Graphical Absorbance-Ratio Method for Rapid

Two-Com ponent Spectrophotometric Analysis

R. C. HIRT, F. T. KING, and R. G. SCHMITT
Stamford Research Laboratories, American Cyanamid Co., Stamford, Conn.

A simple, rapid, graphical method for the ultraviolet For two-component systems, a t a wave length A,
spectrophotometric analysis of two-component sys-
tems uses the ratio of observed absorbances at two AAbsd. = b(U;Ci f U:Cz) (3)
selected wave lengths, one of which is an isoabsorp- and s t another wave length, A,
tive point if possible. This permits a straight-line
plot of the ratio of the observed absorbances us. relative A:bsd. = b(a;Ci f U:Cz) (4)
composition, as derived from Beers law. The method Instead of being solved for c1 or c2, these equations may be
may be applied even though no isoabsorptive point divided :
exists, by use of a plot of calculated ratio us. composi-
tion. I t can be extended to a three-component system
(5)
if desired. Speed and ease in setting up an analysis
are obtained, as knowledge of only three absorptivities
As the same cell length is used for both concentrations, b may
is needed for a two-component analysis if an isoabsorp-
be eliminated from the equation. It may be shown that relative
tive point is used; these values may be readily obtained
concentrations (5, y ) may be used in place of absolute concen-
from the users files, the literature, or another labora-
trations, since
tory.

T \TO-component spectrophotometric analyses generally re-

quire the solution of simultaneous equations, for seldom do
the two components have absorption bands that do not overlap
The equation then becomes

appreciably. Many schemes have been proposed to simplify

the calculations and to lessen the amount of time and effort re-
quired both to set up the method and to use it in the analysis. USE O F ISOABSORPTIVE POINT
I n an effort to simplify further and to speed up these operations
of setting up the method and of using it later, the absorbance- If two spectra, plotted as log a or a us. wave length, have a point
ratio method here described was devised and put into use. of intersection, the numerical values of the absorptivities are
This method has no relation to the similarly named trans- equal a t this wave length; this has been termed an isoabsorp-
mittance-ratio method which makes use of highly absorbing tive point. (An isosbestic point is a special case of an isoabsorp-
blanks in the comparison beam of the instrument ( 4 ) . The tive point, where the two components are interconvertible.)
name absorbance-ratio refers to the ratio of the observed absorb- Figure 1 shows the spectra of 0- and p-nitrophenol (at p H 5.2),
ancies ( 5 ) a t two selected wave lengths using conventional which have two isoabsorptive points. If the wave length of the
blanks. It most nearly resembles the graphical-offset method isoabsorptive point a t 286 mp be chosen as the analytical wave
of Shurcliff and Stearns (? ), though resemblances to other methods length to be used in the denominator of Equation 7, the denomi-
may he noted in the equation%and graphs, for all are based upon nator bwomes a constant equal to the absorptivity value of the
and derived from Beers lav- isoabsorptive point, since x +
y = 1. Equation 7 then becomes:
By proper selection of the sndytical wave lengths, a simple
sti aight-line graph can he drawn t o show the relationship between
the absorbance ratio and the fraction or relative concentrations
of the two components; a simple one-component calculation
hased on Beers lam give?; the total amount present. In many
applimtions, only the ratio of the two components is desired,
:md this latter step may be eliminatrd.

DERIVATION

Beers law may be written in a generalized form for i compo-

nents:

.Iuh,d = bra,Ct (1)

E

or
-4absd. = b?(alCl
z
+ U2CZ $. a3C3 f a4c4 . . )
I (2) !
r
NITROPHENOLS
PH 5.2
- PARA
_____ ORTHO
~

where A is the absorbance ( 5 )(opt,icaldensity), b is the cell length,

millimeters, a is the ahsorptivity ( 5 ) of component i, and c is
the concentration of component i , in grams per 100 ml.
360 340 320 300 280 260 240 mp
1 Present address, Department of Clieiiiistry, Kansas State College,
Figure 1. Spectra of p - and o-Nitrophenols
hlsnhattan, Kan. Showing isoabsorptive points
1270
V O L U M E 26, NO. 8, A U G U S T 1 9 5 4
With the denonlinstor constant, a plot of the ratio ot ihe ob-
served absorbances, A ' / A " , os. I or y is a straight line. The
ends of the line are given by ad/a" and aL/a", where subscript..
o and p , for ortho- and para-, have replaced subscripts 1 and 2.
The use of the isoabsorptive point is the very heart of t h e
a1)sorbance-ratio method, for it permits the setting up of a two-
vomponent analysis from knowledge of three absorptivity valuei
only-i. e., the value a t the isoabsorptive point and the two values
of th(. two pure components a t the other analytii.:il W N V ~ ' length.
I I
/ANILINE
I I
-It
I I
I
I
I
IN 2-ETHYLHEXANOL
I , I l l ~ l , l ~ l
360 3 4 0 320 300 280 260 240 mi
Figure 2. Spectra of Nitrobenzene and Aniline
Showing isoahoorptive pointm
X second c+mnple is sho~viiin Figure 2, n-it11 tht. spectra of
aniline and nitrobenzene, R conilination which had been of
interest in an industrial hygiene investigation (1). Three iso-
absorptive points are s w n ; the center one is selected because of
the high angle of intersection and the greater freedom from stra>-
interferences which might occur with the shortest wave-length
point. .-i siniilar plot may be set up, using the absorptivity values
of anilinc, and nitrobenzene a t the selected analytical wave
lengths.
If no ieoabsorptive point, occurs between the spwt la1 curves
of the components to be determined, the plot of alworhance
ratio us. x is not a straight line, but a curve connecting thc ex-
treme (100%) points which are determined by the ratio of t,he
:ibsorptivit,ies of the pure components. This curve may be
plotted by use of Equation 7 , using the known absorptivity values
and substituting values of 0.05, 0.10, 0.15, 0.20, etc., for x and
t,he corresponding values of 0.95, 0.90, 0.85, 0.80, etc., for y.
If the plot is to be used for many analyses, the extra effort
of plotting this nonlinear curve ail1 be compensated for by the
ease of use.
In general, the criteria for selecting the analytical wave length
which is not. a t the isoabsorptive point are much the same as
for any analytical scheme. Wave lengths as long as possible are
preferred to minimize the effect of possible interferences. -4 large
1271
spread between the curves is dosirable, hut not so large that the
use of absorbance readings in the less accurate very high or very
low ranges of the instrument would he used. (In actual use,
readings that are too high or too low for the instrument's ac-
curate range should not be taken, and a change in cell Irngth
or concentration should be in:tde.)
The use of an absorbanco-mtio gr:iph determines relativc con-
centrations, as fractions, arid anothcv step is necessary to de-
termine absolute concentr:ttiorls. Frequently this second step is
not needed, for only the ratio of the two absorbing materials is
desired. Examples are the examination of a plant product stream
or the effluent from a chromatographic column. I n such rases,
110 weighing of the sampl? or quantitat,ive dilutions are needed;
the concentration and/or cell length need only be adjusted so that
the absorbance readings are in the desired accurate range of the
spcctrophotometer. The US(' of cells of adjustable length, such
; I S micrometer Baly cells ( 2 ) , i-: ptrticularly advantagrow i n
such cases.
UNAVAILABILITY O F 5'L':~NDARDS
The spectroscopist is frequently culled upon to detcrniine
(or estimate) txo-component absorbing systems for which pure
standard samples of either or both components are not rendily
iivailable. If spectra are available in his files, the absorptivit'y
values may be obtained from thwe, and the absorbaric.c-i,at'io
plot made. If bot'h the spectra and the pure samples are not
available, the only recourse is to published data or to thosc com-
municated from another laboratory. With the advent of
punch-card systems, as describctl by Iiuentzel ( 6 ) , and of I:irgc-
-- --7
200t
1601
i ~ ,
I - /
1 1 -
0260 1 ' '
l250-'" 240 " 230~j.1
Figure 3. Spectra in 0.1N Hydrochloric
Acid
- Trimethylolmelamine
-...- - Melamine
Ammeline
scale abstracting programs (such as those of ASTM Comniitt'ee
E-13), very much larger quantities of reliable spectrophotometric
data are becoming generally avui1:ihle to spectroscopists. Such
data may be used with the nlxorlxincc-ratio method when neces-
sary to provide an estimate if i i o t :I determination of the quanti-
ties of the two or three absorbing components.
EXTENSIOV TO TRREI.:COMPONENTS
It is possible to extend the two-component absorbance-ratio
method t o three components, at the expense of some rapidity
and convenience, but still with some advantage over the con-
1272 ANALYTICAL CHEMISTRY

ventional solving 01 thrw siinultaneous equatioiw. The almost research and analytical laboratories and in plant-control labora-
complete lack of true three-component isoabsorptive points re- tories. Two sets of data on known mixtures have been selected
quires the use of plots of absorbance ratio 2s. relative composi- to illustrate the application of this method to the systems used
tion, which are curves calculated from equations like Equation 7 , as examples in this paper. Known samples were prepared
rather than straight lines. Using three srlected wave lengths, by mixing measured volumes of solutions of known concentrations
or usually more conveniently two sets of two s e l w t d wave of the pure components. Compositional data by preparation
lengths, two sets of three curves each may be dra~vn. Thr and by the absorbance-ratio method are given for aniline-nitro-
spectra of melamine, ammeline, and trimethylolmelamine, shown benzene in Table I and for melamine-ammeline-trimethylol-
in Figure 3, prepared for use i n a hydrolysis study ( S ) , may serve melamine (3)in Table 11, to illustrate the two- and the t h r e e
as e x a m p l a The absorbance-ratio curves are shown in Figure romponent :ipplic*n.t ions.
4, and were plotted from values calculated from equations like
that of Equation 7 . Since certain of the analyt,ical wave lengthy
nearly correspond to two-component isoabsorptive points, rer-
tain of t8hesecurves are verj- nearly straight lines. 1::ach curvt: Table 1. . < d i n e and Ni trobenzerie Mixtures
corresponds to a tno-component analysis (the third component By Preparation, % By Absorbance Ratio, &
being zero) for the three possible two-component combinations. Nitro- Witro-
Aniline benzene -4niline benzene
Each ratio of two observed alxorbances then corresponds to two
possible extreme situations. The extremes of composition are 100.0 0.0 100.0 0.0
80.0 20.0 81.2 18.8
read from the plots (like Figure 4) and become two points on a 60.0 40.0 00.8 39.
40.0 60.0 41.4 58.0
triangular composition plot, such as that of Figure 5 . Because 20 0 80.0 19.0 81.0
0.0 100.0 0.0 100.0
Beers law is obeyed, the absorbances are additive, :ind the true
analysis corresponds t o one of the extreme points or to a point
on a straight line connecting them. The desired con~position Table 11. Jlelamine- immeline-Trimethylolmelamine
Jlixtures (3)
lies at the intersection of these two lines, as shown in Figurv 5 .
- . By
~ Preparation.
~ L?a __By Ahvorbance Ratio, 70
Tri- Tri-
Am- methylol- Am- ~netliylol-
hlelamine meline melamine Alelamine meline melamine

2 7 1 53.3%
53.2
0.0
63.1
36.2
22.2
46.7
0.0
49.8
18.4
31.7
19.4
0.0
46.8
50.2
18.5
72 1
58.4
19.6
53.5
54.0
0.0
64.8
34.8
21.8
22.4
46.5
0.0
50.4
19.1
31.1
20.0
58,Z
0.0
46.0
50.6
16.1
34.1
55,2
19.4
22.1 58.3

ADVANTAGES

The graphical absorbance-ratio method for two components

has the advantages of speed and ease in the setting up and use of
the analytical method. Only knowledge of the three absorptivity
values a t the analytical wave lengths is necessary. This allows
a laboratory with a large library of spectra to transmit a minimum
of information-by telephone, for example-to a smaller labora-
tory or plant control unit to enable it to set up and use a plot of
absorbaiice ratio ts. relative concentration. The advent of .
Figure 4. Three-Component Absorbance-Ratio large amounts of reaqonably rrliahle spectrophotometric data,
Plots
A. Ammeline
M. Melamine
T. Trimethylolmelamioe

As in the two-component methods, this gives the relative con-

centrations of the three components. To obtain the total con-
centration, a composite absorptivity value must be obtained
a t some wave length by multiplying each pure components
absorptivity by its fraction present and adding these; this com-
posite absorptivity is then used in a conventional one-component
Beers law calculation to obtain the total concentration. The
absolute concentration of each component is obtained by multi-
plying the total concentration by the fraction present. Clearly
the threecomponent application is clumsier and slower than
the two-component application; this is also true of the solving
of three simultaneous equations as compared to the solving of
two such equations. The graphical absorbance-ratio method is
best applied to the determination of the relative concentrations
of the three components.

ILLUSTRATIVE DATA

The absorbance-ratio method has been successfully applied to Figure 5 . Three-Component Analysis Using Absorb-
a large number of two- and three-component systems, both in ance-Ratio Data
V O L U M E 2 6 , NO. 8, A U G U S T 1 9 5 4
which mill be widely distributed, will further the usefulness of
this rapid graphical method for setting up two-component analy-
ses even when pure standard samples are not readily available
to the user. However, caution must be exercised in the use of
data in such a manner, and a realistic view of the effects on the
accuracy of the analysis must be taken.
ACKNOWLEDGMENT
The assistance Of Iiarl
preparation Of knoTyn
J' and ''
Davis in the
and Of the figures is gratefullv
acknowledged.
Detection and Estimation of Melamine in
Wet-St rength Paper by UIt raviolet Spedrophotometry
R. C. HIRT, F. T. KING', and R. G. SCHMITT
Stamford Research Laboratories, American Cyanamid Co., Stamford, Conn.
The use of melamine resins to impart wet strength to
paper makes desirable a rapid method for the detec-
tion and estimation of the melamine content of paper
samples. A spectrophotometric method has been de-
veloped which makes use of the strong absorption of
the melamine ion at 235 mp. The resin is effectively
extracted from the paper and hydrolyzed to melamine
by refluxing cut-up paper samples in 0.1.V hydrochloric
acid.
A RAPID METHOD for the detection and estimation of the
melamine content of paper samples is desirable in using
melamine resins t o impart wet strength to paper. The strong
absorption of melamine (2,4,6-triamino-s-triazine) in the ultra-
violet (1, 6) near 235 mp makes possible the detection and estima-
tion of melamine by a spectrophotometric method of high sen-
sitivity. The relatively large change in the spectrum of melamine
between neutral and acetic conditions has been utilized for the
determination of the ionization constant (3),and serves as an
additional means of identifying melamine in solution. Refluxing
cut-up paper samples in 0 . 1 S hydrochloric acid effectively extracts
and hydrolyzes the resin t o the melaminum ion, which is then
meawred spectrophotometrically.
APPARATUS AND MATERIALS
X Cary automatic recording spectrophotometer, Model 11, To.
6 i , and fused quartz cells of various light path lengths were used
for examination of the extracts in the range from 260 to 220 mp.
Appropriate adjudment of cell length and/or concentration was
made in order to obtain absorbance readings a t the analytical
wa"e lengths in the accurate range of the spectrophotometer. An
all-glass flask and reflux condenser assembly was used for both
the extraction of paper samples and for the hydrolysis experi-
ments.
The melamine (1, 6) and ammeline ( 4 ) samples were of high
purity and identical with thoqe used in previous investigations
The resin sample was a commercial trimethylolmelamine, a par-
tially condensed resin, called Parez Resin 607 ( 2 ) .
SPECTROPHOTOVETRIC METHODS
The ultraviolet spectra of melamine, ammeline, and trimethylol-
melamine in acid solution have been presented in connection
with the absorbance-ratio method ( 5 ) . The strong band of the
melaminum ion near 235 m p has an absorptivity of 81.0 and may
be utilized in a simple one-component analysis in the absence of
* Present address, Chemistry Department, Kansas S t a t e College, M a n -
hattan. K a n
1273
LITERATURE CITED
Hirt, R. C., and Gisclard, J. B., AXAL.CHEM.,23, 185 (1951).
( 2 ) Hirt, R. C., and King, F. T., Ibid., 24, 1545 (1952).
(3) Hirt, R. C., King, F. T., and Schmitt, R. G., Ibid., 26, 1273
(1954).
(4) Hiskey, C. F., Rabinowitz, J., and Young, I. G., Ibid., 22, 1464
(1950).
(5) Hughes, H. K., Ibid., 24, 1349 (1962).
(6) Kuentzel, L. E., Ibid., 23, 1413 (1951).
(7) Shurcliff, W, .I.,and Steams, E. I., J . O p t . SOC.Amer., 39, 72
(1949).
RECEIVED for review September 16, 1953. Accepted February 11, 1954.
Presented a t the Pittsbureh Conference on Analvtical Chemistrv a n d
Applied Spectroscopy, Pittsburgh, P a . . March 6 , 1953.
interfering materials. A detectability of 4 -( may be obtained with
an observed absorbance of 0.100 (in a 100-mm. cell 31 ml. in
volume), if no other absorbing materials are present.
Because other ultraviolet absorbing materials are extracted
from paper samples by the refluxing with 0.1S hydrochloric acid,
some type of background correction is desirable. Examination of
the extracts of various paper samples did not yield any reproduci-
ble value that could be used as a simple subtractive background
correction. However, because the absorption of melamine falls
off so rapidly toryard longer wave lengths, becoming only
as high a t 260 mp as at 235 mp, all the observed absorption at
260 mp can be taken as due t o absorbing materials other than
melamine. If it is assumed that such absorption does not de-
crease from 260 t o 235 mp, the absorbance a t 260 mp may be sub-
tracted from that a t 235 mp, t o give a first approximation of a
background correction. Because ammeline, the acid hydrolysis
product of melamine, may be present, the analytical x a v e length
used should be 237 mp (rather than 235 mp), which is the iso-
absorptive ( 5 ) wave length for melamine and ammeline. The
absorptivity at 237 mp is 79.0.
HYDROLYSIS OF MELA\.lINE AND OF PAREZ RESIN 607
During the refluxing with 0 . 1 s hydrochloric acid to remove the
resin from the paper sample, hydrolysis of the resin t o melamine
occurs, and t o a much lesser extent, hydrolysis of melamine t o
ammeline. I n order to study these hydrolysis reactions, a two-
component analysis of melamine and ammeline was set up, using
the giaphical absorbance-ratio method ( 5 ) . Because an iso-
absorptive point occurs b e h e e n melamine and ammeline a t
237 mp, this wave length 17-as selected as one of the analytical
wave lengths (the other being 226 mp), so that a plot of the ratio
of the absorbances at the analytical wave lengths us. fraction of
melamine or ammeline would be a straight line. The hydrolysis
of the resin of melamine also involves the presence of measurable
quantities of ammeline in its later stages, so a three-component
analysis, using the graphical absorbance-ratio method, was set
up. The details of the three-component application of this
method have been described ( 5 ) .
-4sample of melamine n a s refluxed in a large excess of 0.1,V
hydrochloric acid and aliquots withdrawn and analyzed spectro-
photometrically a t suitable time intervals, using the two-compo-
nent absorbance-ratio method described. The results are shown
in Figure 1. h similar study was made on the resin, and the
three-component modification of the absorbance-ratio method
was used t o obtain the resin content as a function of time, as
shown in Figure 1.