J. Soc.CosmeticChemists18 207-214 (1967} ¸
1967Society
of Cosmetic
Chemists
of GreatBritain
The distributionand activityof
benzoic
acid in some emulsified
systems
R. A. ANDERSON
and CLARA
E. CHOW*
Synopsis--The distribution of benzoic acid between arachis oil, isopropyl myristate or liquid
paraffin and water or 20 per cent glycerin in water has been investigated. The solute was found
to dimerize in the liquid paraffin phase.
The fungistatic activity against Aspergillus niger of benzoic acid in emulsified systems
depends on the concentration free in the aqueous phase. The benzoate ion seems to have
someactivity. Increasedconcentrationsof benzoicacid are requiredin the presenceof glycerin.
Propylene glycol enhancesthe activity of benzoic acid and at a concentration of 15 per cent
prevents the growth of A. niger in the absenceof benzoic acid.
INTRODUCTION
Many pharmaceuticaland cosmeticemulsionsrequire the inclusion
of a preservativeto prevent microbial deterioration.The activity of a preservativewill be affectedby its interactionwith an emulsifyingagent (1,2)
and by its distributionbetweenthe oily and aqueousphasesof the emulsion
(3,4).
(o Coil
)
Beanetal (4)havecalculated
thepartition
coefficients
Kw-- Cwater
for several preservativesand suggestthat for any given overall concen-
tration of preservativethe concentrationin the aqueousphasemay be
calculatedfrom the expression,
Cw
= c (Kw
(•+1)
ø + 1)
where Cw = concentration
in the aqueous
phase(percentw/v),
C = overallconcentration
(per centw/v),
*Pharmacy Department, University of Sydney, N.S.W., Australia.
207
2O8
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
Kø • oil ' water partition coefficient,and
w
•
=
oil:
water
ratio.
This expressionis not appropriatewhen the preservativedimerizesin the
oil phase,and doesnot allow for preservativeinactivated by ionizationor
interaction
with emulsifier
molecules.
EXPERIMENTAL
Liquid paraffin, arachisoil, glycerinand propyleneglycolcompliedwith
the requirementsof the British Pharmacopoeia
and isopropylmyristatewith
those of the British PharmaceuticalCodex.Brij $5 was used as received.
Benzoicacid was recrystallizedfrom water (m.p. 122ø).All chemicalswere
A.R. grade.Water wasdistilledfrom a Scorahall-glassstill.
Estimationof benzoicacid
Aqueoussolutionswere diluted with 0.01 N HC1 and the absorbance
measuredin a Unicam S.P. 700 spectrophotometerat 274.5 m•. Beer'slaw
wasfoundto holdupto at least1.0 x 10-3moles/i;glycerinandsurfactants
in the concentrations
presentin the diluted solutionsdid not interferewith
the estimations.
The concentrationof benzoicacidin the oil phasewasusuallycalculated
from the decreasein concentrationof the aqueousphase.For liquid paraffin
phasesthis procedurewas shownto be valid by directspectrophotometric
determination
after suitable dilution with isooctane.
DETERMINATION
OF PARTITION
COEFFICIENT
Suitable volumes of oil and aqueousphase containing an accurately
known concentrationof benzoic acid in 0.01 N HC1 were pipetted into
glass-stoppered
bottlesand agitatedbya wrist-actionshakerfor about1hr
at room temperature.The bottleswere then transferredto a water bath at
25 + 0.1ø for at least 7 days,beingshaken3 or 4 times duringthis period,
and then the concentrationof benzoicacid remainingin eachaqueousphase
was determined. Concentrationswere redetermined after shaking and
standingfor a further oneto three daysto establishthat equilibriumhad
been attained.
For arachisoil and isopropylmyristate equal volumesof oil and of
aqueous
phasewereused.The originalconcentration
of benzoicacidin the
aqueousphasewas usually20 millimoles/l,but in someexperimentswas
THE DISTRIBUTION
AND ACTIVITY
OF BENZOIC ACID
209
reducedto 10-15millimoles/1to showthat the partition coefficientwas independentof concentration.For liquid paraffin systemsthe paraffin to
aqueousphasevolumeratio was increasedto 6:1, and becausethe distributionwasconcentrationdependent,the originalbenzoicacidconcentration
in the aqueousphasewas varied from 5 to 25 millimoles/1.In someexperiments,20 per cent glycerinwas includedin the aqueousphase.
Microbiologicalevaluation
A strain of Aspergillusnigerwas maintained at 25ø on slopesof potato
agar containing 2 per cent malt extract, and was transferred every two
months.
The organismalsogrew well at 25ø on a medium containing2 per cent
malt extract and 0.1 M citrate pH 4.2 (or 5.2) solidifiedwith 2 per cent agar.
Attempts to determinea minimuminhibitory concentrationof benzoicacid
in this medium were unsuccessful;
when the experimentwas replicatedit
wasfoundthat the proportionof inoculawhichgrewto form visiblemycelia
and sporesgradually fell from 1 to 0 over a range of benzoicacid concentrations. However, the concentrationrequiredto prevent growth in half of
six or more replicateswas reproducibleand this criterion,referredto later
asFC 50, waschosenasa satisfactorymeansof assessing
the effectsof added
agentson the fungistaticactivity of benzoicacid.
RESULTS AND DISCUSSION
Table
Partition
coefficients
Phases
I
of benzoic acid at 25 ø
0.01 N HC1
20 per cent
glycerin in
0.01 N HC1
isoPropyl myristate
9.87
7.0
Arachis oil
6.14
4,38
Liquid paraffin
2.6
1.7
per cent-•
per cent-t
The partition coefficientsof benzoicacid in arachisoil-water and isopropyl myristate-water systemsare essentiallyindependentof benzoic
acid concentration
and are calculated
as the ratio of the concentration
in
the oil to that in the aqueousphase(TableI). The value of 6.1 for the distribution betweenarachisoil and water is higher than the coefficientof 5.33
210
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
reportedby Garrett & Woods (5); the discrepancyis probably due to the
variable nature of vegetable oils.
When liquid paraffin is the non-aqueousphase,the ratio of the concentrations in the two phasesis not independentof concentration.However,
the ratio of the square root of the concentrationin paraffin to the concentrationin the aqueousphaseis constant (Table II), suggestingthat
benzoicacidexistspredominantlyasdimersin the paraffinphase.Glasstone
(6) found similar behaviourfor the distributionof benzoicacid between
benzene
and water.
Table
II
Partition coefficientof benzoic acid in liquid paraffin-water systems at 25ø
Cw
Initial Equilibrium
Equilibrium
. (M/l)
K=
Co•
-t (mM/1)-t(%)-•
aqueous
concentrationconcentrationCo
concentration
aqueous
phase* inparaffin* Cw
as mM/1
25
20
15
10
(Cw) as mM/1
6.17
5.31
4.53
3.50
(Co) as mM/1
,
3.14
2.45
1.75
1.08
0.51
0.46
0.39
0.31
*Ratio of oil: water ----6: 1
Average
,
9.08
9.32
9.23
9.4
0.287
0.295
0.292
0.296
2.6
2.67
2.64
2.68
9.3
0.29
2.6
When the solutedimerizesin one of the phases,the numericalvalue of
the coefficientdependson the units in which the concentrations
are ex-
pressed
andthevalueof 9.3 M-tl• (TableII) is equivalent
to 0.29mM-tl•
or 2.6ø/o
-t. The last methodof expression,
thoughunusual,is probably
mostconvenientin pharmaceutical
and cosmeticformulation.
The presenceof glycerinin the aqueousphaselowersthe partition
coefficients,and the values in the presenceand absenceof 20 per cent
glycerinare listedin TableI.
FUNGISTATIC
ACTIVITY
The effect on antimicrobial activity of benzoic acid concentrationis
shownin TableIII which recordsthe resultsof eight separateexperiments
in which eachconcentrationwasreplicatedsix times.The estimatedFC50
from eachexperimentis shownat the bottomof the table. Other FC50
valuesgivenin TablesIV andV aredetermined
fromsixreplicates
with the
exception
of the valuefor 20 per centglycerinin the absence
of oil and
surfactant which is basedon 24 replicates.
THE DISTRIBUTION AND ACTIVITY OF BENZOIC ACID
211
The activity of benzoicacidhasbeenshownto vary with the pH of the
system.The FC50sat pH 4.2 and5.2 arelistedin TableIV. The equivalent
concentrations
of unionizedacid(usingpKa = 4.2 in the Hendersonbuffer
equation)have beencalculatedand are includedin the table. Thesedata
Table
III
The determinationof the fungistaticactivity of benzoicacid againstA. niger:at 25ø
and pH 4.2
Total
concentration
of benzoic
Numbers
of positive
results
fromsix
replicates
acid added (%) Expt. Expt. Expt. Expt. Expt. Expt. E
Numberof
positiveresults
of
from total
t. Expt. 48 inocula
1
2
3
4
5
6
•P
8
0.040
6
6
6
6
6
6
6
6
48
0.045
0.050
0.055
5
4
2
6
6
4
6
6
3
6
6
2
6
6
4
6
5
2
6
6
3
6
6
3
47
45
23
I 1
1
0
0
0
1
0
1
4
0
0
0
0
0
0
0
0
0
0.060
0.065
Estimated
FC50
(percent) 0.0530.0570.0550.0530.0570.052
I 0.055 0.055
Table
0.055
IV
Fungistatic activities (as FC 50) of benzoic acid in the presenceof propylene glycol or
glycerin againstA. niger at 25ø
FC 50 (per cent)
pH
Addedagentand concentration
$.2
None
4.2
None
Propyleneglycol 5 %
....
Glycerin
,,
10
15
,,
,,
20 ,,
Concentration
Concentration
acid added
unionized
of benzoic
unbound
0.190
0.019
0.055
0.027
and
0.050
0.035
0.000
0.072
0.040
supportthe conclusion
of EvansandDunbar(7) that lessof the unionized
acidis requiredat higherpH valuesbecausethe benzoateion alsohassome
activity;they donot agreewith the findingsof WinsleyandWaiters(8) that
the antimicrobialactivity dependsonly on the unionizedbenzoicacid over
the rangeof pH 2.2•5.1.
212
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
All other determinationsreported in this paper have been carried out
at pH 4.2.
Propylene glycol or glycerin is often included in pharmaceuticalor
cosmetic emulsionsand the increasedsolubility of preservative in the
aqueousphaseleadsto a reductionin the partition coefficient(TableI). It
has beensuggested(9) that this makesmore preservativeavailablein the
aqueousphasewith consequent
increasein antimicrobialactivity. Table IV
showsthat propyleneglycol augmentsthe fungistaticactivity of benzoic
acid, and that concentrationsabove about 15 per cent have considerable
activity evenin the absenceof any other agent.
On the other hand, glycerin reducesthe activity. The equivalent concentration
of unionized benzoic acid shown in Table IV
has been calculated
usingpKa' value(in 20 percentglycerin)of 4.3 determined
by the method
of Albert and Serjeant(10). This FC 50 of unionizedacid is about 1• times
the FC 50 in the absenceof glycerin.(The concentrationof benzoateion is
only slightlygreaterin the 20 per cent glycerin.)It is suggested
that in the
sameway that the increasedsolubilityof benzoicacidin 20 per centglycerin
reducesthe oil-water partition coefficients,it also reducesthe availability
of the preservativeto the microbialbiophase,and consequentlya higher
concentrationis required.
Brij $5 alsoreducesthe availability of benzoicacid to the oil phaseand
to the biophase.SinceEvansand Dunbar (7) and Mitchelland Brown(11)
have shown that benzoic acid is distributed
between the surfactant
micelles
and the true aqueousphasein a constantratio which is independentof the
overall benzoic acid concentration,it is possibleto calculate from the
solubilizationdata of Andersonand Slade{12) that 0.1 per cent Brij $5
will bind about 3.6 per cent of the unionizedacid presentin the aqueous
phase.When 20 per cent glycerinis presentin the aqueousphasethe proportion of unionizedacid boundby 0.1 per cent Brij is reducedto about 3
per cent {unpublished
data). Thesevalueshave beenused,alongwith the
partition coefficientslisted in Table I, to calculate the concentrationsof
unbound, unionized acid shown in Table V. Although the total concentration of benzoicacid requiredto give equal fungistaticactivity ranges
from 0.062 to 0.23 per cent in the aqueousphase(0.032to 0.115 per cent
overall),the eqhivalent
concentrations
calculated
to be unbound
and
unionizedin the aqueousphasevary over the much smaller range from
0.027 to 0.03 per cent. Similarly the resultsin the presenceof 20 per cent
glycerin range from 0.037 to 0.042 per cent unionized,unboundbenzoic
acid and show reasonableagreementwith the 0.04 per cent found for a
simplebufferedsystem.
THE DISTRIBUTION
AND ACTIVITY
Table
OF BENZOIC ACID
213
V
Fungistatic activities (as FC 50) against A. niger of benzoic acid in emulsified systems:at
25ø and pH 4.2
,
FC 50 (per cent aqueousphase)
Phase ratio
Aqueous
phase
Oil phase
Concentration
w: o
Concentration
of benzoic
unboundand
acid added
unionized
in
aqueous phase
Buffer only
None
0.055
0.027
--
9.1 • Brij 35
None
Liquid paraffin
Liquid paraffin
2: 1
1: 1
0.056
0.062
0.065
0.027
0.030
0.029
Arachis oil
2:
1
0.136
0.027
Arachis
1:
1
0.230
0.028
0.147
0.027
0.072
0.040
0.042
0.041
0.036
0.037
oil
Isopropyl
myristate
3:1
__
20 • glycerin
20 •o glycerin
•.nd
•.1% Brij 35
None
None
Liquid paraffin
1: 1
Arachisoil
2: 1
0.076
0.080
0.145
isoPropyl
myristate
3: 1
0.152
Theseresultssuggestthat the fungistaticactivity of benzoicacid in an
emulsifiedsystemdependson the amountfree in the aqueousphase,and
that the amount required to compensatefor lossesinto oil phasescan be
calculatedif the appropriatepartitioncoefficient
is known. Contraryto the
resultsof Bean et al (4, 13, 14), the phasevolume ratio doesnot seemto
affect the activity, although it should be pointed out that a different
organism,a differentmethodand a differentpreservative
havebeenused
in the presentstudy; in addition the inclusionof surfactantmolecules
which concentrateat the oil-water interface would be expectedto affect
adsorption
of microorganisms
at the interface.
ACKNOWLEDGEMENT
This work has beensupportedby the New SouthWales Pharmacy
Research Trust.
(Received.'
3rd October
1966)
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in Pharmaceutical
Sciences,
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Press, London).
214
JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS
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(4)
(5)
(6)
(7)
Bean, H. S., Heman-Ackah, S. M. and Thomas, J. J. Soc.Cosmetic
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