Simplified
Method
for
Phosphorus
Harry
A simplified
the Estimation
of Inorganic
in Body Fluids
Goldenberg
procedure
is described
and
for
Alberto
the
Fernandez
determination
of inorganic
phosphate
in
body fluids. The method employs two stable reagents
and requires a minimum
number of steps. Serum is deproteinized
with trichioroacetic
acid containing
ferrous ion
and
thiourea.
The
supernatant
is decanted
molybdic
acid. The phosphomolybdate
ferrous ion to produce a blue color
color
is insensitive
thiourea,
Beer’s
and
added
is presented
role
dentition,
and
almost
and
most
cate
that
but
the
not
in health
pressed
in
are
form.
Hypophosphatemia
tile
vated
renal
urinary
failure,
JTeeeived
the
of ten
healing
ill
colorimetric
Bio-Science
for
publication
May
7600
23,
1966;
form
con-
studies
phosphorus
steatorrhea.
the administration
anesthesia
with
to
indiare
de-
Both
in-
of insulin,
or chloro-
ether
ilyperparatllyroidism,
increase.
in
most
of
occurs
in a characteristic
urinary
in
plays
erythrocytes
varies
increased
are
There
mental
generally
are
disease.
reEle-
encountered
in
fractures.
nIetilods
Laboratories,
fluids
tends
is
in the
Clinical
encountered
excretion
levels
the
idiopathic
following
following
and
formation,
phosphate
while
and
and
A comparison
body
blood
phosphate.
Serum
excretion
phosphorus
less
serum,
of body
is also
in
inorganic
to
Recoveries
skeletal
present
The
or
disease.
phosphate
serum
is
organic
coiitent
phosphorus
Numerous
Froln
the
of
similarly
depressed
and adrenalin,
and
that
It
ion, and
(1).
of tile
metabolism,
osteonlalacia,
dexes
glucose,
ports
plasma
all
cell
of
conformity
concentrations.
and SubbaRow
every
volume
ferrous
Excellent
to be quantitative.
of Fiske
piloSphate.
in the
and
rickets,
are shown
a small
reduced
in situ by the
hours. The intensity
of
molybdate,
of phosphorus
that
balance.
phosphate
manner
but
and
organic
exclusively
tain
range
intermediary
acid-base
inorganic
with
supernatant.
throughout
in
mixed
of acid,
serum
and urine
this method
is distributed
important
the
over a wide
to serum
between
PHOSPHORUS
both
in concentration
in decanting
law is demonstrated
of phosphorus
all
to changes
to losses
and
formed
is immediately
that is stable for several
have
Tyrolle
accepted
871
been
Ave.,
for
\all
publication
proposed
Nuys,
for
Calif.
Julie
91405.
28,
1966.
tile
esti-
872
GOLDENBERG
mation
of
usually
treated
inorgallic
subsequently
ployed
by
stannous
ferrous
with
reduced
previous
chloride
sulfate
(5,
most
reductants
of phosphorus.
shelf
life.
& FERNAND
phosphate.
In
these
molybdate
and
the
to molybdeiium
authors
include
6)
in use
are
not
the
phosphorus
phosphomolybdate,
is
formed
is
agents
acid
(1),
(3),
Experience
acid
shown
(4),
that
(7).
completely
ascorbic
has
acceptable
Some
reducing
agents
yield
molybdenum
Others
methods
Chemistry
blue.
The
reducing
aminonaphtholsulfonic
p-methylaminophenol
and p-semidine
(2),
Clinical
are
blue
for
unstable
colors
and
that
the
em-
analysis
have
a short
are
unstable,
deviate
from
Beer’s
law, or are sensitive
to small
changes
in the acidity
of the medium.
lii our study
of various
reducing
agents,
ferrous
ion
was found
to offer
the greatest
advantages
for routine
use. When
employed
in the form
of Mohr’s
salt the compound
appears
to suffer
from
few
or
none
The
veloped
for
of
the
objections
present
method
is based
to provide
the clinical
the
determination
of
the
analytical
approach
appraised.
It is customary
Taussky
and
Shorr
(6)
tions
This
od
to
other
reductants.
on the
laboratory
use
phosphorus
in
be
described
here
body
fluids.
adopted
by previous
to add
the reductant
combined
their
reductant
to give a mixed
reagent
that
would
mixed
reagent
is not stable
for more
to
of Mohr’s
salt
with
a simplified
the
Mohr’s
workers
after
and
is combined
(trichioroacetic
of thiourea.
deproteinized
into
a cuvet
small
volume
Only
two reagents
are required
for the
with
the iron-TCA
and the supernatant
for
color
development
with
inolybdate.
of molybdate
reagent
used,
the sample
decantation
ment.
is shown
no
and
effect
Material
this
on the
stabilized
final
end,
has
been
rethe molybdate.
molybdate
soluphosphate.
in the meth-
with
precipitant
to have
TCA)
was deprocedure
Toward
react
directly
with
than several
hours.
salt
acid,
and
by
the
the
protein
addition
analysis.
Serum
is
solution
decanted
Because
of the
loss
sustained
in
absorbance
measure-
and Method
Reagents
Iron-TCA,
a 500-ml.
(Eastman
sulfate
an
stabilized
volumetric
Kodak
Some
commercial
is purified
dried
distilled
deposit
in
A
water,
is filtered
a desiccator.
and
deposit
of
preparations
by
of
recrystallizing
filter
off
gm.
thiourea
from
45
gm.
dilute
sulfur
if necessary,
on a Buchner
Yield,
50
of
with
distilled
water.
and 15 gm. of Mohr’s
dissolve
bottle.
Thiourea
and
Co.)*
hexahydrate),
amber
of hot
talline
Transfer
flask
to mark
begins
may
water.
and
place
funnel,
trichloroacetic
Add
5 gm.
salt
(ferrous
to
contain
Dissolve
overnight
washed
with
form
small
60
gm.
in
with
of
acid
water.
after
Store
in
a week
anfounts
of
the compound
the
refrigerator.
a little
cold
to
of thiourea
ammonium
but
phosphorus.
in 100
Tile
distilled
ml.
Cryswater,
Vol.
No.
12,
does
12,
not
INORGANIC
1966
interfere
with
6-12
months.
Molybdate
Add
500-ml.
volumetric
22
of
gm.
water,
flask
Phosphorus
if there
1.
with
Place
shaking.
2.
Decant
3.
Prepare
a standard
and
0.2
200
This
for
ml.
of cold
reagent
for
in
of mold
to
water.
in
ml.
of
several
years.
gm.
of pure,
distilled
is added
a
Add
200
0.2197
1100)
is
cooling
distilled
is stable
at
reagent
with
Dissolve
hour
this
dissolved
of chloroform
signs
of
112S04
ml.
an
amount
are
life
previously
5 mg./100
A small
shelf
concentrated
mix.
(dried
for Serum
of
The
containing
standard,
Discard
Procedure
ml.
and
KH2PO4
to a liter.
analysis.
molybdate,
to mark,
anhydrous
tive.
45
ammonium
dilute
dilute
the
873
PHOSPHORUS
water
as
and
a preserva
growth.
(or Plasma)
ml. of serum
Let stand
for
the
in a test tube
and
add
10 mm.
and centrifuge.
supernatant
into
a blank
containing
containing
0.2
ml.
a clean
5 ml.
of
iron-TCA
tube.
0.2 ml. of water
of 5 mg./100
+ 5 ml. of iron-TCA,
ml. P + 5 ml. of iron-
TCA.
4.
Add
version.
within
5.
0.5
The
2 hr.
Read
versus
(A8)
wavelength
ml.
of
color
molybdate
reagent
develops
rapidly
the
each
tube
is measured
the absorbance
the blank
at
660
m
serum
unknown
(Klett
filter
No.
between
and
750
mjj.
in
the
640
of
to
and
and
mix
by
in-
after
20
mm.
or
and
standard
at any desired
(An)
66)
or
Calculation
The
the
amount
of
phosphorus
serum
or
plasma
is calculated
by
formula
A.
mg.
phosphorus/100
ml.
serum
-i--
=
X
(1)
5
Comments
In
decanting
the
centrifugate,
few drops
of solution,
since
analytical
error
of only
1%.
The procedure
as described
be readily
iron-TCA,
adapted
centrifuge,
it is unnecessary
a loss
of
above
to other
volumes.
decant,
and
0.5
ml.
requires
For
add
(10
0.2
0.1
0.3
to
ml.
ml.
transfer
drops)
ml.
of
the
last
produces
serum
of serum:
of molybdate.
Use
an
but
can
3 ml. of
The blank
contains
0.1 ml. Qf water
+ 3 ml. of iron-TCA
+ 0.3 ml. of molybdate;
the standard,
0.1 ml. of 5 mg./100
ml. of P + 3 ml. of iron-TCA
+
ml. of molybdate.
Read
in a photometer
and use equation
1. For 0.5
of
serum:
Use
10 ml.
of
iron-TCA,
centrifuge,
decant,
and
add
0.3
ml.
1 ml.
874
GOLDENBERG
of
molybdate.
TCA
+
10 ml.
tion
The
1 ml.
blank
1. It may
(from
0.5
The
speed
able,
+
be noted
ml.
the
standard,
of serum)
As
analysis.
a loss
an
iii
decanting
solutions
volume
of molybdate.
are
filtered
P +
0.9%.
as
may
an
ml.
by only
well
as
withdraw
one-tenth
a centrifuge
and
iron-
supernatant
accuracy
analyst
of
in Equa-
the
reading
with
If
ml.
substitute
for
the
it
10
Chemistry
of 5 mg./100
and
photometer
and
mix
development.
deproteinized
1.
tile
+
0.5 ml.
Read
alternative,
the
supernatant
reagent
for color
the
its
an
volume
of
is not avail-
aliquot
mixed
with
for Urine
To
TCA
2.
water
is recommended
a one-tenth
Procedure
Clinical
of
of 1 ml.
decreases
of
ml.
of molybdate.
that
procedure
of
0.5
1 ml.
decantation
aliquot
molybdate
contains
of molybdate;
of iron-TCA
& FERNANDEZ
0.2
ml.
and 0.5
A blank
of urine,
diluted
ml. of molybdate
and standard
1 to 20 with
reagent.
are prepared
of water
and
0.2 ml. of phosphorus
the diluted
urine.
3. Zero the photometer
with
the
setting)
and measure
the absorbance
standard
water,
add
5 ml.
Mix by inversion.
in like manner,
standard
blank
of
iron-
using
0.2
ml.
in
place
of
respectively
at
the
of
660 m
(or other
urine
unknown
selected
(An)
and
(A8).
Calculation
The
amount
of phosphorus
111g.
in the
phosphorus/100
urine
ml.
is calculated
=
urine
X
--
by
the
formula
100
(2)
Comments
The
this
urine
may
dilutions
be varied
Urine
specimens
most
at the
commonly
discretion
containing
used
of the
protein
A detailed
sis
The
were
except
was
study
made
reagent
similar
for
in
of the
order
parameters
to
establish
1 to
20 or
1 to
10,
but
analyst.
will
addition
of iron-TCA.
If this
happens,
15 mill.
before
centrifuging.
Decant
the
(0.5 ml.)
in tile usual
manner.
Experiment
are
develop
allow
the
supernatant
a turbidity
protein
and
to
add
upon
settle
for
molybdate
Results
involved
the
in the
optimum
concentrations
and
procedure
to those
described
above
under
the parameter(s)
under
evaluation.
phosphorus
conditions
analyof
assay.
used
in these
experiments
“Material
and
Method,”
Vol.
12, No.
12,
Molybdate
1966
INORGANIC
PHOSPHORUS
875
Reagent
The
influence
of variations
on the
intensity
of the
a maximum
at
a plateau
a reagent
through
concentration
iii
color
is shown
concentration
the
highest
of amnionium
in Fig.
of
value
1. The
about
tested,
molybdate
absorbance
2.3%
viz.,
reaches
and
10%.
remains
on
Molybdate
is
0.4
0.3
Fig.
1.
Color
phosphorus
amillolliuni
centration.
700
mi
in
intensity
produced
(16
tg.)
molybdate
as
Readings
were
a
by
function
reagent
Beckman
0
z
of
con-
taken
DU
Lu
0.2
at
lfl
spectro-
photometer.
0.1
% AMMONIUM
employed
be decreased
intensity.
reagent
ciple.
in
This
Unlike
Mohr’s
dilution
when
dependent
as 50%,
As seen
on
is
the
the
stock
reagent
at a concentration
of 4.4%,
which
almost
one-half
or doubled
with
no effect
on the
insensitivity
to changes
in concentration
of the
is mandatory
phorus,
is
the
by
for
the
substances
salt,
mixed
on
a valid
the
and
intensity
application
present
in
thiourea),
with
tile
sample,
decantation
double
call
of
the
the
TCA
decantation
prin(phos-
undergoes
a large
a fluial
A ma,or
the
tolerate
can
color
color
supernatant
molvbdate
yielding
loss.
would
approximately
in Fig.
1, the system
The ability
intimately
MOLYBDATE
concentration
decantation
fiuial niolybdate
this
increase
that
loss,
such
concentration.
with
no effect
of color.
of the
related
concentration
system
to tolerate
to a factor
not
of acid
iii
the
increased
previously
mixture.
If
amounts
of molybdate
considered,
namely
insufficient
acid
is present,
the molybdate
may
undergo
reduction
to form
molybdenuni
blue in the
absence
of phosphorus.
The amount
of acid
required
to give a colorless
blank
increases
with
tile amount
of molybdate
employed
for analysis.
The iron-TCA
solution
prevent
spontaneous
volume
acid,
as
molybdate
of molybdate
the
solvent.
to iroii-TCA
contains
development
used
contains
10%
of
(0.GN)
color
distilled
TCA;
even
water,
However,
increasing
tile
to 1 :5 results
in a colored
this
when
rather
ratio
blank
is sufficient
the
one-tenth
than
sulfuric
of unacidified
whose
intensity
to
876
GOLDENBERG
deepens
eliminated
on
standing.
by
adding
The
& FERNANDEZ
possible
sulfuric
Clinical
development
of
acid
(approximately
molybdate
reagent.
When
prepared
in this
reagent
has a shelf
life of several
years.
The
effect
of sulfuric
acid
on the
color
varying
corresponds
the
contribution.
in any
of
(0.53N)
acid
is
included
in the
rapidity
and
effort
Mohr’s
days.
sulfuric
varies
reaction
0.53
color
If the
the
to
molybdate
was
studied
by
from
0 to 6N,
which
exclusive
of the TCA
acid,
from
the
is
the
intensity
acidity
were
noted
due
to TCA
permissible
range
1.06N
or
when
stable,
ferrous
ion
increasing
of
higher.
Reagent
Sumner
(5) noted
phosphomolybdate
employed
Mohr’s
the
in
blanks
to
3N)
manner,
reagent
mixture,
differences
a 2-hr.
period.
with
system
than
2% in 2 hr.
to be 2 hr. The
was
also
found
an
of the
molybdate
0.55N
in the final
No significant
tubes
over
the
used
lron-TCA
to
acidity
to 0 to
colored
Chemistry
as
This
He reported
the
ferrous
molybdate
to be unstable
stability
was
salt
salt
of color
a primary
with
TCA
not
obtained
and
shelf
life of his ferrous
reagent
of Taussky
after
several
hours.
development
made
to establish
is comparatively
was
antioxidants
the ferrous
tory
since
that
the color
is instantaneous
obtained
conditions
for
stable
toward
standard
provided
satisfactory.
sulfate
reagent
and
Shorr
(6)
In view
of the
using
ferrous
its stabilization.
oxidation
by
in quantitative
a reagent
with
reducing
were
therefore
tested
as possible
ion. Hydroquinone
and
diphenylamine
on aging
they
imparted
yellow
or pink
ion,
air,
analysis.
a shelf
life
Miscellaneous
is added
no more
and
is
Combining
of several
agents
and
stabilizing
agents
for
were
not
satisfaccolors
to the reagent.
Other
agents
were
also
tested,
including
hydroxylamine,
hydrazine,
and sodium
sulfite,
but in each case the compound
underwent
oxidation
to produce
a marked
yellow
or green
color.
Thiourea
was found
to give
the most
satisfactory
results.
Its principal
oxidation
product,
sulfur,
precipitates
from
solution,
hence
the
reagent
remains
colorless.
The
stabilized
iron-TCA
reagent
but this is largely
an illusion
was found
to be usable
for
The
optimum
concentrations
termined
first
study
by
the
assumes
a yellow
appearance
due to the sulfur
precipitate.
periods
up to 1 year.
of thiourea
and
ferrous
studies
that
are
summarized
Mohr’s
salt
was
fixed
from
0 to 5% in
obtained
in 10-40
trations
up
to
definite
increases
1%.
the iron-TCA
mm.
with
At
in
the
readings
no
reagent
apparent
higher
in
3%
at
(Table
effect
concentrations
were
noted,
on
The
ion
aging
reagent
were
de-
Tables
1 and
2. For
the
and
the thiourea
varied
1). Stable
by thiourea
of thiourea,
approximating
colors
were
at concensmall
2-4%
but
over
Vol. 12, No. 12,
the
was
1966
indicated
selected
INORGANIC
30-mm.
interval.
On the basis
as optimal.
Table
2 illustrates
ferrous
ion in the presence
of a 1%
clear
from
the first
line
in the table
molybdate
in the absence
of iron,
but
Other
concentrations
eral
results.
The
this
is
Mohr’s
of
effect
apparent
at
salt
probably
is
about
4%
regarded
intensities
as
agent,
2 hr.,
the
thiourea
3, 5, and
optimum
the
with
serving
as
shelf
using
2%
salt
and
ion
an
the
a
Mohr’s
The
stability
that
ferrous
same
genthe color;
10%.
Five
concentration.
to maximize
color
intensity
compared
whether
reductant.
1) suggest
1% thiourea
of increasing
were
also
tested,
with
the
Mohr’s
salt is to stabilize
chosen
in
as
of these
data,
the
effect
concentration
of thiourea.
It is
that
thiourea
reduces
phosphothe color
formed
is not
stable.
of
the
been
(increase)
arises
the
(Table
with
has
in
Mohr
‘s salt.
The
question
thiourea
of adding
concentrations
represents
ever,
a 3% solution
reagent.
The
change
salt
877
PHOSPHORUS
per
life of the
3%
Mohr’s
increase
or
for
thiourea
is
magnitude
of
is the effective
antioxidant
and
cent
How-
5%
to
be
the color
reducing
supplementary
reductant.
Spectrum
Absorption
The
mum
molybdenum
between
700
blue
and
800
spectrophotometer).
Table
1.
chromophore
The
o
EFFECT
m
with
has
a peak
absorbance
THIOUREA
ON
a broad
at 725-750
decreases
COLOR
(%)
Table
#{149}
reading
10
20
salt
maxi-
(Beckman
90%
of
=
20
DU
its
Mo.)
(rain.)
40
0
252
253
252
0.5
250
251
252
1.0
252
250
251
3.0
260
262
266
5.0
263
266
273
2.
EFFEcT
05’
FERROUS
ION
ON
CoLoR
Klett
Mohr’s
m
to
(PHOSPHORUS
INPENSIPr
Ktett
Thiourea
absorption
(%)
(PHOSPHORUS
INTENSITY
readi
ng
=
20
Mo.)
(mm.)
10
20
30
40
0
268
276
280
284
0.5
237
247
253
256
1.0
244
249
253
255
3.0
251
251
252
253
5.0
263
263
262
264
10.0
269
269
271
269
peak
GOLDENBERG
878
value
at
660
m
and
to 84%
450 m.
Wavelength
tivity
for phosphorus
also
and
acceptable
offer
the
length
limits
Phosphorus
were
tested
20-30
Each
m
Clinical
before
above
700
However,
they
of
most
to Beer’s
Conformance
640
settings
analysis.
because
advantage
of
at
& FERNANDEZ
it reaches
mt
provide
settings
at
do not
involve
being
compatible
clinical
a minimum
maximum
660 or 640
much
with
Chemistry
loss
the
at
sensim
are
of sensitivity
upper
wave-
photometers.
Law
standards
under
the
ranging
routine
in 3 different
instrument
provided
in concentration
conditions
of
photometers.
a linear
up
assay,
to 20 mg./100
and
read
ml.
after
The results
are given
in Fig.
response
with
n
indications
Fig.
2.
method
Evaluation
for
of
conformity
to
2.
of
phosphorus
Beer’s
law.
Lu
0
z
H
H
0
8
16
24
MICROGRAMS
deviation
from
Conformity
Beer’s
to Beer’s
containing
5%
Recovery
Studies
94.3
was
(diluted
obtained
to 100%.
The
40
law
law
Mohr’s
Phosphorus
or urine
coveries
32
PHOSPHORUS
was
at
the
also
highest
phosphorus
obtained
using
amounts
(0.2
an
concentration.
iron-TCA
reagent
salt.
added
in 10-pg.
1 :20)
and
analyzed
in
from
6 serums
averaged
phosphorus
recovered
ml.)
to 0.2 ml.
of serum
the routine
manner.
The
97.0%,
falling
in a range
from
6 urine
specimens
varied
reof
Vol.
12,
No.
12,
1966
from
95.0
from
added
serum
and
in amounts
to
Comparison
INORGANIC
101%,
to Reference
A series
cedure
and
an
3, the
ference
of
the
Table
yielded
mg./100
average
phosphorus
was
extended
to
of
ml.
the
value
urine
for
analyses,
serum.
the
COMPARATIVE
recoveries
pilosphorus
was
the present
As indicated
proin
When
present
OP
sample
(mg./I0O
the
Present
a mean
to
comparison
method
gave
(Table
SERUM
ml.)
with
is equivalent
values
RESULTS
by
(1).
results,
difference
Fiske.S,LbbaRo,v
Sevom
Quantitative
when
comparable
This
Fiske-SubbaRow
3.
98.0%.
also
obtained
16, and 20 g.
was analyzed
in duplicate
of Fiske
and SubbaRow
2 methods
0.03
than
average
Methods
of 12 serums
by the method
Table
higher
with
urine
were
of 4, 8, 12,
879
PHOSPHORUS
4)
and
PHOSPHORUS
the
difference
ANALYSES
De’iat
ion
(mg/ICC
nil.)
ml.)
1
3.61
3.53
-0.08
2
3.36
3.27
-0.09
3
3.94
3.91
-0.03
4
3.78
3.75
-0.03
5
4.64
4.51
-0.13
6
3.80
3.79
-0.01
7
4.03
4.12
+0.09
8
3.23
3.26
+0.03
9
3.70
3.64
-0.06
10
3.92
3.99
+0.07
11
3.28
3.22
-0.06
12
3.75
3.71
-0.04
3.75
3.72
-0.03
MEAN
Table
4.
COMPARATIVE
RESULTS
op
URINE
of
PHOSPHORUS
ANALYSES
Deviation
Fiske-SubbaRon’
Urine
sample
(mg/ICC
ml.)
method
(mg/ICC
ml.)
(mg/ICC
(%)
ml.)
3
50.3
53.2
+2.9
4
69.7
72.6
+2.9
5
6
34.2
35.5
+1.3
60.1
63.8
+3.7
7
48.5
50.0
+1.5
8
93.6
98.8
+5.2
9
49.2
50.9
+1.7
10
78.2
80.6
+2.4
+
+
+
+
+
+
+
+
+
+
11
8.9
10.1
+1.2
+13.5
47.1
48.6
+1.5
+
+
1
59.4
62.7
+3.3
2
48.4
50.0
+1.6
12
%
Present
MEAN
DEVIATION
the
of methods
results
5%
method
(mg/100
dif-
1%
5.6
3.
5.8
4.2
3.8
6.2
3.1
5.6
3.5
3.1
3.2
5.1
880
& FERNANDEZ
GOLDENBERG
was
statistically
significant
study
The
at a later
small
but
analyses
prompted
cedure
same
by
date
led
significant
the
to
the
t test
similar
differences
use
of
a
the use
method
the
present
of ferrous
of Fiske
one
ion
and
suggested
that
the
above
is attributable
The
origin
of this
Effects
in
< 0.001).
findings.
noted
second
of Taussky
and
Shorr
(6)
results
as the present
method
is unlike
(p
Clinical
in
a number
as the reductant.
SubbaRow
is
5% discrepancy
to the difference
discrepancy
has
Repetition
the
reference
was adopted
(p > 0.5).
of
Chemistry
and
This
respects,
of this
urine
phosphorus
method.
The
found
to
reference
but
pro-
give
the
method
resembles
The
reductant
aminonaphtholsulfonic
it in
employed
in the
It is
acid.
in urine
phosphorus
in reductants
used
not been
determined.
values
noted
in the methods.
of Anticoagulants
The
effect
tested
of
using
anticoagulants
phosphorus
on
the collection
of plasma
samples.
sodium
citrate,
and
disodium
were
added
in 1-5-mg.
quantities
standard
or
these
agents
serum.
in the
the
standards
There
amounts
phosphorus
and
serum
determination
before
Sodium
heparin,
potassium
ethylenediamine
tetraacetate
(0.1 ml.)
to 0.2 ml. of
was
no evidence
of
used.
Their
influence
was
proceeding
interference
on plasma
with
oxalate,
(EDTA)
phosphorus
by any
phosphorus
of
values
was evaluated
by comparison
with
serum
analyses
on the same
blood
specimens,
which
were
distributed
among
5 tubes.
The
anticoagulants
were
added
in the following
amounts:
heparin,
0.2 mg./ml.
of blood;
oxalate,
2 mg./ml.;
citrate,
5 mg./ml.;
and EDTA,
1 mg./ml.
The results
obtained
Plasma
prepared
lower
This
plasma
with heparinized
with
oxalate
and
phosphorus
values
than
did
effect
by citrate
and
oxalate
components
and
may
be
plasma
citrate
and
tended
serum
(average
is a fairly
attributed
to
serum
to
were
yield
difference,
common
a dilution
shift
in water
from
erythrocytes
to plasma.
Using
coagulant,
erratic
differences
were
noted
between
values
in a small
series
of tests
(6 blood
specimens).
identical.
somewhat
5%).
one for
caused
EDTA
plasma
other
by a
as the antiand
serum
Discussion
The
bining
cantation
simplicity
the
of the
reducing
principle
eliminating
control
studies
to the
reliability.
analysis
These
and
(8),
volumetric
indicate
of
phosphorus
method
deproteinizing
by eliminating
flasks
that
phosphorus
observations
for
these
has
agents,
unnecessary
been
by
final
adjustment
changes
in the
involve
no sacrifice
have
been
confirmed
achieved
by
com-
employing
dilutions,
the
and
to mark.
classical
Quality
approach
of accuracy
by 4 major
deby
or
lab-
Vol. 12, No.
oratories
private
l,
1966
INORGANIC
in New
York
communicatioiis
procedure
can
alkaline
It is
phosphatase
recognized
cantation
be
readily
chemist,
by training
pipets
for transfer
for
measuring
out
for
less
of
further
chance
adapted
may
881
are now
using
the method.
Dr. Julius
Carr*
and Albert
to
in serum.
that
the
analyst
principle
centration
that
from
PHOSPHORUS
have
tile
who
determination
is
in
sampling.
of random
the
mixture
Decantation
contamination,
its
to
tile
acid
with
about
the
offers
of
unfamiliar
reservations
and
experience,
is inclined
of samples.
Pipets
or related
serum
and
precipitant,
but
phosphate
According
Haiiokt,
and
the
de-
validity.
The
toward
the
use
of
devices
are necessary
upon
fixing
the
con-
pipet
becomes
superfluous
greater
speed
and
accuracy
than
pipetting,
surpassing
the
sensitivity
of the clinical
photometer.
The
acceptability
of sampling
by decantation
is contingent
upon
a
number
of requirements
(8).
Foremost
among
these
is that
the supernatant
must
not be diluted
appreciably
before
the absorbance
measurement.
If the color
reagents
are added
in infinitesimally
small
volume,
theoretically
with
no
the
effect
99%
on
of the supernatant
could
be lost
during
the analysis,
provided
enough
sample
photometric
analysis
one-tenth
reading.
the volume
the volume
Under
the
of color
reagent
of supernatant.
conditions
of
(molybdic
As shown
decantation
remained
the
for
phosphorus
acid)
used
is finite,
viz.,
in Fig.
3, the analytical
5.
z
5.
Fig.
3.
Analytical
from
application
ciple
to
errors
of
z
resulting
decantation
prin-
of
phos-
r
I,
determination
phorus.
Serum
S ml.,
molybdate
=
0.2
ml.,
=
0.5
serum
iron-TCA
0
=
ml.
20
% LOSS
error
resulting
about
one-half
a 2% error.
volume-i.e.,
loss
of
Figure
*Methodist
tBronx
sample
3 was
Hospital,
Municipal
from
a 10%
If
would
result
plotted
in an
from
Brooklyn,
Hospital
decantation
the molybdate
one-twentieth
N.
Center,
loss
40
DURING
is 1%;
DECANTATION
a 20%
loss
were
double-strength
and
the volume
of supernatant-a
analytical
Equation
N.
error
3, given
Y.
Bronx,
30
OF SAMPLE
Y.
of
below,
only
yields
used
in
20%
1%.
and
confirmed
882
by
GOLDENBERG
experiment.
reagent
In
this
(molybdate)
The
validity
of
equation
alid
Analytical
(%)
is
ease
of calculation
it is assumed
the
calls
Suppose
lytical
that
20%
of the
error
of 20% would
(1 ml.)
were
diluted
tometer.
On the
were
mixed
with
It may
cal
not
for
error
equal
be seen
from
made
purely
20%
8 ml.
color
mixture
(0.2/9.0)
X
from
Equation
for the decantation
3 by
into
an
factor.
the
analytical
of
For
10 ml.
color
and
reagent.
during
(8 ml.)
transfer.
and color
before
reading
An anareagent
in
that
with
of color
of sample
loss,
but
instead
to 9.0
100 =
ml.
with
2.2%.
1 ml.
the
a pho-
excess
similar
analyti-
reagent
corresponds
A
to
color
result
V, 8 ml. for
X 20 = 2.2%.
is
diluting
reagent.
The
is obtained
V,1, and
20%
intuitively
by following
the
or it may
be derived
by ele+ Vd),
is referred
to as the
For
example,
when
the
supernatant,
a decantation
of about
example.
considerations
substituting
1 ml. for
error
as follows:
(1/9)
error
an
would
be zero if the 8-mi.
sample
to give a final volume
of 8.8 ml.
The decantation
formula
can be derived
reasoning
used
in the example
just
given,
mentary
geometry
(8).
The ratio,
Ve/(V(.
telescoping
one-twentieth
color
(3)
is exactly
volume
volumetric
sample
by
volume)
is lost
sample
error
reagent
of
(%)
error
supernatant
a prescribed
in mixing
to the
the 8.8 ml. of
error
is clearly
to
volume
Chemistry
superiiatant.
checked
(one-tenth
supernatant
result
if the
other
hand,
the
0.8 ml. of color
the
decantation
easily
tile
1 ml.
the
of
X
expression
Clinical
represents
volume
=
that
procedure
V
V1 is the
error
this
& FERNANDEZ
volume
error
of color
reagent
of 40%
is telescoped
is
2%.
References
I.
2.
Fiske,
C. 11., and
Chests.
66, 375
Kuttner,
T., and
chloride
4.
5.
6.
7.
8.
The
Y.,
R.,
micro
The
Micro
estimation
colorlinetric
determination
colorimetric
studies.
of
phosphate
of
I.
and
A
calcium
J.
phosphorus.
molybdic
in
acid,
pus,
Biol.
stannous
plasma,
and
J. Biol.
Chem.
75,
517
(1927).
Gomori,
G., A modification
of the colorimetric
phosphorus
determination
for
use with
the
photoelectric
colorimeter.
J. Lab.
GUn. Med.
27, 955
(1942).
Lowry,
0. H., and
Lopez,
J. A., The
determination
of inorganic
phosphate
in the
presence
of labile
phosphate
esters.
J. Biol.
Chen,.
162,
421
(1946).
Sumner,
J. B., Method
for the colorimetric
determination
of phosphorus.
Science
100,
413
(1944).
Taussky,
H. H., and
Shorr,
E.,
A microcolorimetric
method
for
the
determination
of inorganic
phosphorus.
J. Bud.
Chem.
202,
675
(1953).
Dryer,
R. L.,
Tammes,
A. B.,
and
Routh,
J. I.,
The
determination
of phosphorus
and
phosphates
with
N-phenyl-p-phenylenediamine.
J. Biol.
Chem.
225,
177
(1957).
spinal
3.
reagent.
SubbaRow,
(1925).
Cohen,
H.
Goldenberg,
1003
fluid.
H.,
(1956).
Decantation
as
a precision
step
in
colorimetrie
analysis.
Anal.
Chesn.
28,