method and the one still most widely used today.
‘Using a dye solution of 2.6 dichlorophenol indophenol, only the reduced
form of ascorbic acid was measured. Later workers introduced.modifications
when sulfhydryl compounds (6), ferrous and ferric compounds (1) reductones
(25) were found to interfere. Other dye solutions were used also such as
l'auber's method (36) with Prussian blue and Lund's (23) method using methy-
lene blue. When photelmeters were introduced, other methods of determinat-
ions were developed mking use of them such as the methods of Mindlin and
Butler (24), Bessey (2), lNelyn (8), and lbsssner (38).

All the dye titration methods, haever, measured Just the reduced
form of vitamin 0. Dehydro ascorbic acid could be determined by reduction
with hydrogen sulfide to the active form and then titrated with the dye sol-
ution. The procedures (8,?) which have been published are contradictory
and not adapted to routine procedure. Therefore, the dehydrorascorbic acid
content materials has been fairly cmsistently disregarded as too unstable
to be measured (4). In 1944 a new chemical method was published by Bee and
Oesterting (31) which was specific for dehydro and.which.opened this field
for investigation.

Not satisfied with the dye methods of measurement as being specific
enough, Boo, a biochemist at George washington‘University, became interested
in developing'a method based on a principle other than oxidation-reduction.
He found tint by using 2, 4 dinitrophenyl hydrazine, an osazone derivative
of dehydroascorbic acid would be fanned and accordingly published his first
method in 1939 (29). Later modifications of the original method were de-

veloped for blood and urine in 1943 (30) and for plant tissue in 1944 (31).

 

Ill’l’l'lf vi" |Ill i.‘

The osasone method is suitable for routine work and is widely used in
laboratories at the present time.

Iith the advance in methods, the comparative value of foods has
becone comnon knowledge. Mustard greens, peppers, cauliflwer, cabbage,
parsley, and strawberries were found to have high ascorbic values and
nutritional requiranents for lnnnans were also determined. the unfavorable
effect of heavy metals especially copper (12), of high taperature (19),
cf exposire to air (15.17.38), of dissolved oxygen (19) of enemas (37)
and many other eondit ions has been deten'nined. Favorable methods (16,ld,d)
of retaining the vitamin have also been worked out. Previous work would
fill several volumes and even though frank scurvy itself has alm0st disap-
peared, surveys have shown that subclinical vitamin C is still widely
prevalent. The work of the past must be continued in the tuture until proper

nutrition has eradicated vitamin 0 deficiency entirely.

PRELmINARY EXPER IMENTLL WORK

sas t

The color react ion as published by Bee and Kuether (30) determines
active and dehydro ascorbic acid in 4 percent trichloroacetic by oxidation
with norit. The activated charcoal, norit must be freed fran traces of
iron and other interfering metals by repeated washings with 10 percent
hydrochloric acid and distilled water. An aliquot of the norit treated
filtrate was stablised fran further oxidat ion by the addition of one drop
of a 10 percent alcoholic solution of thiourea. 2, 4 dinit rophenyl
hydrazine reagent then formed the red osasone crystals when held at 37° 0.
for three hours. At the end of this incubation period, the solutions are
iced and 85 percent sulfuric acid is slowly added to dissolve the osasones.
The intensity of the color produced is the measure of the ascorbic acid
content as read in a photeloneter with a suitable filter. 111 photelometer
work was done on the dance. Sanford, and Sheard type using a green filter
of 525 wave length.

The chemistry cf the method is as follows:

 

e=°
“h “N
nos 0 mi 0 a c o
l
H; _J Oxidat ion a? J
300)}: soon
3303 egos

l - ascorbic acid dehydro ascorbic acid

 

G a O H
)\ (,3 r. n . n
0 3 O N
I o s n o = o\ oz
0 = c r—n - us a ) o
I J -n - n = o
no no, r {J 1102
I + ——-—) 30
soon 1102 |
I soon
soon so; \
a \ noon
2,4 dinitro 302 H
phenyl hydrazine
dehydro asccrb is Osasone

acid

The follmving modifications d the original procedure were investi-

gated:
1. Effect of tenperature

Because 6 the desirability of decreasing the time of the determin-
at ion, temperature variations were made. Pure ascorbic acid solutions in
four percent trichloroacetic prepared at the same time were placed at 37° 0.
for three hours and 55° 0. for various ”t imc periods. Repeated experiments
with standard solutions incubated in a water bath at 55° 0. for one hour
show the same results as B” O. for three hours as seen in table 1.

Since the results appear to be consistent, a one-hour incubation
period in a 55° 0. water bath was adopted rather than the original three

hour incubation period at 37° 0.

2. Effect of oxidizing agents
Since norit is of uncertain origin and carposition, the extent of its
oxidation would be difficult to prove. Aerosol, iodine, copper chloride,
and potassium permanganate decolorised with hydrogen peroxide were tried

in an attempt to oxidize the active ascorbic acid present further than the

-8-

dehydro form. Paget and Berger (26) feimd that permanganate oxidized
ascorbic acid to oxalic acid. If any oxidation products beyond dehydro-
ascorbic acid such as diketogulonic acid, 1-threonic acid, or oxalic acid
fanned osazones, the method would not measure true physiological activity.
The results obtained are shown in table 2.

It is evident than that other oxidising agents allow osasone formation
to the same extent or more than norit itself. Since ascorbic acid has been
foind to be easily oxidized and thereby destroyed (12), the osasone forma-
tion is difficult to explain when coPper chloride or permanganate are used.
However, the only breakdown product available in pure form is oxalic acid,

solutions of which gave no osasones when tested.

3. Effect of various amounts of norit
When standard runs were made weighing the norit analytically to see
if any appreciable amount of ascorbic acid was absorbed, tests show that
absorption did not occur unless the quantity was increased to three times

the amount called for.

4. Effect of acidity
Norit oxidises only in acid solutions having a pH of two or less.
A reagent such as acetic or trichloroacetic is necessary, metaphosphoric
acid standard solutions forming no osasones. Acetic acid is absorbed by

the norit to give active oxygen needed for rapid oxidation of ascorbic acid.

5. Effect of agar
Since sugars form osazones, dextrose was added to standard ascorbic
acid solutions and the osasone procedure followed through to determine if

increased photelcmeter readings would be obtained. Pure dextrose and cans

sugar solutions of two and ten percent do form osazones, which can be read
as ascorbic acid on the phot elometer. However, when a five percent sugar
solution was combined with ascorbic acid in trichloroacetic, no increase
was obtained until the standard reached 24 gamma per milliliter. After this
level was reached, the increase was unmistakebbly large showing that anger

in high concentrations would interfere.

23:9 ascgbic agid 39133193;

Previous to any sample determinations it was necessary to know how long
the ascorbic acid content would be measureable by the osazone method. To

obtain this information the following effects were studied.

1. Effect of tanperature
1. 25° 0.

The results are shown in table three.

While the dye titration showed conplete disappearance of vitamin C in
distilled water in two hours, the osazone method showed no change and even
increased on standing. Evidently the ascorbic acid forms a stable compound
as measured by the osazone method.

2. 55° 0.

Since ascorbic acid is readily destroyed by heat (16,21,22), standard
solutions in water were incubated at 55° C. to see if the osasme method
would bring this out. Results of unusual stability are shown in table 4.

There is then considerable destruction of pure ascorbic acid in water
solution when held at 55° C. for 24 hours especially in the higher concen-
trations. Little or no further decrease is found when the same samples re-in
at roan tenperature for six days. If ascorbic acid it self in water solutions

is stable for this length of time, samples would be suitable for testing if

held in acid solutions at five° G. overnight.

3. Effect ofaeration on standard ascorbic acid solutions.

In 1941, Strohecker and others (34) found tint the stability of ascorbic
acid was more affected by the oxygen of the air than by temperature. A
series of aeration experiments using. standard ascorbic acid solutions was
undertaken using the osazone method of measurement. The results appear in
table five.

Since the stability was greater than would be expected, aeration of
pure ascorbic acid in water was repeated many times using different samples
of vitamin 0, different distilled water, and even vacuum to suck in roan air.
Results revealing unbelievable stability by the osazone method were also
verified by the dye titration method. I

In contradiction to Keys work also (17), the reason for pure ascorbic
acid in water to disappear by dye titration measurement in two hours but
ranain 70 percent after two days aeration is unknown.

4. Effect of hydrogen sulfide and nitrogen treatment
on ascorbic acid oxidized by norit

Only reversibly oxidised solutions of ascorbic acid are anticorbutic
acid and therefore physiologically valuable. Further oxidation products be-
yond dehydroascorbic acid are neither reversible by reduction nor active in
disease prevention. In using the osazone method it was extremely important
to know if the resulting norit treated solutions were reversibly oxidized.
Positive results given in table seven are one of the strongest facts in favor
cf the specificity of the osazone method as a true measure of active and
dehydro ascorbic acid.

The procedore for reducing dehydro ascorbic acid to active ascorbic

-11-

acid by Bessey (2) was used. Pure ascorbic acid solutions were oxidized to
the dehydro form and buffered to a pH of 3.5 with.a citrate buffer. Hydrogen
sulfide was bubbled through the buffered solution for 30 minutes and after

24 hours nitrogen was bubbled through the same solutions for two hours. The
hydrogen sulfide reduces the dehydro ascorbic acid and the nitrogen drives
off the hydrogen sulfide. All the ascorbic acid is then present in the re—
duced form and is measured by'dye titration. Iodine is the oxidising agent
in common use for converting ascorbic acid; hence it served as a comparison

to norit.

25212.11 Photelometer readings of standard solutions of ascorbic acid by
the original osazone method.

 

 

 

m. sg° o.+ 55° 0.

:’°l°!"1° 3 hr. 1 hr. 2 hr. 5 hr.

0.0 99 99 ' 99 99

4.5 w 95 91 92 92

5.0 90 57.5 57.5 55.5
11.2 ' 55 as 52.5 as
15.0 79.5 75 75 75
24.0 72 7o 57 55
32.0 54 54 59 5s

40.0 58 5.5.5 J L

 

 

 

Tab}: 2. The effect of oxidizing agents on standard solutions of ascorbic
acid as shown by photelcmeter readings with the osazone method.

 

 

 

gm. Oxidising agents

an”: norb 1. Norit Nothing 0u012 Aerosol mm 3202
0.0 99 98 99 99 99
8.0 88 89 88 88 88

16.0 77.5 80.0 66 .5 78.5 80

24.0 69.5 74.5 58 .5 71 71

 

.13-

T.p;g_§‘ Comparison of standard ascorbic acid solutions of five milligrams
per 100 milliliters by dye titration and osazone method.

 

Time in hours Osasone method

 

of standing ascorbic acid int idscorbic acid Ascorbic acid in
at 25° di til d.wa wa c
0 88 glfigg 11.2
0.85 12.9
0.60 9.0
0.75 5.8
1.0 88 3.6 15.1
2.0 88 0.7
3.0 88 9.2
4.0 89 6.9
5.0 4.1
24 85
48 81
72 81

 

7.—

W The stability 1 ascorbic acid in water by the osazone method.

 

”minivans per pH of bosinnms ___£srs.sntass_lass
100 ml. assayed solutions of water 55° 0. for 55° 0. for 24 hours

 

 

 

at the beginning 24 hours and 6 days standing*at
salsa
0.53 5.08 92 92
2.2 4.31 60 59
3.2 4. 01 66 66
6.4 3.60 67 58

10.0 3.40 53 47

 

-14-

ggble g, The stability of ascorbic acid in water after aeration as
measured by the osazone method.

 

 

 

 

Milligrams per PW
100 m1. of ascorbic 24 hours aera- 24 hours aeration plus
apid originally p3 3199 “3 gm g; 25° 9.
0.55 5.39 25 38
2.13 4.29 5 17
3.22 3.92 7 20
6.40 3.62 20 30
10.00 3.38 10 12.5

 

MILL Aeration results of pure ascorbic acid in water using the dye
titration method of measurement.

 

lilligrams per Beginning dye

 

 

d8!

 

 

._—m__ 4229.39.11—
Dye titra- % lose fron Dye titra- i lose

 

 

100 ml. in titration in

w New.
s,e 15.5 11.7 12 9.1 50
4.0 51.5 25.4 10 25.2 25
5.0 45.5 55.7 as 52.7 50

 

2.15..

[able 1, The reversibility of oxidized solutions of ascorbic acid when
treated with Mrogen sulfide and nitrogen.

 

Oxidising
.__££:aisf
control

Iodine

Horit

Dye titration

Percent rec overy
Dye tit rat 1 on

Percent recovery
Dye titration

fiercent recovery

Milligrams per 100 ml. ascorbic acid in

 

1&9:

 

1.0
0.0

2.2
0.0

0.9
0.0

 

 

4%211122102599119

£29. 429
19.1 35.3
18.1 35.3
100 100
15.5 30.7
14.4 25.4
51.0 50
17.0 29.7
15.1 25.0
95 51

 

-16..

EIPERIMEMAL

W

ren grams of vegetable solids were extracted with a strongly acidic
solution by grinding in a mortar and pestle with sand. The mixture was
then centrifuged and the extraction repeated. After three extractions the
volume of the extracted material was made up to 100 m1. and assayed. Fruit
and vegetable Juices were assayed as such by simply preparing a one to ten
dilution. Samples were made in duplicates and assayed by each method. the
acid extracting solutions were necessary, eight percent triohloroacetic for
the beginning work and five percent metaphosphoric acid for the later work.
htraction of vitamin c was found to be equally effecient with either solu-

‘10ne

Win!
The determination of vitamin 0 by titration with 2, 6 dichlorophenol

indophenol originally suggested by Tillmans (3’7) was used throughout these
experiments. Utilizing the reducing ability of ascorbic acid, the dye is

changed from a blue to a pink color.

“Q“ ___1_;_; Mfr _:

2, 6 dichloro 1nd0phenol

 

Dye solut ions were made by dissolving specially prepared tablets in dis-
tilled water at 80° 0. Because of the instability of the dye solution.
standardization with pure ascorbic acid previous to sample determinations

was necessary. The faintest pink color lasting for 15 seconds was taken

-17..

4

as the endpoint in all titrat ions. An example of dye standardization and
the calculation of ascorbic acid content is a sample is given in table 8.
Although the dye titration method is a rapid means of measuring the

ascorbic acid content. it determines only the active or reduced form. In
addition the method lacks specificity since any reducing canpound such as
glutathione, cysteine, and phenolic compounds present may produce the same
effect as ascorbic acid on the dye. Highly colored solutions obscure the

Photelometers have therefore been

endpoint and cause erroneous results.

adopted to make the dye determinations more accurate.

d t h
Bessey's modification (2) of the method of Mindlin and Butler (24)
followed in this assay wait has many decided advantages. Eliminating the
personal factor of endpoint titrations, the assay of highly colored, turbid
solutions becanes possible. Differing slightly chemically frcm the dye

titration, ascorbic acid changes the dichloro indophenol to the colorless

leuccbase.

030

~ 15/“"

110011 Ind0phenol dye red
I in acid, blue in
HCOH alkaline solution

ii

Let ive asc orb io
acid

:25 + 5 5

u
o
'8

..I\ . 0.
0:0 ° + ’1 \
HCIJ—J \ H
a \n

ROCK
| Leucobaee
H0 w c 01 orle as
H

Dehydr o a so orb ic
ac id

325;, 8. the standardisation and calculations of ascorbic acid content
by the visual dye titration method.

Dye solution - ten tablets
Standard ascorbic acid - 1o gamma/ 1111111115512

 

  
 

ML. stand- Final Dye titration Average dye Titration m. dye
_ . .. V 1n n" ’ _ 1

 
    

         

_ 1: on ._ .
O 25 1.3 1.2 1.2 0.0
5 25 4.7 5.3 5.0 3.8 .76

10 25 9.3 8.9 9.2 8.0 . .80

15 25 13.8 13.3 13.5 12.3 .82

80 25 18.8 17.5 17.5 16 .3 .815

 

mummies/100 1111. in standard = milligrams/m1 dye - 100

for examplfl h 3 [0123

On milliliter dye solution equals .0123 milligrams per
100 vitamin 0.

Saple cal culat ions

 

Juice Original 1111. dye dye titration Results
-' 1 .: - “1101' ti.“ = =-_ , a ~15 111410,: - 4-2] .- -,:_ '1' o .
0181180 1 " 1° 3 24e6 23.3 4e66 . e01“ 58.5
‘ 10 ' 100 3
Quote 1 - 10 10 20.8 20.8 1.90 ' .0123

' 1° . 100 ‘3 24e1

 

-19-

when a solut ion of five percent meta phosphoric acid buffered to a pH
of 3.5 is added to the dye, an intense pink color is formed. The presence
of ascorbic acid decreases the intensity of the pink color as measured by
the photeloneter. Pure ascorbic acid in five percent meta phosphoric acid
served to standardize the dye and unknown samples are assayed for vitamin 0
content by referring to the standard curves. Turbidity and interfering colors
are eliminated by taking an initial photelcmeter reading and a final photelc-
meter reading in which all the dye has been coupletely reduced by a crystal
of ascorbic acid, only residual color renaining. The method of deriving a
standard curve is given in table nine.

The 10garithm of the final reading minus the initial reading is used
to plot the standard curve. Typical curves are seen in the following chart.
(Figure 1)

To calculate the ascorbic acid cmtent of any sample, the logarithm
tained. This number corresponds to the milligrams per 100 milliliters as-
corbic acid as read from the standard curve. By correcting for the dilution
factor the final result was obtained. To follow through the calculations,

a earnple of tomato Juice was diluted one to ten with five percent meta-
phosphoric acid. Five milliliters of this dilution were taken for assay,
diluted with metaphosphoric to 25 milliliters and buffered to a pH of 3.5.
The buffered sample was made to 100 milliliters. Using four milliliters of
dye and four milliliters of buffered sample, the initial photeloneter read-
ing was taken. A small amount of ascorbic acid crystals was added and a
final photeloneter reading taken of the residual color. To calculate the

amount of ascorbic acid in the original material, the formula given above was

85... «3.8 332
2e a: r. 38 52.35.. .3 833223... a: .5 .258 2.33. 235 .2 an

In“: Had“ a non ugql undue!— neuaunu «6 no."
an. on.

on.

 

 

 

 

on

on

“outrun! cot «d was ore-too" :9 mum

‘81-

m Standarization of dye and calculation of samples in the photelc-
meter method.

10

EMmmer of dye tablets in aqueous solution
Number of milligrams per 100 ml. ascorbic
acid in five percent metaphosphoric acid 3 8

 

 

 

m1. stand- Total Final buf- milli- Log. of final
and solu- acid v01.fered vcl- grams perlnitial Final reading minus
tion in ml. ume. pH 100 ml. reading reading original reading
marble

0 25 100 3.5 0.0 58 98 .2337

5 25 100 3.5 10 50 99 .2147

10 25 100 3.5 20 63.5 98 .1941

15 25 100 3.5 30 65.5 98.5 .1739

20 25 100 3.5 40 68 .5 98 .1577

23 u 100 3.5 50 7.2 99 .1361

 

-22-

applied. Thus:

105135-5— _ 1532.39:- .2249

.2249 on the standard clart represents five milligrams per hundred
milliliters. Multiplying by the dilution factor, the original sanple
contains Eta—49% or 10 milligrams vitamin 0 per hundred milliliters. In

this way, all sam;:les assayed by the dye photelcmeter method were calculated.

The gsazgng me 5M

The procedure followed for the osazone method has already been given.
Originally eight percent trichloroacetic acid was used. Standard curves
were made with pure ascorbic acid solutions such as the ones shown in Fig. 2.
Calculations of samples were made by reading the milligrams per 100 ml. from

the chart and multiplying by the dilution factor. An example would be as

follows:
Orange Juice

Original dilution with trichloracetic = l - 10
Mls of original assayed = 3
Final dilution in mls I 25
Milliliters of oxidized portion assayed = 4
Photelometer reading 3 69
llilligrams per hundred frmn chart . 2.55

W a 53.1 milligrams per hundred ascorbic acid

The modified osazone method (31) used five percent metaphosphoric acid,
one percent thiourea and 10 percent acetic acid as the extracting solution.
Standard curves were prepared as previously described and this was used for
sample calculations. The chief advantage of the modification was the intro-
duction of a new chemical assay for dehydroascorbic acid alone. Using meta-

phosphoric acid, an aliquot of four milliliters was taken before norit

-23-

osidation. The same procedure as for total ascorbic acid was then followed,
that is, the addition of dinitrOphenyl hydrazine, the incubation at 55° 0.
for one hour, and the photelcmeter readings. A separate chart was prepared
for dehydro ascorbic acid by oxidizing a pure ascorbic acid solution with

branines. The curve is seen in Fig. 2, and the sarnples were calculated the

same way as shown in the previous example.

on;

Into-u Jet-micron! so witness

co.”

.m
can»... uses-so an» up 33 5388.. no 8323—33 on» .8.“ eerie 2.3.8 .3 «a

32. .28... «a .8352... at .55

 

00...",

 

 

om

 

or

00

con

lessees aeselnteao'ld

-23-

RESULTS

rative studie with dr itrat on a d e r a as math

Both methods of assay were run in duplicates on the same sample ex-
tracted on eight percent trichloroacetic. The dye titration was carried out
on the first day after extraction and the osazone method on the following
day, the extracted sample being held at 5° 0.

As shown in table 10 the two methods gave about the same ascorbic
acid content in the canned vegetables assayed. Theoretically the dye tit-
ration which measures only the reduced fonn, would give slightly lower
values than the osas one method which measures both active and dehydroascorb-
ic acid. This was not found to be true, however, since the osazone values
were sonetimes higher and sometimes loner. In table 11 showing comparisons
with canned tomato Juice samples, the osazone results showed some decrease

after five days holding.

Qfloarativg studies with dye t itrat 193, dye mgtelgpetgg,
a_n_d mgified gsazgng mghgg

Table 12 shows the results obtained by three methods of assay using
the same sample extracted in five percent metaphosphoric. The dye deter-
minat ions were run on only the metaph05phoric solutions, acetic acid and
thiourea being added for the osazone determination. The inaccuracy of the
dye titration method using a visual endpoint in contrast to the dye phote-
loneter method was not observed. In general, both methods gave conparable
results with the same samples. ‘Apparently the solutions were not highly
colored enough at the dilution used to interfere with the pink endpoint of

the dye titration. The modified osazone method in general gave conparable

-25-

results with the dye method, green snap beans being an exception. Even in
the modified method, however, the results were not consistently higher due
to the snail amount of dehydro ascorbic acid present, nor were the results
obtained for total ascorbic acid any noticeable improvement over the original
method. When the same samples were held at 5° 0. and retested after seven
days, the dye titration showed a decrease in all cases while the osazone
results were inconsistent. In some samples, a slight decrease was observed
while other samples remained the me or showed an apparent increase in

vitamin 0.

h dr so rbi id 1 1
With the modified osazone method it was possible to determine the
amount of dehydroascorbic acid present in the foods tested. Seeking an
answer for the unusual stability of the osazone amples, dehydro determina-
tions also were repeated after seven days.) Decided and consistent increase
in dehydo is shown in table 13. Evidently ascorbic acid in plant tissue is
oxidized to dehydro on standing and rernains‘stable in this fonn longer than

would be expected from previous work (4,9,28).

.27..

 

”.5 0.0 ¢.¢ Now
0.» 0.0 0.0H I!
H.¢ o o.n 0.0
H.m b.b 0.0 n.»
o.o 0.0 o.m o.»
o.» o.ma o.mH c.0H
o.m m.H b.a ¢.N
O.HH n.m o.m 0.0
”.0 o.¢H m.NH n.m
n.nH n.5H m.o m.o
o.NH o.>H o.¢H o.nH
b.>H o.wH o.oH . o.ma
«.mfl o.mfl n.9H N.HH
a. a. 2n... has 2n...

 

neofiah

gnaw caucus: non assuming:

sesamm.

enouuno

aueem

Henson edema, tonne
Henson oHons.i spec
Aeneas»

goddamn

annen.musu neeuc
some: moon geese
snoop muse neeuu
«"8334 £de
uaMnnad..aseu

weeks .a cam

pecan .msem

nuance

cook

 

.usOaunnHauoucc can no owsnops i neapnpcmop cognac nu vans cannoenm

msfisdanouoc no» cameos esousno Hasawduo nan nodunnoau one no unannon evanescence

.OH Oman“

-28-

xablg 11. 0onparat ive results of dye titration and original osazone method
for determining*ascorbic acid in canned tomato Juice - average
of duplicate determinations.

 

 

 

 

 

Number Pounds vacuum ‘Dye Qrigiggl gsazgng
tismm‘mflwwamd
1 0 6.4 5.0 4.3
2 8 10.3 9.0 6.8
3 0 6.1 3.7 3.7
4 4 9.4 8.5 6.6
5 3 9.1 ‘ 7.5 6.9
6 7 4.8 4.5 4.9
7 8 11.2 10.9 8.2
8 7 12.5 10.9 9.2
9 2 7.9 11.0 5.9

 

 

‘89-

 

 

 

 

 

0.0 .Hn...od 0.0 0.nH .Hna...d ..0anq
0.0 Canon 0.0 050..” Canon ..gfion nuoon
5.. 0.0 a.» 0.0a n.0 ..0.50 .5...
0.0 0.0 0.0 «.n 0.0a .0aac. mosses
n.nH 0.0a 0.0a 0.0« n.nn ..0asq
0.0. a... a... 0..“ 0.0. .0220. .ounsna
0.0 0.0 0.0 0.0 H.« ..oasq
0.0 0.0 0.0 0.0 H.« .02... ..0...0
0.0 0.0 0.0 0.0 n.« ..oanq
0.0 s.0 0.0 0.0 e.« .0HH0. .nnnano
0.0« 0.aH n.«H a... 0.«« ..0asn
n... 0.2. 0.. 0.06 a... .02... nonnadn
0.0» 0.5» an an 00 .00..“
0.0a ».n« 0.0» 0..« 0..» .02H0.no.saan
0.0 0.0 «.H n.« 0.« ..0asn .5.05
mean
0.0 0.0 n.H «.n n.« .0“... stone
0.0 «.« 0.0 0.» n.« ..o..« .5...
Anne
0.. n.H n.0 «.n n.« .eaao. 0.5.0
0.0. 0.. 0.0 a... n.HH ,..0aaq ...d
«.0 0.n a... 0.0. 0.0 .0“...
”..: r3 n.3,. 56 o4. :33. neon
n.n 0.» «.0 a.» 0.0 .eaflo.
0.0 0.0 0.0. 0.0. 0.. ..oanh ..cm
0.0 0.» n.» a.» 0.0 .0nao.
... .... 6. 1.4.... .1: ... H ”4.. «4.8..-. .0 M ..: H. ”:1.-. COLA. 4.. .4. 38“». 4..
g venues
were» mango: seams neamsdm cease! eczema manna; cosh
t1Iaaaamumuummquuuqugamumqqmaa

 

3.8333893. 03» m0 amuse; 1 3335mm» 08:3 3 33 03.803 meanness»;
new campus accuses 058308 use .nouonnaoponm 93 .3393: one B 0335.. 05093980 3

:30-

 

 

 

 

 

 

0.00 0.00 0.00 0.00 0.00 00000 0000000000
0.00 0.00 0.00 0.00 0.00 00000 0000000000
0.0 0.0 0.00 0.0 0.0 00000 000000000
0.0 0.00 0.00 0.00 0.00 .0000 000000000
0.00 0.00 0.00 0.00 0.00 .0000 000000
0.00 0.00 0.00 0.00 0.00 .0000 000000
0.00 0.00 0.00 0.00 0.00 00000 000000
0.00 0.00 0.00 0.00 0.00 .0000 000000
0.00 0.00 0.00 0.00 0.00 .0000 000000
.2002 302...: 0. W a :0. 00.0... 3 3.; - 20. L. .. -1 a." 0.3 In
. 0100:0000. 000.00
H050 $0030: 000mm 00.3080 0030000 000200 03000.0 00000.0
x1100mm00400000000001000000000:0 .

 

.0003“. 30.3 008000 5 300 03.00000 90300»
.03 0003000 0000000 000.3000. 0.00 000000500003 05. 5030.33 000 «0 00.0000.— 05 00.00.0050 03» 0.0

gablg 14. Dehydrcasccrbic acid results on canned vegetables, vegetable and
fruit Juices - average of two determinations.

 

 

 

 

 

Milliggggg per 1133er m.
Food Freshly cpened Sample after holding
gegted samplg Egggg day: a; §° 0.

solids 1.3 4.0

Peas Juices 0.0 0.0
solids 1.3 4.5

Peas Juices 0.3 2.6
solids 0.0 4.8

Peas Juices 0.0 3.8
Green solids 0.0 1.6
snap beans Juices 0.0 1.0
Green solids 1.0 2.6
snap beans Juices 0.0 1.2
solids 2.6 8.0

Spinach Juices 0.0 6.3
BOlid’ 4.3 8.9

Spinach Juices 1.9 7.1
IOlld. 0.0 1.6

Carrots Juices 0.0 0.0
solids 0.0 1.0

Carrots Juices 0.0 0.0
.011d' 0.0 4.0

Tomatoes Juices 0.5 3.5
.Olid. Oeo 2.0

Kidney beans Juices 2.5 4.5
solids 0.0 0.0

Beets Juices 0.0 0.0
Tomato Juice 1.3 9.3
Tomato Juice 0.6 7.8
Orange Juice 2.3 9.3
Orange Juice» 2.1 7.5
Orange Juice 4.2 7.5
Pineapple Juice 0.0 4.5
Pineapple Juice 2.2 3.5
Grapefruit Juice 1.3 5.9
Grapefruit Juice 1.3 5.9

GENERAL D ISCUSS I ON

It is evident from the results obtained that further verification of
the csazone method is necessary. In general, the results are canparable
to the dye methods, but they are not consistantly higher to the amound of
dehydro present as they should be. For total ascorbic acid content. the
original (30) and modified (31) methods are equally comparable to the dye
determinations. The real advantage in the modified method (31) is that it
is now possible to determine dehydro chemically.

Since the oxidized form, dehydro. is no more stable than the reduced
tom, the results obtained from holding the samples are difficult to ex—
plain. Apparently under these testing conditions, reduced ascorbic acid
forms a more stable compound. Dehydro results on the fireshly prepared
nuplee verify the fact that plant tissues contain little or no dehydro.
The very consistant and decided increase of dehydro ascorbic acid on stand-
ing, however, needs facts for explanation which are not available at

pf. ”Ht e

43-

SUMARY

Results obtained fron the comparative studies on fruits and vegetables
of reduced and oxidized ascorbic acid show that

l. A modification of the osazone method reduced the time of incubation
tron three hours at 37° 0. to one hour at 56° 0. with conparable results.

2. The original osazone method gave the same general values for the
ascorbic acid content of foods as the dye titration method. "'

3. The modified osasone method was no improvement over the original
osazone method as a measure of active and reduced ascorbic acid.

I. The dye titration method gave values which approximately equalled
the dye photelcmeter method. Evidently the vegetables when diluted were
not highly colored enough to interfere with the pink endpoint of the dye
titration.

5. The osazone method gave approximately the same value for ascorbic
acid as the dye methods.

6. (m storage, the osaz one method showed (less decrease in sec orbic
acid cmtent than the other methods studied. Under the conditions of the
osazone test, ascorbic acid reamined in a stable form much longer than it
did under the conditions for the other tests. If storage is necessary. the
osazone method of measurement voild be invaluable.

7. Dehydroascorbic acid as measured by the osazone method was present
only in small amounts in the original plant tissue.

8. 0n storage dehydroascorbic acid mas found to greatly increase. in-

dicating that reduced ascorbic acid on storage changes to the dehydro four.

I

BIBLICBRAPHY

1e Basu. Ks Fe and Hath, Mece

Interfering compounds of the Indophenol Dye Titration Method for
Measuring Ascorbic Acid

J. Indian Chan. Soc. 153133 9(1938)

2. Bessey, A.0.

A Method for the Determination of Small Quantities of Ascorbic Acid
in Turbid Solutions in the Presence of Other Reducing Substances.

J. Biol. Chem. 126:771 (1938)

3. Bessey, A.0. and King, 0.0.

The Distribution 6 Vitanin O in Plant and Animal Tissues and Its
Determination.

J. Biol. Chem. 1033 687 (1933)

4. Bors00k, Davenport, Jeffreys, and Warner

The Oxidation of Ascorbic Acid and Its Beduct ion in Vitro and in
V1V0e

50 Check, HO 811d Ema, no“.

The Distribution Among Foodstuffs of the Substances Required For
the Prevention of Beriberi and Scurvy.

”OOe Boy. SOOe Landon B 90844 (1917)

6. Dnnerie, A.

Sulfydryl Compounds; A Source of Interference in the Dye Determin-
at ion of Ascorbic Acid.

Biochem. J. 283268 (1937)

—35-

7. (Mario A. and van Echelon, ll.
Sane Critical Banarks on the Determination of Ascorbic Acid.
Biochem. J. 2831161 (1934)
30:25 (1936)
8. Evelyn, K.A. Malloy, H.T., and Rosen C.

The Determination of Ascorbic Ac is in Urine With the Photoelectric
Colorimeter.

J. Biol. Chem. 1263645 (1938)

90 F01, F.W. md Levy, Lop.

hperhnents Confirming the Antiscorbutic Activity of Dehydro Ascorbic
Acid and A Study of Its Storage.

BiOChHMe Je 303211 (1956)

10. Gannon. 0.3. and Mc Govern, T.

A Study at Pyridinium Gunpounds Which Interfered in the Detennination
of Ascorbic Acid.

P106. SOCe Exptle BiOle Med. 388267 (1938)

110 GOOdmn, LO 811d 311MB, ‘0
The Pharmacological Basis of Therapeutics

The Macmillian 00. New York 1941 pl 263

12. Boss. AJ. and Unger, I..J.

The Distraction of the Antiscorbut ic Vitamin in Hill: by the Catalytic
Action of Minute Ammnts of Copper.

Proc. 300. Ethl. Biol. Med. 198119 (1921)

15. lelt, 30L.
The Structure of Ascorbic Acid

J. Soc. Chem. Ind. 523221 (1933)

-36-

14. Holst, A. and Frolich, T.
Experimental Studies Relating to Ship Beriberi and Scurvy

J. Hyg. 7:634 (1907)

15c Hapklns, FeGe and Crook. EJ-i.

hperimental Observations on the System Ascorbic Acid-Glut iathione-
Ascorbic Acid-Oxidase.

16. Dean. 3.1.. and Roe, J.H.
The Preservatim of Ascorbic Acid in Drawn Samples of Blood.
J. Biol. Chen. 1333579 (1940)

1'7. Keys
Stability in Citric Juices

New Zealand Med. J. 403379 (1941)

18. K1118, 00G.
Vitamin C. Ascorbic Acid

Physiol. Rev. 163238 (1936)

19. Kohman, SJ.
~ The Protection of Vitamin C in Foods

Ind. Eng. Chem. 15.273 (192:5)

20. Kruse
Chemical Methods for Determining the Plasma Level of Vitamin 0.

Am. J. Pub. Health 3131079 (1941)

21. 103 M9,, VeKe , Campbell, HeLe and Shemm, 11.0.

The Effect d Temperature and of Hydrgen Ion Concentration Upon the
late of Distraction of Anti scorbutic Vitamins.

Proc. Soc. EIPtl. Biol. Med, 183122 (1921

.37—

22. La Bier, Campbell, and Shaman

The Effect of Temperature and Concentration of Hydrogen Ion Upon
the Rate of Distruct ion of the Antiscorbutic Vitamin.

Je he Chem. SOOe 445172 (1922)

230 Land, H. 8111 Lieck. H.

A Specific Reaction for the Qualitative and Quantitative Determination
cf Ascorbic Ac id in Serum.

Nature 1373784 (1936)

24. Mindlin, 3.1.. and Butler, Adi.

The Determination of Ascorbic Acid in Please; a Macromethod and a
Micranethod.

J. Biol. Chem. 1229673 (1937)

25. Nelson. 1.x. and Brown, C.A.
The Preperties and Chenical Constitut ion of Glucic Acid

J. Am. Chan. Soc. 613830 (1929)

26s Pagot. Me and 361389,, Re
Manganese Oxidation of Vitamin C

Compt. Rand. Soc. Biol. 1293960 (1938)

27. Tmberg He and K191119139 TeSe
A Method for the Quantitative Determination of Ascorbic Acid

28. Roe. J.H. and Barnum, 6.1..

is

The Antiscorbutic Preperties of Reversibly Oxidized Ascorbic Acid
and the Observation of an Enzyme Which Reduces the Reversibly Oxidized Vita-
min.

J. Butrition 113359 (1935)

-38-

29. Res, J.H. and Hall. JtM.

The Vitamin 0 Content of Human Urine and Its Detennination ThrOugh
the 2-4 DinitrOphenylhydrazine Derivative of a Dehydroascorbic Acid.

J. Biol. Chem. 1283329 (1939)

30e ROG. JeHe and KUBther, CeAe

The Detennination of Ascorbic Acid in Whole Blood and Urine Through
the 2-4 Dinitrophenylhydrazine Derivative of Dehydroasccrbic Acid.

J. Biol. Chem. 1473399 (1943)

31c 30.. JeHe and Oesterting. MeJe

The Determination of Dehydroascorbic Acid and Ascorbic Acid in Plant
Tissues by the 2-4 DinitrOphenylhydrazine Method.

J. Biol. Chem. 1523511 (1944)

32. Silva, 8.8.
The Influence of the Stability of the.Antiscorbic Factors.

Lancet 13478 (1921)

35. 311V8, SOS.

The Influence of the Oxidation of the Antiscorbutic Factor in
Lemon Juice.

Biochem. J. 173410 (1923)

34. Strohecker, Buses, and Wbinreich, A.
The Effect of Oxygen of Air on Ascorbic Acid
2. Untersuch.Lebensm. 813126 (1941)

36. Ssent-Gyorgyi,.A. *

0n the Function of Hexuronic Acid in.the Respiration of the Cabbage
Leaf.

Je BiOle Chem. 902585 (1931)

~39-

36. Tauber, H. and Kleiner, I.
An Enzymic Method for the Estimation of True Vitamin C.

J. Biol. Chem. 1103559 (1935)

37 . Tillmans, J.

54333 (1927)

38. Woessner, 17.17., ElvehJem, C.A. and Schuette, H.A.
The Determination of Ascorbic Acid in Canmerc ial Milks

J. Nutrition 183619 (1939)

 

 

t:

. v

 

 
 

 
 

 

 

   
 

       

 

MITITWHHTIMIWIWNW “I”
3 1293 03084

9354

W“