e FISHERIES RESEARCH BOARD OF CANADA

Translation Séries No. 2725

Investigation of colorimetric methods for the ' quantitative analysis of phosphor

by Misae Furukawa, Masako Taneda, YasukiChi • Nakamura, Seiji Kasuga and Narutoshi Yoshikawa

Original title: Hishoku-ho ni yoru Rin Teiryo ni tsuite no Kento

From: Seikagaku (Journal of the Japanese Biochemical Society), 24(1). : 76-82, 1953 •

Translated by the Translation Bureau(EHH) Multilingual Services Division

Department of the Secretary of State of Canada

Department of the Environment Fisheries Research Board of Canada

Marine Ecology Laboratory Dartmouth, N. S.

1973

24 pages typescript

DEPARTMENT OF THE SECRETARY OF STATE SECRÉTARIAT D'ÉTAT

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AUTHOR — AUTEUR

Misae•FURUKAWA, Masako TANEDA,' Yasukichi NAKAMURA, Seiji KASUGA & Narutoshi • YOZHIKkUA

TITLE IN ENGLISH — TITRE ANGLAIS

Investigat:on of colorimetric methods for the quantitative analysis of • Phosphor

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Journal of Japanese Biochemical Society

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INVESTIGATION OF COLORIMETRIC METHODS FOR THE

QUANTITATIVE ANALYSIS OF PHOSPHORUS .

Misaet FURUKAWA, Masako TANEDA, Yasukichi NAKAMURA

t - • * Seiji KASUGA and Harutoshi YOSHIKAWA

In recent years, coupled with the introduction of the radioactive

isotope P 32 for metabolic research, the relation bdtween biological processes

and the metabolism of phosphorous compounds has received increasing attention.

• Today, the tracing of phosphorous compounds through the metabolic process is

• becoming an important aspect in metabolic reseàrch.

For this type of research, a primé requirement is the separation and

quantitative analysis of phosphorous compounds within the histological

structure or fluids. However, both procedures have problematical points which

we have been forced to analyse. The following report describes the results of us,

our investigations on the quantitative analysis of phosphor/which were based

mainly on our research data. Hoping that it would be of some use to other

workers, we hereby present the results.

uNF.Dur:D TRANSLATION For informa'ion only

TRADUCTION NON REVISEE Information seulement

* Faculty of Biochemistry, Tokyo University t Fivnounciation Uncertain. EHH

505-200-10-31

71530-21-020-5332

The colorimetric method is employed at present for the quantitative

analysis of phosphorus,First, the desired component containing phosphorous

compounds is separated from the histological structure or fluid and then

converted to an inorganic compound by the so-called "wet ashing" process.

The inorganic phosphonMs then subjected to colorimetric analysis. us,

The process of converting the organic phosphon/to inorganic phosphonfand the

process of quantitatively analysing the inorganic phosphote both contain key

points that could create unexpectedly large errors. We intend to report on

the separation procedures at another opportunity and describe here the results

on the investigations concerning the two key points in the quantitative

analysis procedure that were mentioned above.

1. THE CONDITIONS REQUIRED TO PRODUCE A CHROMATIC REACTION FROM INORGANIC

PHOSPHORUS.

Ortho-phosphoric acid is either made to react with molybdic acid under

a constant level of acidity to produce phosphomolybdic acid or a suitable

reducing agent is made to react with this t and the resulting blue coloured

f compound is subjected to measurements by a colorimeter or a photometer.

Bell and Doisy1)

were first to apply this method to quantitative

analysis. The reducing agent they employed was hydroqUinone. However, other

workers pointed out the shortcomings of this method as a' procedure in quanti-

tative analysis. The shortcomings were, the long time lapse required for the

chromatic reaction, the possible presence of many substances that interfere

with the chromatic reaction and the sensitiveness of the chromatic reaction

to ambient conditions. Subsequently, Kuttner2) employed SnCrt as the reducing

agent and Fiske & Subbarow3) employed 1-2-4-aminonaphthol-sulfonic acid as

the reducing agent in attempts to improve the method. But.even with these

t The original text is not clear what "this" refers to. EHH

3

improvements, the method was not considered definitive from the point of the

stability of the chromatic reaction and a number of new methods were reported

later. The basic principles of the later reports were identical to previous

reports but differed only in the choice of the reducing agent. In the mean-

time, the measurement technique itself progressively improved from the Dubosq

Colorimeter, Stufenphotometer to the photoelectric photometer and accurate

quantitative measurements of microscopic amounts became possible. This fact

has generated an increasing concern over the measurement errors introduced

by the instability in the chromatic reaction.

When national and international literature is surveyed, the Fiske-

Subbarow method appears to be most widely employed for the quantitative

analysis •of phosphor. The results of our investigation of the method are

shown in Section 1. The instability of the chromatic reaction has been

greatly reduced when compared to the SnCkand hydromehnone methods. • However,

it is obvious from the results that great care must still be exercised when using

this method. (The original paper was published in 1925 and the Dubosq colori-

meter was employed at that time.)

We further investigated the Allen4) and Gomori5) methods and wish to draw

attention to the fact that each method has its characteristic points.

THE FISKE-SUBBAROW METHOD

Reagents

i) Molybdic Acid Solution: An aqueous solution of 2.5g/d£ of

ammonium molybdate was used.

ii) Reducing Agent: A 500 mg mass of 1-2-4 or 1-2-6 aminonaphthol-

sulfonic acid was first dissolved in 195 mi2. of 15%

aqueous solution of NaHS0 3 . 'Heat was

applied if the acid was.difficult to dissolve.

A 5 mk volume of 20% Na 2SO

3 was added to this

solution. The solution could be stored

for several weeks before use if it was

stored in a brown glass bottle. Depending

on the ambient temperature, some p6Wcipi-

tates could appear. However, in such cases,

the supernatant fluid can be used.

iii) 3N Aqueous Solution of H 2SO4

OPERATIONAL PROCEDURES

A 10 m2, volume of the solution to be quantitatively•analyzed was first ofelsnary

placed in a graduated test tube which had an-eeagefi stopperf. Next,1 m£ of

3N H2SO4 s olution, 1 ne of molybdic acid solution and - 0.4 mZ of reducing agent

were ndxed together in the stated order and water was added then to bring the

'volume up to 10 ne. This solution was mixed into the test sample and the

temperature of the sample was maintained . for 15 minutes at a temperature

between 20 0C to 25 °C by placing the test tube in a water bath. Colorimetric

measurements were taken immediately after this procedure.

4 EXPERIMENTAL RESULTS 1 •

a) Absorption Spectrum:

Figure I displays the dependence of the extinction coefficient

on wavelength. An aqueous solution of potassium phosphate vs,

(KH2 PO4 ) that contained lOy of phosphontwas the test sample.

The chromatic reaction was induced by the Fiske-Subbarow method

and the measurements were taken at various wavelengths on a

Coleman Spectrophotometer. According to Fig. I, the most suitable

t The od tex oes ciarj-fri-wha.t.--e--Meat--by a t tube eat has a co ion share

s tppr. ne-test....tape mge,have tw rancheeMing tp,n

ommon stpri5er, o two bein.ches.iitercone-Cctedfr ,ough q,cônimon stop5Ock e,--Enii •-

l•

5 III•

light source to use for photometric measurements is a source

that emits primarily in the 740 to 780 nm wavelength region.

For the Stufenphotometer, the most suitable filter to use

is the S 72 filter. t

h) Acidity:

In order to indu.ce a chromatic reaction, a specific level of

acidity is required. Figure 2 shows the relation between the

reaction and acidity. The extinction coefficients of solutions •

containing equal amounts of phosphoileWere measured under

identical conditions while the concentration of the sulfuric

acid was varied in the reagents that were added to the test

solutions. According to Fig. 2, the concentration of sulfuric

acid clearly influences the chromatic reaction. The tolerance

range of the sulfuric acid concentraiion lies between 0:1 and

0.6 N in terms of the terminal concentration after completion

of the reaction. The original method certainly employed an

acidity level within this range.

When trichloreerdoacetic acid tt is used as a peecipitating

agent for protein, the acidity of this agent is added to the

acidity of the sulfuric acid. In fact the pH value decreases,

but no influence is seen in the extinction coefficient values

as long as a 10% aqueous solution is used.

c) The dependence of the strength Of the chromatic reaction on

temperature and the elapsed time after the colorimetric

reagents have been added to the test sample:

t No reference is given in the original text. EHH

tt Translated ZiteraZZy. EHH

.1

c) (cont s d)

Figure 3 displays the variation of the extinction

coefficient with time at room temperature (24 °C)

after the colorimetric reagents have been added to

the test sample. With. passage of time, the value

of the .extinction coefficient increases. However,

a somewhat stable state is observed during the time

interval of 15 to 20 minutes.

Figure 4 shows the values of the extinction coefficient

measured 15 minutes after the colorimetric reagents had

been added to the test samples.. The samples were

placed in a constant temperature water bath during the

15 minute period': The.temperature was systematically

varied from sample to sample. It can be seen that the

chromatic reaction . is affected by temperature as well.

There is-a stable region in fhe temperàture range of

20 °C to 25°C.

From the preceding results, it can be said that if reliable

results are desired, before a measurement is taken, the test

sample should be placed in a water bath with a temperature

between 20 °C and 25°C for a duration of 15 minutes after the

colorimetric reagents have been added to the sample. The

original method gives the requirement that the reacting test

sample be left standing for 10 minutes at room temperature.

However, 4ecial precautions must be exercised for laboratories

such as ours where the room temperature can differ by more than

20 °C between summer and winter.

t The original text is unclear. It reads..."placed in a water bath during the period where a.temperature was added". EH!!

d) The Highest Concentration that can be Measured:

. Beer's law is obeyed up to a phosphotOncentration of 80y

when the final test sample volume is set at 10 mit. as

described in the original method, and when a 5mm cuvet for

the photometer is used. That is, the relation of the

èxtinction coefficient and concentration is linear. Therefore, vs,

in actual measurements, the concentration of phosphorishould be

measured only to the neighbourhood of 80y.

If precaution is exercised on the points mentioned above, errors may be

limited to the neighbourhood of 1% even for routine work such as clinical

examinations t . However, the question arises as to whether there might be a

method that is more stable with respect to ambient conditions. We describe

Allen and Gomori's methods next.

2) THE ALLEN4)

METHOD i•

The points that differ in Allen's method when compared to the Fiske-

Subbarow method, are the use of amidol (2-4 diaminophenol) as the reducing

agent and the use of perchloric acid (HC£04 ) as the acid in place of sulfuric

acid. The reason for using HC9,0 4 does not lie in the chromatic reaction but

lies in the wet ashing process which employs HC04 . The HCZO4 used in the

.ashing process was merely utilized for the chromatic reaction.

Reagents

i) Molybdic Acid Solution: An 8.3% aqueous solution of ammonium

molybdate was used.

t Original text reads as "clinical bed examination". It could mean "clinical bedside examination". EHH

. .

-8

ii) Reducing Agent

A 2g mass of amidol and 40g of sodium hydrogen sulfite

(NaHSO4 ) were dissolved in water and the total volume

brought up to 200 mt. The solution was stored in a

black glass bottle. Even so, exchange with a new

solution was required once every 10 days.

iii) Per:chloric Acid: A 60% (specific gravity 1.54) solution was

used.

OPERATIONAL PROCEDURES

tos, A 25 m2, tyw volume of the test solution containing phosphoas placed in a

graduated test tube with a common stopper and 2 mt of thé aqueous solution of

HCZO4' 2 mt of the amidol solution, 1 mt of the molybdic acid solution was added

to the test sample in the given order. The test sample was kept in a water bath

maintained at a temperature between 20 °C to 25° C and measurements were taken

later within the time interval of 5 to 30 minutes.

The original paper describes how the ambient conditions such as

temperature and time affect the chromatic reaction. Table 1 reproduces the

results given in the original paper where the variation due to the time lapse

is given. The test sample was left standing with the ambient temperature at

room temperature, which probably was approximately 25 0C. The method is des-

cribed as being more stable with respect to temperature when compared to the

Fiske-Subbarow method. However, in our experiments, a temperature variation

from 10 °C to 25°C produced a 3% to 20% difference. Figure 5 displays this

relation.

Therefore, after the reagents have been added to the test sample, a

constant temperature should be maintained and measurement& should be completed

during the time interval of 5 tu 30 minutes. The fact that the chromatic

9

reaction is swift and stable is an advantage(to be discussed later) when

quantitatively analysing the inorganic phosphoeontained in a biological

sample. This point is superior to the Fiske-Subbarow'method. But the slightly

higher cost of the reagents and the fact that amidol is relatively unstable

when compared to aminonaphthol-sulfon, may be an inconvenience in some cases.

Also, Beer's law is obeyed up to a phosphoeOntent level of 350y in a

25 re sample. Therefore, compared to the Fiske-Subbarow method, the Allen

method is superior for its capability to measure a wider range of phosphorus

concentrations.

3) THE GOMORI 5) METHOD

This method employs elon (methyl-P-aminophenol sulfate), sometimes

known as metol, as the reducing agent.

.i) Reagents

1) 6N H2

SO4

2) Molybdic Acid Solution:

A 2.5% aqueous solution of sodium molybdate was used.

3) Reducing Agent:

4 Elon lg was dissolved in 100 mt of 3% aqueous solution

of NaHSO3'

OPERATIONAL PROCEDURES

A 25 nit or 15 mit volume of the test solution containing phosphoras

placed in a graduated test tube with a common stopper and 2.5 nit of the

molybdic acid solution, 1 rut of sulfuric acid and 1 mit of elon solution were

added in the stated order. The test tube was then filled with water to the

marker line. The colorimetric measurements were made during the time interval

of 45 minutes to 90 minutes after the solution had been prepared.

- 10 -

The stability characteristics of the chromatic reaction produced by

this method differs from that of the Fiske-Subbarow and Allen methods.

That is, Allen's method was stable during the time interval of 5 minutes to

30 minutes but the Gomori method became stable after a time lapse of 30

minutes. (At room temperature, no change is_ observed between the time interval

of 30 minutes to 120 minutes.) This means that measurements are best taken

during the time interval, 45 minutes to 90 minutes after the chromatic reagents

have been added to the test sample. However, the solution is left untouched

during this periodl and the possibility that an increase in the inorganic

phosphorntent could take place through hydrolysis, particularly when organic

phosphorusthat are especially susceptible to hydrolysis a're present in the us,

test sample. When the phosphorlin the sample has already been converted to

inorganic phosphoehe problem does not arise. In such cases where organic us„

phosphfflis present, this method is not suitable. But since the temperature

effect is smaller than in the case of Allen's method, this method is advantageous

provided the material has been converted into the inorganic form beforehand.

When the final concentration of phosphorlexceeds 100y in a 25 m£ sample,

Beer's law is no longer obeyed. Therefore, from the point of the range of

possible measurements, Allen's method is superior. US?

II QUANTITATIVE ANALYSIS OF THE PHOSPHORYCONTENT IN BIOLOGICAL MATERIALS

The following procedures are followed:

1) Blood Serum

One volume of blood serum is mixed thoroughly with 4 to 5

volumes of a 10% solution of trichlortexteecacetic acid which has

t The original text is unclear as to what time interval is meant here. Most Zikely the waiting period from 0 minutes to 45 minutes is the time interval referred to here. Effll

- 11 -

been cooled beforehand. The mixture is then maintained at 0oC for

10 minutes to 15 minutes and then filtered. A fixed amount of the

filterate is taken and subjected to the measurement. Blood serum

subjected to the quantitative analysis outlined above usually yield

phosphoruXmtent values of 2 to 3 mg/dt. Therefore, in practice, it

is sufficient to mix 2 mI of blood serum with 8 mt of trichloredele_

-oacetic acid and then subject 5 mt of the filterate to the measurement.

2) Histological Structures:

Immediately after separation, the test sample is immersed for

several minutes in 'a 0.1% aqueous solution of NaCt that has been

chilled to 0°C. After a thorough cooling, the test material is spread

over a filter paper in order to remove the water content. With minimum

delay, the test material is weighed. A homogenate (usually at a 10 to

20% concentration) is then prepared by using a fixed amount of 0.1%

aqueous solution of NaCt. The preparation is carried out under chilled

conditions. Next, the homogenate is subjected to quantitative analysis

as in the case of the preceding case for blood sera. However, in the

case of histological structures, the pancipitates produced by the tri-

chloridated acetic acid is removed first by centrifuging. The

re supernatant fluid is then removed and the peycipitate is subjected to

a second rinsing in an ice cooled 5% solution of trichlortdamicacetic

acid. The supernatant fluid from the second rinsing is usually

combined with the first for the final measurement. Also, the amount

of homogenate that is to be used in the measurements differs for the

different types of histological structures. If liver is taken as an

example, 1 to 2 mt is considered to be sufficient.

- 12 -

• In performing the operations described above, the point that

. must be considered carefully is the possibility of the measured

value of inorganic phosphor being raised during the operations

through the hydrolysis of organic phosphorus.The action of enzymes

could be one of the causes. In order to avoid such action, the

•operations must be carried out quickly at low temperatures after

the subject material has been separated. Also, in the case of blood,

it is recommended that the blood sera be separated soon after the

sample volume of blood has been obtained. A second cause of con- 'is us

version of organic phosphon/to inorganic phosphoeis hydrolysis

caused by acids. This occurs by the action of the trichlortC=IL

-acetic acid used for the removal of protein as well as by the acids

contained in the reagents for the chromatic reaction. Caution must

be exerçised when organic phosphore(Phospho-creatine is an examplé).

At 15°C, 25% of the material is hydrolized in 1 hour when subjected

to a 10% solution of trichlor*lem*acetic acid. At 0 °C, the reaction

hardly proceeds.)susceptible to hydrolysis are subjected to the

analysis. Therefore, for such cases, during the process of immersion

in the trichloridated'acetic acid solution, cooling by ice is a

mandatory requirement. Also, Allen's method is advantageous because

the time lapse before commencement of the measurement is short

compared to other methods. However, even when such precautions are

exercised, hydrolysis cannot be avoided. When an absolute value

for inorganic phospho es required, a -ecipitant for inorganic

phosphor should be used and measurements taken on the inorganic re 6)

phosphorusithsat have been so ptecipitated . As an alternative, the

Lowry-Lopez method7) could be employed. This method uses a low

concentration of sulfuric acid in the reagents and ascorbi• acid is

- 13 -

employed as the.reducing agent. It is reported that this method

gives results that are very close to the results obtained from re

methods that employ the parcipitating technique. In fact, we have

also compared the general method and Lowry and Lopez's method and

found that_the latter method gives 5 to 10% lower values. The tests

were carried out for the quantitative analysis of inorganic phosphorus

in liver.

Because phosphocreatine is subject to acidta hydrolysis in

a special way when molybdic acid is present, the {true inorganic

phosphor + creatine Pl content can be obtained if the molybdic acid

solution and an acid is first added to the test sample and left for

a 20 minute period and then a measurement taken after a reducing

agent has been added to the test sample.

Different numerical values, as show h above, of the inorganic • #<,-

phosphor ,,trontent can, on occasion, be obtained depending on the

conditions of the measurement. This fact must be considered carefully.

III QUANTITATIVE ANALYSIS OF ORGANIC PHOSPHORUS.

Occasionally, the measurement of the total phosphor/content of histo-

logical structures, blood samples and other samples is required. The phosphorus

content of the separated components of the samples mentioned above is also

required in some cases. As mentioned previously, such requirements are met by

us, decomposing the organic phosphorehrough oxidation (wet

t ashing process) and by

quantitatively analyzing the phosphoras inorganic phosphor.

There are many problems associated with the separation of phosphorous

compounds. However, we will not discuss the matter here. Generally speaking,

t Original text reads loam ashing process". It must be a misprint of 'Iwet ashing process". EHH

- 14 -

the phosphorous compounds can be classified into the three classes of Acid

Soluble Phosphon‘Lipld Phosphol4nd Acid Insoluble Nonlipid Phosphorus.

.( Kaplan and Greenberg

9) have divided the acid soluble phosphor

0eurther into

us/ finer classes. The insoluble nonlipid phosphoribas been separated into nucleic

us/ 10) acid phosphoeand protein phosphon/by Schneider .. If Thanhauser and Schmidt's 11)

method is employed in the analysis, the nucleic acid phosphoean be separated

further into ribonucleic acid and desoxy ribonucleic acid.

There are two methods for the wett ashing process of organic phosphor>'One

method employs sulfuric acid as the main component while the second method employs

perchlorate as the main ingredient. Both require hydrogen peroxide to assist in

the oxidation process. The first method is that of Fiske and Subbarow 3) while

the second method is that of King 12) who reported the method in 1932. In 1942,

.Allen4) improved upon King's method.

1) The Sulfuric Acid (Fiske-Subbarow) Method

A suitable sample volume of liquid containing organic phosphoric

acid is first placed in a Kjeldahl oxidation tube. A le volume of

5N H2S0 .k.added_to the - sample and the tube heated by a micro burner.

- The distance between the bottom of the oxidation tube and the top of

. the flame must be maintained at approximately 2 cm during the heating

operation. As an alternative, satisfactory results are obtained if

the temperature is maintained between 130 °C and 160°C. The contents

of the sample volume will turn black and white smoke will begin to

emerge from the sample. After thorough blackening of the sample,

the burner flame is weakened and a drop of H 202 or HC£0 4 is added to

the sample. The heating is continued further. (In the original

report, HNO 3 was used). If the blackness has not been cleared by the

t Original text reads as "warm ashing process". It must be a misprint of lioet ashing process". EH!?

- 15 -

first drop, a second drop is added. The step is

repeated until the sample volume becomes clear. In most cases a

single drop is sufficient. Even if the sample has become clear,

the heating must be continued for at least 1 minute longer. The

ashing process is thus complete (30 min to 60 min), but a small

amount of water is added to the sample.at this point and the oxidation

tube containing the sample is placed in a boiling water bath for 10

minutes. This step is required partly for the purpose of reconverting

the pyro-phosphoric acid to ortho-phosphOric acid. After completion

of the process:the sample volume is transferred to a test tube

graduated at 10 m2, for quantitative analysis. As required by the

Fiske-Subbarow method, 1 m9, of the molybdic .acid solution, 0.4 m2

of the reducing agent is added to the sample together with water to

make up a total volume of 10 m2. The quantitative analysis for in-

organic phosphorVneed only be carried out from this point.

A large quantity of organic matter may be present depending

on the separation procedure that is employed and decomposition

through oxidation may be difficult to complete in many cases. In

such cases, the flame must be positioned close to the oxidation

tube and the oxidation process must be promoted vigorously. One

fact to be kept in mind here is the irrecoverable loss of phosphorus

during such an operation. This has been pointed out previously by .

Mart1and13) Baumann 14) and Whitehorn 15) . Of course, the amount of

phosphoehat is lost depends on the degree of vigour employed in

heating as well as the amount of sulfuric acid added to the sample.

However, if the conditions are kept fixed, the amount of irrecoverably

lost phosphorytan be correlated to the duration of the heating

operation. Therefore, a correction can be made to account for the

lost phosphoe Figure 6 shows the decrease of the extinction

- 16 -

coefficient with the increase in the heating time period for the

conditions of our experiment. .

2) The Perchlorate (Allen)Method

The separated sample volume of liquid containing organic

phosphoric acid that is to be quantitatively analysed is placed

in a Kjeldahl oxidation tube. Heat is applied to reduce the liquid

volume by évaporation to a small a volume as possible. (The tri-

chlor cacetic acid, alcohol and ether used as solvents are all

organic compounds.) A 2.2 11-19, volume of 60% HC£04 is added to the

sample and heat applied. When the contents begin to turn black the

sample tube is removed from the heat and cooled thoroughly. One

drop of H20 2 is added to the test sample and heat is applied again.

The process is repeated until the sample becomes clear and colourless

which indicates completion of the oxidation process. (In most cases

1 or 2 drops are sufficient). The sample is then transferred to a

test tube graduated at 25 m9, for quantitative analysis. A 1.0 mit,

volume of molybdic acid solution and 2.0 m9, of Allen's reducing 4 1 •

agent is added to the test sample. The remainder of the operation

is identical to that of the quantitatfve analysis for inorganic

• phosphorus.

Among the operations in the procedure outlined above, careful attention

must be paid to the heating temperature. That is, since the boiling point of

HC9,04

is 200oC, the optimum condition for decomposition through oxidation is

provided when the temperature is maintained just below this boiling point.

Under such a condition, white smoke is emitted rather profusely and the

optimum condition may be judged from the emission of this smoke. However,

the white smoke must not be permitted to escape from the oxidation tube. If.

0.8 m£ 60% NM)4

Amidol solution 0.8 m9.

- 17 -

u5y. these conditions are observed, no HC£04 is lost and no phosphorvis lost either.

Table 2 shows the comparison of the extinction coefficients of two samples

before the heating operation, and after heating followed by a 30 minute cooling U5

period. The two samples had phosphorvtontents of 200y and 400y each. In most

cases, oxidation is completed within 30 minutes if the preceding conditions

are satisfied.

Thus, the use of HC204 for decomposition through oxidation is a process

that has less sources of error when compared to the process that employs

sulfuric acid. Also, the HC£04 process is faster but the cost of HU°4

is

slightly high. Therefore, we use smaller amounts of reagents and employ a

final test volume of 10 m£.

. Total volume is made up

Molybdic Acid Solution 0.4 mt j . to be 10 m.q,

For the ashing process, 0.9 re. of HC£04 is used.

It is also possible to employ the Gomori method to produce the chromatic

reaction for quantitative analysis after perchloric acid has been used to

decompose the sample by oxidation. For such samples that have been converted

to inorganic form, the Gomori method is advantageous because the reaction is

stable during the time interval of 45 minutes to 90 minutes after the addition

• of the colorimetric reagents. However, in this procedure, 5.0 m£ of 2.5%

solution of molybdic acid must be added to the test sample. (This is required

because, if only 2.5 mt was used as in the quantitative analysis of inorganic

phosphoe-Éhe chromatic reaction can be blocked by the HC£0 4 .) A 1.0 mt

volume of reducing agent is required in this test procedure. The standard

calibration curve for this method is obtained from the following reagent

mixture.

- 18 -

P - Standard Solution

HCO4t 60% 2.0 mt

2.5% molybdic acid solution 5.0 m9„

Elon solution 1.0 mft

In the wèt ashing process, there are occasions where the silicic acid

contained in the glass of the test tube is dissolved into the test sample

and reacting to produce a chromatic reaction. The result is a larger

numerical value for the quantitative analysis of the phospho*Ontent. (c.f.

the section on retardation of the chromatic reaction.)

IV MATERIALS THAT RETARD THE CHROMATIC REACTION

When the following chemicals, (NH4 ) 2504 ,NaC2,,NaNO 3 ,NaF,FeC£2 ,CC2,3 COOH

and others are common in the solution, the chromatic reaction is more or less

retarded. However, when Na2 S 1 0 3 , CH3 CH 2OH are common in the solution, the

chromatic reaction is accelerated.

In some cases, an aqueous solution of ammonia is used to neutralize the

4 1 sulfuric acid that was used in a wet ashing process. Of course, (NH4 ) 2SO4

is formed in such cases and care must be exercised.

When the substances mentioned above are present in the test sample the

chromatic reaction must be investigated by a blank test.

If a Potter-Elvehjem type homogenizer is used to homogenize the histo-

logical structure, there is a possibility that Na2 SiO 3 is dissolved into the • use sample. Also, when organic phosphoryls subjected to the wet ashing process,

depending on the glass composition of the oxidation tube, SiO3-- may be

dissolved into the test sample. In both cases, the chromatic reaction is

stronger. Gomori has reported that good results aré obtained if 0.5 in of

t ProbabZy à misprint of HC9,04 . EH!!

- 19 -

10N sulfuric acid is added to his chromatic reaction reagent because the

chromatic reaction by the silicate is suppressed.

The trichloreeetecacetic acid used as a protein pgcipitant has no

effect on the colorimetric reaction provided sulfuric acid with approximately

3N concentration is used in the molybdic actd solution. However, if highly

concentrated trichlortd=eleacetic acid, or sulfuric acid with a concentration

higher than 3N is Used, the colormetric reaction is suppressed.

CONCLUSIONS

We have investigated the Fiske-Subbarow, Allen and Gomori methods for

quantitatively analysing phosphor contents of samples. Evaluation of the

methods was based mainly on the experimental results obtained by the authors.

The éonsideration was carried out for the categories of, chromatic reaction

conditions for inorganic phosphoeihe method for quantitatively analysing the

vs„, inorganic phosphoricontent of histological structures or blood serum, the

method for quantitatively analysing the organic phosphorycontent of a sample

and the materials that retard the chromatic reaction.

4 To summarize the results briefly; the Allen method requires only a short

time (5 min) before the chromatic reaction is stabilized after addition of the

reagents. The colour is also stable for a duration of 20 minutes to 30 minutes.

Therefore, the Allen method is suited for the quantitative analysis of inorganic

phosphorvs.The use of perchloric acid in the wet ashing process is faster and

• has fewer reasons to produce errors when compared to the use of sulfuric acid.

However, the Allen method is rather expensive and amidol is unstable. Regarding

the Fiske-Subbarow method; the stability of the chromatic reaction is poor when

compared to the Allen method. The reaction speed of the colorimetric reaction

t Original text gives "tri hydrochloridated acetic acid". From the context it must be a misprint of "trichioridated acetic acid". EHH

- 20 -

is also slower than the Allen method. (The time required to reach a suitable

interval where measurements can be taken is 15 minutes.) However, the

aminonaphthol sulfuric acid is more stable than amidol.

Regarding the Gomori method; the chromatic reaction is stable during the

time interval of 40 minutes to 120 minutes after the colorimetric reagents

have been added to the test sample. The method is unsuited for the quanti-

tative analysis of inorganic phosphoZ(ihat coexists with the type of organic

phosphotat is susceptible to hydrolysis because the time lapse to

stabilization of the chromatic reaction is too long. However, the method is us_(

advantageous if the organic phosphortis converted to inorganic phosphontefore

the quantitative analysis.

Extinction Coetliacient

0.010 mg 0.100 mg

0.498

0.025 '

0.531 2.0t0

2.070

0.e11 2,070

0.531 - 2.070

0.532 2.100

0.535 2.130

0.5À8

2.220

0.400 -4 P

0.049

0.055

0.055 0.065

0.056

0.057

0.066

M m • o • 11.1 e ."0 • 0"' M • 0- CD

n. rt

P. CD

P .

M ce CD (D

rt. co

1. 1.5 2 3 4

15 30

40

66

120

Table 1.

Tale 2.

Extinction Coefficient

Before Heating

0.670

1.71

_

After Heating

0.671

1.70

200y

400y

Ms

oat)

0.28

0-26

0 24

Extincti

on C

oef

fici

ent

700 800 500 600 . 5 10 15 20 25 39

Wave length (nm)

Figure 1. Absorption spectrum

Time (min.)

Figure 3. Variation of the chromatic . reaction with time.

• i,L;j" 1.0 1.2 1 .:4 1.6 P 1-1 1. ...-___L -1.0 . 0.6 0.3 0.1 . (N) density of

sulfuric acid

• 0.30 rt •

r3 rt

0 0.20

o rà (D

n• 0.10 (D

rr

Figure 2. The relation between the Chromatic reaction and density of sulfuric acid.

• e 2e 2v 3V •

Temperatilre(acy

Figure 4. - The influence of temperature - on the chromatic reaCtion.

lue

-pT

gr

op

uoT

louT

qx3

U28

027

9r .0

c,» • I ie 2»•,-

/01

10 30

lUaT

ZT

33

a0D

11

0T

1D

UT

1X

3

0.600

ae,

0 300

0.200

0.100

1/ • ,t-

4

0 10

Time ultin.)

Figure 5. The relation of the chromatic reaction to temperature and elapsed cime.

m 0.20 '

m 019

P. • 018

1-+1 017 1-t)

P. 01(i (1) P

20 30

Time(min.)

Figure 6. The reduction of phosphoreontent with time at the boiling point

of sulfuric acid.

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19'20

2) Kutner, ;1'.,.C&')hen, H., J. BioL Chem. 75, 517,

1927 •

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•

9) Kipljn, N. O., Greenberg , D. V., J. Biol. Chem.

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(Received: 30/5/1952)