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ANALYST, NOVEMBER 1987, VOL. 112 1619

Spectrophotometric Determination of Polyquaternium-I with Trypan

Blue by a Difference Procedure
Larry E. Stevens and Jill 1. Eckardt
Analytical Chemistry, Alcon Laboratories, 6201 South Freeway, Fort Worth, TX 76134, USA
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A spectrophotometric method is described for the determination of polyquaternium-1 in a pharmaceutical

solution. This method is based on a bathochromic shift of the trypan blue absorption which occurs when it is
complexed with polyquaternium-I. The difference in absorption is measured at 680 nm with an apparent
average molar absorptivity of 1.5 x 105 I mol-1 cm-1. Linearity is observed over the range 5-15 pg ml-l.
Keywords: Polyquaternium-1 determination; trypan blue; difference spectrophotometry; pharmaceutical
preparations

Polyquaternium-1, co- {4-[tris( 2-hydroxyethyl)ammonio]- Apparatus

but-2-enylpoly(dimet hylammoniobut-2-enyl) }tris( 2-hydroxy-
ethy1)ammonium polychloride (I), is a polycationic antimicro-
biological agent used in a variety of applications ranging from
water treatment to pharmaceutical preparations . 1 The mole-
cular mass of polyquaternium-1 ranges from 5000 to 10 000.
Trypan blue,2 tetrasodium 3,3’-(3,3’-dimethyl)-1,l’-bi-
phenyL4,4’-diylbis(azo)bis( 5-amino-4-hydroxy-naphthalene-
2,7-disulphonate) (II), is an anionic dye that forms a water-
soluble ion pair with polyquaternium-1 and is a good
chromophore. This paper describes a spectrophotometric
method which uses this complex for the determination of
polyquaternium-1 in a pharmaceutical preparation by taking
advantage of the bathochromic shift that occurs in the
absorbance of trypan blue from 658 to 700 nm. The
concentration is determined from the absorbance at the
wavelength where the maximum in the difference spectrum
occurs (680 nm).
A Perkin-Elmer 559 A double-beam spectrophotometer with
1-cm path length cuvettes was used.
Procedure
Determination of polyquaternium-l in a pharmaceutical soh-
tion by difference spectrophotometry
Pipette 5.0-ml portions of each of the polyquaternium-1
standard solutions, distilled water blank and samples into
separate 15-ml test-tubes. To each of these test-tubes add 3.0
ml of the buffer solution and vortex to mix well. Add 1.0 ml of
the trypan blue solution and mix well again. Measure the
absorbance of each solution at 680 nm, subtracting the blank
in each instance. Calculate the sample concentrations based
on the slope and intercept obtained from the three standard
concentrations.

Results and Discussion

Experimental
Reagents and Solutions
Analytical-reagent grade or spectroscopic-reagent grade
materials were used.
Trypan blue solution, 0.05% mlV. Prepared in distilled
water.
Polyquaternium-1 standard solutions. Approximately
0.0005, 0.001 and 0.0015% mlV in distilled water.
Buffer solution. Prepared by dissolving 30 g of sodium
chloride in 1000 ml of 0.5 M sodium phosphate solution (pH
3.5).
Sample solution. OPTI-SOFT brand contact lens soaking
solution.
Polyquaternium-1, which does not have a chromophore that
absorbs above 200 nm, can be determined spectrophoto-
metrically by the formation of a water-soluble complex with
trypan blue. The formation of an ion pair between the
quaternary ammonium and sulphate ions increases the elec-
tron delocalisation in trypan blue and produces a batho-
chromic shift of about 40 nm (Fig. 1). The absorbance of the
complex is then measured at its maximum difference (AA)
from a blank at 680 nm.
Effect of Concentration of Reagent and Time
The amount of trypan blue necesary to obtain a linear graph
for a sample concentration range of 5-15 pg ml-1 was studied.

(HOCH2CH213& -CH2CH= CHCH2

( n + 2)CI-

Na0,S S0,Na

NH2 OH OH NH2
CH, CH,
II
 View Article Online
1620 ANALYST, NOVEMBER 1987, VOL. 112

Table 1. Recovery of polyquaternium-1 from product samples with

0.5
known concentrations
0.4
Recovery, 70
Polyquaternium-l/
-6 0.3
F
pg ml-1
5.0
Day 1
101.9, 100.1
Day 2
100.4, 98.6
Day 3
99.5, 101.2
0.2
7.5 98.7, 95.5 - - - -
0.1
10.0 99.5, 100.7 98.7, 99.7 100.2, 100.8
12.5 101.1, 100.6 - - - -
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15.00 100.4, 99.6 100.2, 99.8 98.7, 100.1

0
Mean+s.d. . . , . 99.8 f 1.76 99.6 f 0.76 100.1 k 0.90

Fig. 1. Absorption spectra for 5.8 X 10-5 M trypan blue in (A) buffer 0.15
and (B) buffer with 1.1 X 10-6 M polyquaternium-1

0.10

q
a
0.4
0.05
q
4 0.3

0.2
0.1
0 10 20 30 40 50
Polyquaternium-1/pgml-1
Fig. 2. Effect of trypan blue polyquaternium-1 concentration ratio.
(A) 0.02 mg ml-* trypan blue; (B) 0.05 mg ml-1 trypan blue; and ( C )
0.1 mg ml-1 trypan blue
Solutions which contained from 0 to 500 pg of poly-
quaternium-1 were prepared and mixed with various amounts
of trypan blue in 10 ml of buffer. Absorbance values obtained
at 680 nm are shown in Fig. 2, from which it can be seen that
0.05 mg ml-1 of trypan blue is necessary to achieve linearity
over the concentration range 2.5-7.5 pg ml-1. This corre-
sponds to a range of 5-15 pg ml-1 for a 5-ml sample aliquot. A
slight excess over this amount can be used; however, this also
raises the blank absorbance and decreases the sensitivity.
A shaking or vortex time of 5-10 s immediately following
the addition of trypan blue to the sample - buffer mixture is
necessary for good precision. The complex is stable for 24 h,
after which slight precipitation occurs at higher concentra-
tions.
0
0 1 2 3 4 5
NaCI, Yo
Fig. 3. Effect of sodium chloride on absorbance at 680 nm for 10
pg ml-1 of polyquaternium-1
Analytical Data
Linearity was demonstrated over the range 5.0-15.0 pg ml-1
of polyquaternium-1. The apparent molar absorptivity was 1.5
X l o 5 1 mol-1 cm-1 at 680 nm. The linearity between the
absorbance difference at 680 nm (AA) and concentration (c,
pg ml-1) was observed on three different days and was found
to be expressed by the equations AA = 0.0326~- 0.0138, AA
= 0.0334~- 0.0212 and AA = 0.0243~- 0.0154, with
correlation coefficients of 0.999, 0.998 and 0.999, respec-
tively. The mean recovery for a set of 20 independent product
samples with 10 pg ml-1 of polyquaternium-1 was shown to be
100.3%, with a coefficient of variation of 1.2%. Recovery
values for product samples with polyquaternium-1 concentra-
tions ranging from 5 to 15 pg ml-1 on three different days are
shown in Table 1. The mean recoveries and coefficients of
variation for the graphs ranged from 99.6 to 100.1 and from
0.9 to 1.8%, respectively.

Effect of pH
The effect on the absorbance values of varying the solution pH
from 2.0 to 7.0 was studied. The resulting absorbance values
indicated an optimum pH range of 3.5-5.0; however, the
values differ by only 3% throughout the pH range 2.5-7.0.
The absorbance value at p H 2.0 was 8% less than that
obtained at pH 3.5. A buffer concentration of 0.5 M was found
to be sufficient to bring the p H into the optimum range.
Effect of Ionic Strength
The absorbance of trypan blue increases to a maximum with
increasing sodium chloride concentration. Fig. 3 shows that a
3.0% sodium chloride concentration in the buffer solution is
necessary to obtain the optimum absorbance. This eliminates
any significant difference in absorbance (less than 1%)
between a standard and sample with different ionic strengths.
Interferences
The effect of additional sample components was also studied.
These included 1 mg ml-1 of polyvinyl alcohol, 1 mg ml-1 of
hydroxyethylcellulose, 1 mg ml-1 of ethylenediamine tetra-
acetic acid, 1 mg ml-1 of polysorbate and 10 pg ml-1 of
benzalkonium chloride. Of these components, only benzalko-
nium chloride showed a slight interference which was ob-
served as a 2% increase in recovery for a solution concentra-
tion of 10 vg ml-1.
Conclusion
Trypan blue was chosen as a complexing agent with the aim of
obtaining a water-soluble ion pair which would have a
measurable bathochromic shift in a wavelength region above
potential solution interferences.
The described procedure is simple, fast, accurate and
precise in determining polyquaternium-1 in a pharmaceutical
preparation. This method has also been studied in other
sample matrices and appears to work well in the presence of
 View Article Online
ANALYST, NOVEMBER 1987, VOL. 112 1621

polyvinyl alcohol, polysorbate, hydroxyethylcellulose and 2. Windholz, M., Budavari, S., Stroumtsos, L. Y . , and Fertig,
ethylenediaminetetraaceticacid. M. N., Editors, “The Merck Index,” Ninth Edition, Merck,
Rahway, NJ, 1976, p. 1255.

References
1. Petrocci, A . , Clarke, P . , Merianos, J . , and Green, H., Paper A61387
“Development In Industrial Microbiology,” Volume 20, Received October loth, 1986
Society of Industrial Microbiology, Arlington, V A , 1978, p. 11. Accepted June 5th, 1987
Published on 01 January 1987. Downloaded by University of Illinois at Chicago on 01/09/2013 08:53:57.