Partition (distribution) I

Cotfficient of Berberine in n-Hexane,

Kerosene and Paraffin

ABSTRAct

Emul5\ion liquid membrane tech­

nology is the plloeel>s in which an extraction

process occures! by means of selective liquid

separating ph3ise; liquid membrane. The

selection of the; membrane (solvent) should

favor the distribution of solute from the

external phase. ,In this work, effect of three

organic soJven(s (n-hexene, kerosene ,md ,

paraffin) on berberine extraction is discussed.

The experimerltal result was shown that ,

kerosene was rh~ seleeted .solvent which was

suitable solven~ to berberine extraLtion on

ELM process.

Sirilml Chum;awang 1

Chirakarn llIuangnapoh" 2

Chada PhisaJaphong 3

Keyword: panilion (distribution)

coefficient, Emulsion Liquid Membrane

(ELM), Berberine

INTRODUCTION

The emulsion liquid membrane

(ELM) process was fIrst developed by N.Li

in 1968. Many studies have since been

carried out using ELMs for separation of

heavy metals, phenols, organic aeids (Draxler

and M;m, 1988; Zhang, Liu and Lu 1937;

Ma and Shi 1989), bioseparalion (Cussler

and Evans, 1980) and phanoacy (Tang, Ma

and Liu, 1990) ete.

Lecturer, Dtparrment of Chemical and Proces." Engineering, Faculty of Engineering

Rangsit University.

2 Lecturer, Department of Chemical Engineering, Faculty of Engineering, Chulalongkom ,

University. !

3 Research an~ Development Inslituti(m, The Governmenl Phannneentical Organization.

102 JrJ.Jn,'}:}:IRJ IS :1Ji7.

In emulsion liquid membrane

technology, the organic phase is referred to

as the membrane phase. The feed phnse and

[he stripping phase are as external and

internal phase respeelivity (see Figure 1).

The mass transfer of solute occurs 10 all

phases. The solme in the edemal phase

transfers across the external-membrane

interface, then diffuses through the mem­

brane phase. At the membrane-internal

interface with the internal phase, the solute

transfer into the small droplets or the

internal phasc. Any reaction al lhis interface

is usually assumed to be instantane{lus

because of the high specific interfacial area

of the small droplets (Lorbach and Marr,

1987). As mentioned above, the membrane

phase confers selectivity on emulsion liquid

membrao(' pro('esses and consequently the

mechanism of solute transport across this

phase is of primary importallCe.

MEMBRANE

PHASE ••

I

I •

EXTERNAL PHASE

Figure 1 Schematic Representation of an Emulsion Liqnid lUembrane

The function of the memhrane is

to allow se!e('tive transport of the solute from

the external stream ioto the stripping phase.

In order to make this process valid, it must

al.so prevent the physical contact between

the extcrnal phase and stripping phase. The

aliphmic diluents are generally preferred as

Ihe membrane solvenl (Likidis and Schugerl,

1987) beeau.'!: of their lower solubility in

water.

In this work, effect of organic

solvent on Berberine extraction is discussed.

The three org<:mic solvents were nsed, namely

n - Hexane, Kerosene and Paraffin. The k('d

phase W<1.s Berberine which is an alkaloid,

being used in the tre;)lmenl of gastrointestinal

disorder and used as antimicrobial action

which covers Ihe range of organisms from

fungi and prorozoa to bacteria (Bhakuni.

198,1). The feed phase came from two

,wtJ'U IHV - Wl}Flfimuu

, sources. The first was synLhetic Berberine

and the secon4 was Berberine derived from

herb:;.

The ~)bjeetive l)f this work were

to determine: the partition (distributiou)

coefficient in brgrJOic solvent as mentioned i ~

before. b\' usihg crude berberine le-'i.lracted"' i

from Khall1i~ Khrue::J.j. The obt(lin;Jbk

result was a]so eomparse with synrtletie

herberine sl)lll'tioll.

THEORY

The emubil)ll liquid membrane

(ELM) procfss is unique and different

fHlm Ihe Orilej' membrane process. In ELM,

membraue i, a liquid phase involving:

double emuHion configuration. This kind

of memhranq is somelirnes called liquid

surfactanr ~embranes or surfactant

liquid memb~ncs. There are Iwo types of

ELM, i.e. O/~/O systems an WIOIW systems.

In case ()f o/r10 systems, the w;Hcr phase

separating th4 organic phases is the liquid

membrane. Ftprw/olv. system:'., [he organic

phase is Ih~ liquid membrane that is

between two !\v;Uer phases.

ThciELM consists of three phases,

The mcmbra~e phm.c ~eparares the <'Ol':IP­

sulated imerntl droplets in the emulsion from

Ihe extern<lJ (continuous) ph;tsc as seen in

Figure 1. Fo~ unfacilitarioil. rhc membrane

ph.1se consist~ only a diluent (Ind :mrfactam

w stabilize i the primary emulsion. No

extractam is reeds for this type, bur mem­

brane pha::\~ ;ean be comidered to be an

extracting s~lvellt phase. The imporlanr

:JH.Jn~S:JFfJ7," ;JJ!7. / (/:1

part of EL~I system was membrane phase.

(ienerally, solntc run be more OT less

dissolved in different solvenL by panirion

into solveur. At equilibriulll thc ratio

beLween clmcentration of sohlte in different

phases at eonst:ml temperature can be

expressed as:

Kj) = C~ICI

where KIJ 1.'­ [he paniti'Jtl (distribUTion)

cocfficieu[

c! is the concentration of soluLe in

[he raffinatc phase

C~ is the concentration of f>olute in

the extxaction ph;tse

Berberine is Ihe on~ derivative of

a group of alkaloids wllieh occur in a wide

variety of bOlanical fal11illies. Berherine has

t\"'O fOffilS. namely salt ;lnd free base fnrm

as "hown in the hgme :'.The salts are mO:'.lly

yellow in colour and crystallized well: the

hydroch lpride dihydrate as small yellow

needle,: nitrate as green-yellow needles and

Ihe sulphate as slender yellow needles. The

phosphate se.~qnihydrate is a brigJll yellow

and al~() crystalline. Its main use in Western

medicine is as bittle Ionic and stomachie.

It h(ls some tryponocidal a aelion and had

been used as all adjunct to quinine in the

tre;tlment of malmia. The sulphate in

coni.:entraLion of l -") mg/ml decrease the

anticoagulant action ,.If hepmin in god and

human blood in vito (Glashy. John, S., 1975)

•• •

wlvff ·J·5 ,Js::vYD 2541

8) BERBERINE (FREE BASE)

b) BERBERINE CHLORIDE

CI.nH 02

c) BERBERINE HYDROCHLORIDE

Hgure 2 a) Chemical Structure ofBerberine (Free Base)

b) Chemical Structure ofBerberine Chloride

c) Chemical structure of Berberine Hydrochloride

MODEL SYSTEM

Tn the extraction equilibrium study,

the two .~otute were used a) synthetic

berberine solutio{l and b) eTUde berberine

solution from Khamin Khruea ('lJn'WU''I1fl) at

vrious pH hom pH 8 to pH 12. There are

three organic solvents; n-hexane, kerosens,

and paraffin which were used.

There are two fonns of berberine

as mentioned before and it can be changed

between two forms by acide/base reaction

as follows:

+ + + ~

R 4 N X + Na OH ---7 R J N OH + Na X

R 4 N OH +H CI ~ R~N Cl +H2 0

MATERIALS and METHODS

In the eJ<;traction equilibrium study,

there are three kinds of organic solvents; n­

hexane and parafrine (Mallickrodt Specialty

Chemical Co., Ltd.) were from Nirin

Supply Company. Kerosene was kindly

supplied by Petroleum Authority of

Th<liland. Synthetic berberine chloride

from Sigma Chemical Co., Ltd. and crude

berberine ex~acted from Khamin Khruea

which W:;lS plltchasedeorm Boun-Zae folk­

loric medicine! shop.

300 ;ml of 0.05 gil synthetie i

berberine solution was pouTed into a

l,OOO ml of beaker, To this beaker, 30n ml

of n-hexane ~'as also added to give 1

by volume h~ using a mechanical stirrer

(lKA., model Eluroatar Digi-vis), Ihe mixrure

solution in Ih~ beaker was agitated at 2dO

rpm at 2S0C for a peTiod of W: hours to

ensure equilih1\1uffi condition wa~ reached.

The pH of aqrous phase was adjusted lo

pH 8, 9, 10, 11 and 12 respectively with

concentrated scl:iium hydroxide solution. The

phases were aI]owed to settle under gravity.

The sample wlls collected with respect to

lime until 96 1II0urs. The aqneous solution

was then assaykd for berberinc concentra­

tions and the pH were then measured. The

cOl1cenlratiom,pf herberine in the organic

phase wcre determined by tile difference of

berberine conceptrations in the aqueous phase

between tile initial and final Mages. The

berberine concentrations .in the aqueous phase ,

JJ1JIlf>S.1)t.fflf> .1)11 10:)

were analyzed by using HPLC (Water™

600,717,996) on octyl column (l5 em).

The mobi(e solution was prepared by

mixing 70''/0 acetonitrile III O.l % H~ P0 4 which

was adjusted 10 pH 6 with concentrated

arrunonia solution. This mobile solution was

passed through the column at the flow rate

of 1 mUmtn. Berberine detection (,UV

detector) was achieved at 345 nm. The pH

of the aqueous phase solution was measured

by using pH meter (Schott, CG825),

The experiments '.'..eTe repeated at

Ihe same experimental condition,~ by usmg

kerosene <lnd paraffin, respectively.

The experiments were also repellted

at the same experimental wnditicms for crude

berherine solution at concentration of O.ll

gil (lOCO g Dry sm~lI pieces of Khamin

Khruea was soaked in distilled water for

72 hours. The solution was filtered and the

filtrate '.'ias diluted to 80JO ml with distilled

water to form a mother liquor for the

succeeding tests;.

The experlmcnl~ were can-ied out

under the conditIons as shown in Table l.

Table 1 The Experimental Conditions at 2SoC for Extraction Equilibrium

Organi Phase

,

Il-Hex~ne or

Keros¢ne or

Paraffin

Aqueous Phase

synthetic berberine or crude berberine

solution ladjustcd to pH 8, 9, 10, II

and 12;

rpm

2<30

Time

9t h

lor"'! )FfJn::;,<;:JJi1J7.'J V17.

RESULTS AND DISCUSSION

1. The El1'ect of Organic Solyents

on Extraclion Equilibrium of Synthetic

Berberine

The effect of organil: solve'nls on

extr<:letioll equilibrium of synthe'li;,,; berberine

was investigated by mea~uring the dist.ribution

(partition I of berberine solute oclv....een aqueous

solution and organic solvents.

Figure ;. shows the distribuTion

coetfieient of berberine between synthetic

berberine solution and II-hexane ar variou~

pH of al.Jueous rhase, where the X-direction

is time (h) and the '{-direction is distribu­

rion coefficient, Kf). IL shows lhat when the

basicity of aqueom phase ""a~ increased from

pH 8 to pH 12, the distribution coefficient

was aJsCl increased. But for each pH value,

the distributiCln coefficient increases rapidly

at the' iniLial time interval <lnd becomes

sIigluJy conSLaOI when the lime passed.

Figure LI descrihes the clistrihution

coefficient or berberine between synthetic

berberine solution <1nd kerosene <It varions

pH of aqueous phase, while Figure 5 depicLs

the disrribution coefficient of berberine'

between syntheli,-' berberine solution and

paraffin at various aqueous phase pH.

Apparently thc~e resuhs are' the silme a~; In

rigure 3. but at ditTercnt KJ)

Figure 6 shows the comparison of

distribution coefficient of berberine between

~ynlh<.::tic berberine solution and organic

solvents (n-heX<:lnc. kerosene <:lnd p~u'affin)

at pH 12 of aqueous phase. It was shown

that K 1, of bcrberine/n-hexane, herberine/

kero'iene and berb<.::rine/paratfin werc ar.out

0.152, O.l136 abd 0.0288 This means

that berl:oerine :-olute was more soluble m

n--hexane than kero"ene and paraffin.

From thc physical properties of

n-hex<:lne, kemsene and paraffin, it w".~ found

thilt n-he:...anc is ~'Jightly soluble in ""'<:ller

(comp<:lre TO kero:-enc '.llld paraffin). The

molecular weight of n-hex.me' is th.: lowest

while the molecular we'ight of paraffin is

the highest. n-he'xane is more volatile when

compared with kerosene and paraffin.

Although the distribution coefficient of

synthetic berberine/n-hexane was thl:'

highest. bUI n-hexalle was not suitable for

u:-ing as organic solvent on ELM due 10 its

SOlubility rroperty.

Compariiion between kerosene

and paraffin, the di.<.tribution eoeffieie'nr

of synthetic berberine/kcmsene was higher

than the distribulioo coefficient of syutbetie

berberine/paraffin. So kNoseIle W<:lS selecled

as organic solvent for ELM system. Tbe

p<:lr:1ffin was not chosen because of its high

viseosiry.

'~Lm7.F1JJ - ~iJwrfinwu JfTJIlS.S:u,wJ;) JJ17. 107

016

0.14

0.12

0.10

>2 o.oB

0.06

0.04

0.02

0.00

-~~~----- ~---~--~---~-------- -_.__._~._~

.. x

IiI •

GPH~ __• __ ~H9 & pH10 x p~~z pH12 I

o 20 40 60 Bo 100

Time (h)

i Figure 3 jDistribution Coeflident of Berherine in Syulhetie Berherine solution/

in-Hexane System at VOiriOW; pH of Synthetic Berherine Solution

0.1B ,

0.16 ! z

0.14

" '"

0.12

0.10

o.DB :&

z

• z

• z ,

, o.DB

0.04

0.02 •• • pHS • pH9 .. pHlO X pHll z PHli]

0.00

0 20 40 60 BD 100

Time (h)

Figure 4 Distribution Coefficient ofBerherine in Synthetk Berherine Solution/

Kerosene System at Various pH of Synthetic Berberine Solutiou

0.08 S ~HB • pH9 .. pH10 )( pHl1 S PH12­ll

0.06

I " Q 0.04 " '" ..

• I • •002 " ..

o ­

0 20 40 60 80 100

Time (hI

Figure 5 Distribution Coefficient of Berberine in S)'nthetic Berberine Solulion/

Paramn System at Various pH of S.J'nthetk Berberine Solution

I I, I

0.2

• 0.16211 0.15

• 0.1416

0.1 tlO • O.ll!':6 •I 0.1186>2 0.1 0.1056

.. 0.073 • 0.06;19

0.05 0.02118

•

o 20 40 60 60 100

I I

•

Time (h)

1_._~_H_'~._",_P_H_'_2 __._"'_,_,,_ene pHl~. .6. Pof8ffin PH~ /

Figure 6 Comparison to Distribution Coefficient of Synthetic Berberine at the

Different Organic Solrents (pH 12)

• • • •

0.14 ,. ~

0.12 ; x ~

x ~

0.10 ~

x 0.08

,f ~

•006 . •

0.04 ;

0.02 . I • pHS • pH9 .. pHl0 )( pH11 :c PH121

0.00,

0 20 40 60 60 100

Time (h)

Figure 7 ! Distribution Coef'fjcient of Crude Berberine/a-Hexane at Variuus pH

of Crude Berberine Solution

------~--0.14',

~o12 ­ )( l~OlD I I

I • 0.08

c

0.06 "" t0.04

c;; · pH9 )( pH11 I002 .. pH10 :I ·pH12

0.00

0 20 40 60 60 100

Time (h)

Figure B I JJislribution CoeJllcient oJ" Crude HerberinelKerosene at \/arious pH

of Crude Berberine Solution

• •

0

/ 10 JiDJ1SS;)J(iJ75 :JJn u:vvt! 35

0,15 •

I • pHS • pH9 • pHl0 )( pHl1 '" pHI2 n 0,10 !

I I

><

0,05

:0:

I •.. I 0,00

0 20 40 60 80 100

Time (h)

f1gure 9 Distribution Coemcient of Crude Berberine/Paramn at Variou.'5 pH of

L'rude Berberine Solution

Time (h)

~ ':=1 L_~_ n~Hexane pHl~ • kerosene,,,:,p_H_'_2__.--,p_,.,.{fi_n pH12 i

Figure 10 Comparison to Distribution Coefficient of Crudr Berberine at the

different Organic SolYents (pH 12)

2. Extra~tion Equilibrium of

Crude lBerberine Sotution ,

In this study, the aqueous phase of

extraction equilibrium ha~ been changed from

synthelic bqrberinc solution to crude

berberine solJ'ion (mother liquor of Khamin

Khuea). The ~xperimcntal methods have been

done in the ~arnc way in order to see the

effect of orgJic solvents Oil crude berberine_

The experirn~ntal conditions controlled at ;

25°(' were 0.11 g/ I of initial crude berberine

solution at pl-I 8 to pH 72 \vhich was the ,

aqueous pha~e and lhe organic phase wa~

changed int~ Ihree solvents; n-hexane. - !

kerosene and: paraffin. ,

Fig~re 7, 6 and 9 ~h()w the djstri-·

bution cocffidienl of berberine hetween crude

JIlJnSS:JJ,jJ-1S .Un. l' 1

berberine solution and n-hexane, crude

berberine solution and kerosene. and crude

berberine solution and paratfin at various

aqueous phase pH r(:spectively. The X­

L1irection if; time (h) and Y-direction is K Il ,

The near similar results of these experiments

arc obtained in section 1. The man the

basicity in aqueous phase was increased from

pH 8 to pH 12, distribution eoeffieien! was

also increased.

Figure j 0 shows [he comparison (If

distributil'ln coefficient of berberine bell,',een

crude b(:rberine ~'olution and organic

solvents (n-hexane, kerosene and pamfIin)

at pH l;: of aqueous pha.f;e. The X -direction

is time (h) and Y -direction is Kn It was

"hown that K l ) of berberjne;n-hcxane was

the highest l,','hile K[l of berberine/paraffin

was the lowest as follows:

Kpof bcrberirje/n-hexane (0.1 :345) > berberine/kerosene (0,1 04C') > berberine/pamffin (O.02L19)

Thq comparison of distribution !

coefficient pf berherine from synlhetie ,

berberine sq,lution and crude berherine

solution at Ivarious organic solvents at !

pH 12 went shown in Tabk 2. II: was

found that I'ID of berberine [rom synthetic

berberine solution was higher (han K[l of

crude berberine solution at various organic

:-olvents.

Owing to many derivatives in crude

alkaloidal extract from Khamin Khruea,

therefore all the derivalives and berberine

could partition into org::Hlic !>olvcnts.

T:Jble 2 C(lmparison or Ko between SJnthetic Berberine Solution to Crude Berberine

,Solution

rll~!i_~;:;nthetiC Berberine KD of Crude Berberine Orga ic Solvents

I' Solution Solution

n"'hexane 0.l520 <).l345

kerosene rJ.ll86 O.lOAO

paraffin 0,0288 Ci.2J70

112 JFfJnSS:JJi5T7.S :JJlI,

CONCLUSION

In this ~xperiment, the .suitable

organic solvent will be used as membrane

phase in ELM process. The membrane phase

should not dissolve in the two aqueous phase.

The KD value of berberine/n-hexane was

the highest and berberine/paraffin was

the lowest. The selected solvent whieh

was suitable solvent to ELM process, was

kerosene.Due to the solubility property of

n-hexane and high viscosity of paraffin.

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Liquid membranes for separatiom and

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Draxler. J. and R.J. Marr 1988.

Emulsion liquid membranes:partl . phenom­

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