1955_bowman_4309_1.pdf

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4,0['-
so
a0
to
o
IaSD
ACTIVATION
SPECTRUM
the
wavelength
of the
activating
light
tion
spectrum.
The wavelength of the
exciting
light
is
determined
from
the
--
FLUORESCENT
SPECTRUM
/%k
calibrated against
the lines of dial
mercury
monochromator
stand-
ardized
a
curve
designated
as a
yields
the activa-
"
arc. The
location of the maximums
in
the curves presented
in Fig. 2
are
accu-
rate
to -+5 ml_,
but
their
shapes are
slightly
deformed
as a
result of
several
minor optical
defects.
Activation
and
fluorescence spectrums
O
i
_"
j
573
40
a0
2o
I0
_
_
....
0
r
"
'_
.
_
_!
_¢i¢_eeI
.
#*g
_
3;_
"
(49ss
)
401
__
I
200
J\
I
I
300
400
m_o
I
soo
tryptophan and
5-hydroxytryptamine
(serotonin) are
presented in Fig. 2
(2).
f
These
spectrums
are typical of
those of
other
indoles
and
5-hydroxyindoles.
Over
therangeofpHfrom2toll,
indoles
are maximally
indoles are activated
5-hydroxy-
activated
at 295
at 275
mp. and
'
ACTIVATION SPECTRUM
A
i
_i _
/
i
;tO
aol___..._
0
40
_o
z0
I
o
0
I
200
FLUORESCENT
SPECTRUM
fluoresce at
360 mrt.
All
the
indole
and
ml*
1. fluoresce at
fluorescence maxi-
Table
and
Activation
and 330
mlh
whereas
mums
of some organic
compounds.
Me-
dium
pH
2-11
pH
2-11
pH
2-11
pH
2-I
1
pH
2-11
pH
2-If
ration
(mEt)
275
Acti-
275
275
275
295
295
cence
( mta)
360
Fluores-
360
360
360
330
330
Spect
rophotofluorometric
Assay in the Visible
and Ultraviolet
With
the commercial instruments now
available, fluorescence assay
has been
ap-
plied
to
many compounds
that are
visi-
bly fluorescent. This
paper (1)
describes
an experimental instrument that extends
the scope of fluorescence analysis to per-
mit the excitation of compounds and the
measurement
of the resulting
fluores-
eenc¢
throughout the ultraviolet and
visible
regions.
The instrument, which
we call a
spectrophotofluorometer,
has
heenlapplied
to a number of problems
invoking both identification and quantio
tativ_ assay of organic compounds. Data
obtai0ed with indoles and 5-hydroxyin-
dole/ illustrate the
usefulness of
the in-
strmhcnt,
The
apparatus (Fig. 1) consists of a
125-_,vatt
xenon arc
to
provide uniform
_,
XENON
!
MOTOH _
AP¢
Compound
Tryptamine
Tryptophan
Indoleacetic
acid
Indole
5-Hydroxy-
tryptamine
5-Hydroxy-
tryptophan
5-Hydroxy-
indoleacetic
acid
Epinephrine
Norepine-
phrine
Dihydroxy-
phenvla-
lanine
Tyrosine
Morphine
o-Amino-
phenol
m-Amino-
p-Amino-
phenol
phenol
o-Hydroxy-
acid
m-ltydro-
benzoic
acid
I
300
I.
400
It,/4,
I
500
'
Fig. 2. Activation and fluorescence
spec-
trums of tryptophan
(top)
and 5-hydroxy-
tryptamine
(bottom).
The
units
on the
ordinates are arbitrary.
light output from the ultraviolet through
the visible, together with a Bausch and
Lomb grating
monochromator
to select
the activation wavelength. The sample,
about 1 ml, is placed in a 1-cm 2 silica
cuvette. The fluorescent light is analyzed
pH
2-11
0.0IN
H._SO,
0.01N
H=SO,
295
275
275
330
320
320
;
_IILP!I!!_:TO
by
passing it
through a
modified quartz
prism microspectrograph equipped with
a mechanical scanning device containing
an ultraviolet-sensitive
photomuhiplier
.ONOC.RO"ATOR
type 1P28.
The photomultiplier
output
is coupled to the vertical axis of a cath-
*AVt_tNDTH
ode-ray
oscilloscope,
and the
output of
ORUM
NELIPOT
ICALISPATEO
MOTOR
a
potentiometer coupled
to the
wave-
axis.
length cam is
applied
to the horizontal
In operation,
the
phototube scanning
the emitted fluorescent
light
plots a
0.0IN
H=SO,
275
0.0IN
H.oSO, 270
0.0IN
H._.SO,270-290
4N H._SO, 265
320
300-330
365
310
[_
\
P.O,6_,__/,,
,,P_,t,
.........
_.
",
\
t.OVt
NT_OVETEii'.
_fl.' .......
.
".
I
_
--I_l
_
_APTE
CONDENSING
4NH=SO,
pH
8
pH 5-6
0.1N
NaOH
265
295
290
310
365-370
420
t
P_
rolau
0
:
wavelength versus
intensity
diagram on
a Du Mont type 30411 cathode-ray oscil-
SPECTROGRAPH
loscope. This is
designated as the fluores-
_:_CT.06.,
RAY
cence spectrum. The same signal may
IIATTERV
OSC,LL0aRAP"
"'E_¢,T,HD
also
be
supplied to a pen-and-ink
re-
TIP
WAVELENGTH
c LEER
POTEHTmWETER
corder
or to
a
galvanometer. A
wave-
'
be°
oi
acid
benzoic
315
420
p-Hydroxy-
0. I
N
NaOll
pll9
280
290
330
360
:-
:_;/im-su,-_
_
WAVE
L t RGTH
'LUORESCENT
length
information
signal
is
also pro-
POTENTIO_TINTided on the
input monochromator
so
Ch_TTEPT
that
when the fluorescence
analyzer is
put,
can I)e
set
tothe incident waveh'ngth
peak
out-
the wavelength of the
varied
through
the
visibleand uhraviolet,
Fluorescence
intensity plotted against
Aniline
Lysergicacid
diethyla-
mide
arrant_ement
t I'i_ I.
Srhematic
x-,
ntCOADE_ of spectro-
:q)hotolhmronwter and block
diagram of
,'h'_triccompon_.nts.
i
3-'
pil9
315
SCIENCE.
440
VOL.
l_
5-1;vdroxyindole compounds
ex,tmined
/
Table
1) fluoresce with sumcient
in-
wn_ity
that
0.1
to
0.4 tag/ml can bc
:_
measured.
This
sensitivity
has made
it
",
possible
to
develop a fiuorimetric pro-
_ cedure, described elsewhere (3),
for
thc
_i_ determination
of 5-hydroxytryptamine
in
blood. This compound
is
found
in
human blood to the extent of about 0.1
to
0.2 Lag/ml.
=/i Fluorescence
evoked by ultraviolet
'
radiation below 365
m;_
is not peculiar
=_,,.
the indole
compounds but occurs with
,.
to
':'_]'a
.
largc number of organic
compounds.
_:t
_._7.
.
.
_ lhc
results
of a prehmmary survey of
organic compounds
that show
both visi-
bh" and ultraviolet fluorescence are pre-
seuted
in
Table 1.
The
instrument
described
is
intended
:'_only
to provide
information
about the
_.Utility and design of a spectrophoto-
_i_fluoro|neter.
A
more practical
form
of
_=.this
instrument is
currently being dc-
._'.'-_;{ned
".
ROBERTL. BOWMAN
!_/
_._1_'_"
_
PATRICIA
SIDNEY
A.
CAULFIELD
UDEN
FIUEND
,Laboratories
of Technical Development
and Chemical Pharmacology, National
;_ _Jeart
Institute, Bethesda, Mar_'[and
_i_"
_
i.
We
ard
this
wish
References
to
express
and
our
Notes
appreciation
instrumental
was
and
made
Upjohn
in
to
Bern-
|erring
by
_:_'_:
_,2,
_',
_
B. Brodle,
who
was
study
under
way.
Laboratories
5-Hydroxytryptamine
_
Abbott
as
the
creatinine
tryptophan
and
available
Laboutlories
sulfate
complex.
5-hydroxyindoleacetic
5-Hydroxy-
acid were
•synthesized
by A.
indole
compounds
that
we_
shown
pure.
3.
_
_.
4 April
S.
Udenfriend.
Biol.
1955
Chem..
Ek and
B. Witkop.
The
other
were
commercial
samples
to
be
chromatographically
C.
in
T.
press.
Clark,
H.
Weis_hach,
,_'_

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