Shoaf, W


Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
58 

 

 

                  

                                
                                 Al-khwarizmi 

           Engineering  
            Journal 

 

Al-Khwarizmi Engineering Journal, Vol.2, No.1,pp 85-97, (2006) 

 

 

 

 

 

 

Extraction of Chlorophyll from Alfalfa Plant 

 
     Isam Kamal                 Dr.Ahmed Jawad             Ali Khalid Khudair Al-Jomaily 

     Chem. Eng. Dept                Biochem. Eng. Dept                           Chem. Eng. Dept   

     University of Technology         KhwarizmyCollege of Eng           University of Technology 

    University of Baghdad 

 

 

 

 (Received 5 December 2005; accepted 4 April 2006) 

 

Abstract:- 

The extraction process of chlorophyll from dehydrated and pulverized alfalfa plant 

were studied by percolation method. Two  solvent systems were used for the 

extraction namely; Ethanol-water and Hexane-Toluene systems . The effect of 

circulation rate, solvent concentration, and solvent volume to solid weight ratio were 

studied. In  both ethanol water, and Hexane-Toluene systems it appears that solvent 

concentration is the most effective variable.  

 

Keywords: Chlorophyll, Extraction, Percolation, alfalfa 

 
1.Introduction 

Chlorophyll, so defined, is a mixture of 

green pigments, which is always 

combined by other pigments, 

principally those of the carotenoid 

group [1]. Alfalfa is usually used as a 

green plant source for the extraction of 

chlorophyll because of its availability 

along all seasons of the year and 

because of its low price. Chlorophyll is 

relatively labile and during isolation it 

is necessary to protect it from 

degradation. As a general precaution, it 

is advantageous to work in dim light 

and low temperature to avoid pigment 

loss[2].  

For extraction of high quality 

chlorophyll, there are many laboratory 

methods [3-10]. The most popular 

applied process is the extraction of 

chlorophyll by hexane or ethanol 

where both batch (percolation) and 

continuous processes (CSTR) were 

used [11,12]. 

No data appeared in literature for the 

evaluation of process parameters for 

the extraction of chlorophyll. Hence 

the purpose of this study is to 

investigate the process parameters for 

the extraction  of chlorophyll from 

dehydrated alfalfa plant using 

percolation method. These parameters 

are namely; circulation rate of solvent, 

type and concentration of solvent, and 

volume of solvent to solid weight ratio. 

Different solvent systems were used 

for the extraction, namely, ethanol-

water, and hexane-toluene blend.  

 

2.Experimental Work Material  

The alfalfa used for extraction was first 

dried in an oven at 45-500C for 1-2 

hours[11]. Then the dried alfalfa was 



Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
58 

 

 

finely ground in a ball mill and then 

screened in a  50-mesh screen[11]. The -

50 mesh size cut  of alfalfa was used in 

the extraction experiments. Ethanol 

(95%, near isotropic concentration 

obtained from atmospheric distillation 

) used  in ethanol-water system was 

supplied by General Company For 

Drinking and Food Industry. Hexane is 

supplied by Tagi gas plant while 

Toluene (99.9%) was supplied by 

Arabian Company for Detergents 

Chemicals. 

 

3. Description of Equipment 

Extraction experiments were carried 

out in QVF glass rig (Fig. 1) [13]. The 

extractor is a QVF cylindrical vessel of 

21 cm in diameter and  58 cm height. 

In the extractor a cloth pocket is 

introduced filled with alfalfa. Liquid is 

distributed on the alfalfa pocket  by a 

porous glass distributor.  Solvents were 

circulated from a 20 liter QVF 

spherical vessel. A centrifugal 

pump(Q=2160 l/h, Head = 3m)  were 

used for solvent extraction. A QVF 

Rota meter (0-400 l/h) was used to 

control the volumetric flow rate of 

solvent. The batch distillation system  

for the recovery of chlorophyll and 

solvents consists of QVF cylindrical 

vessel, QVF condenser with double 

coil of 8 cm in diameter and 50 cm 

high. A 148 kW electrical heater 

(Stabilag, England) was used in batch 

distillation.  A digital temperature 

indicator controller was used for 

controlling temperature in batch 

distillation within 0 – 400 0C.  A 

chiller (MGW LAUDA) was used for 

cooling of water necessary for the 

condensation of solvent vapors in the 

condenser. A ball mill (Siemens) was 

used for grinding dehydrated  alfalfa 

meal. 

4. Experimental Procedure 

For the extraction system, the cloth 

pocket is filled with 1 kg of pulverized 

dehydrated alfalfa meal and then 

placed in the extraction cylindrical 

vessel. The spherical circulating vessel 

is filled with a certain volume of 

solvent. Before starting the 

experiments the solvent is heated to   

350C and kept constant at this 

temperature throughout the 

experiments using a temperature 

controller in order to compare the 

different operation conditions at 

constant temperature . Then the 

circulating pump was switched on to 

circulate the solvent through the 

extractor and the spherical vessel. A 

needle valve was used for adjusting the 

volumetric flow rates of solvent at the 

desired value as indicated on the 

Rotameter. The circulation of solvent 

was continued for a maximum duration 

of 3 hours. Every fifteen minutes, a 

sample was taken for the solvent by a 

pipette. The concentration of 

chlorophyll in the sample was 

measured by atomic absorption 

spectrophotometer at the university of 

technology by measuring the ratio of 

absorbencies at 654 nm (A645) and    

663 nm (A663).              The 

concentration can then be calculated  

using the following equation[ 17 ]:   

Total Chlorophyll (mg/L) =  20.2 A645 

+ 8.02 A663    ------------------ (1) 

When the  concentration of chlorophyll 

remains constant, the extraction 

experiment was stopped. 

Batch distillation is used for the 

concentration of the product and for 

the recovery of solvent. The 

temperature of distillation of solvents 

must not affect the color of the solute 

(chlorophyll).  This temperature was 

900C for ethanol-water system and 80 

0C  for hexane-toluene system. This 

process for the separation of 

concentrated chlorophyll and recovery 

of the solvent were proceeded for 5-6 

hours. 

 5. Results & Conclusion 
The effect of operating variables on the 

extraction rate of chlorophyll for 



Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
58 

 

 

ethanol-water and hexane-toluene 

systems from alfalfa were  studied in 

this work. 

The percentage of extracted 

chlorophyll was calculated using the 

following relationship : 

                                             Amount of 

chlorophyll dissolved 

% extracted chlorophyll =   -------------

---------------------------------------- 

                                                     Total 

amount of chlorophyll in        

                                                           

Dehydrated alfalfa meal   

                                      

6. The Effect of Circulated Rates of 

Solvent  

 both system  (Figs. 2 and 3 ), the 

percentage of  extracted chlorophyll 

increased with increasing circulation 

rates of solvent. The two main steps of 

mass transfer in the extraction process 

are diffusion of the fluid through solid 

and diffusion through film surrounding 

solid particles. Since solid particles are 

pulverized,   the resistance to mass 

transfer  by  molecular diffusion of the 

solute from solid internal pores to the 

film surrounding  solid particles will be 

smaller   than the solute mass transfer  

across the film surrounding the 

particles to the solvent bulk. Hence, the 

expected  rate controlling step is the 

diffusion of solute through film 

surrounding solid particles to the 

solvent bulk.  

It is well known that extraction 

efficiency increased with increasing  

velocity of solvent. This phenomena is 

applicable for laminar flow (131 < Re 

<431) , since in turbulent  flow 

increasing of velocity of solvent have 

little effect on extraction rate [ 14 ]. In 

our case, the flow inside extractor is 

streamline (laminar), so the effect of 

velocity is noticed. 

7. The Effect of Solvent 

Concentration 

In ethanol-water system (Fig. 4  ),  it 

can be noticed that the percentage of 

extracted chlorophyll increased as 

concentration of ethanol increased. 

This behavior can be explained as 

follows; higher concentration of 

ethanol means lower viscosity of 

solvent. This will lead to higher 

wetting of solids  and hence increase 

extraction rates[16]. It also appeared in 

Fig. 3 that lower concentration of 

ethanol (50%) indicates lower 

extraction efficiency of chlorophyll. 

Hence, the minimum concentration 

used for the extraction of chlorophyll 

must be not less than 70%. This result 

is in agreement with the conclusion of   

Othmer [15]. 

      The effect of blending of hexane 

and toluene on the percentage of 

chlorophyll extract is shown in Fig. 5. 

The percentage of extracted 

chlorophyll when using a pure hexane 

did not exceed 77%. At toluene 

concentration of less or equal to 21% 

by volume,  the extraction efficiency is 

increased, while the extraction 

efficiency is decreased at toluene 

concentration greater than 21% [13].  

This behavior can be explained as 

follows[9] ; the phytol group 

(C20H39OH) is a long-chain alcohol 

separated from the structure of 

chlorophyll and when the extraction 

process started, the phytol group is 

freely mobile in the liquid phase. This 

layer of phytol is impeded the non-

polar solvent (hexane) to penetrate 

through the chlorophyll [9]. But  when a 

certain volume of toluene (21%) is 

blended with hexane,   a reaction 

occurs between toluene and phytol 

group which produced aromatic 

alcohol (benzyl alcohol). This aromatic 

is considered a more stronger solvent 

than pure hexane. On increasing the 

percentage of toluene above 21%, the 

physical properties of toluene as a 

solvent will be the controlling step, 

hence the extraction efficiency 

dropped. 



Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
55 

 

 

8. The effect of solvent volume to 

solid weight ratio 

As shown in Figs. 6,7, the percentage 

extracted chlorophyll increased with 

increasing volume of solvent for both 

system. This behavior could be 

explained as follows; as the volume of 

the solvent increased, the concentration 

difference (which is one of the most 

effective driving forces for mass 

transfer) will be increased and hence 

the extraction efficiency will be 

increased. But on increasing volume of 

solvent with respect to solid weight, 

the cost of solvent recovery will be 

increased with respect to volume of the 

distillation unit and the power applied.  

 

9. Conclusion 

1- The extraction rate of 
chlorophyll increased when; 

increasing the circulation 

rate of solvent in the two 

systems, increasing the 

concentration of ethanol, 

blending less than 21% of 

toluene in toluene-hexane 

system, and increasing 

solvent volume to solid 

weight ratio. 

2- For ethanol water system, 
The effect of ethanol 

concentration on percentage 

of extracted chlorophyll was 

found to be most effective 

compared to circulation rate 

of solvent and volume of 

solvent to weight ratio. 

While the minimum 

concentration of ethanol used 

in the extraction must not be 

less than 70%. 

3- In hexane-toluene system, 
the effect of hexane toluene 

blend is the most effective 

parameter on percentage of 

extracted chlorophyll 

compared to the effect of  

circulation rates and solvent 

volume to solid weight ratio. 

While the optimum 

concentration of blend was 

found to represent 21% 

toluene and 79% hexane.    

 

10. References 

1- Dobrota, G.L., Katz, J.J., 
Thomas, M.R., "Separation of 

chlorophyll pigment from 

plants", Agr. Food Chem.  8, 4,        

3 (1987). 

2- McFeeters, P.S., and Kempster, 
H.L., "The chlorophyll", 

J.Plant Physiol. 17, 3, 600-

618(1971). 

3- Schertz, F.M., "Isolation of 
chlorophyll, carotene and 

xanthophylls by improved 

methods", Ind. Eng. Chem. 30, 

2, 1073-1075(1938). 

4-  Iriyama, K., Ogura, N., 
Takamiya, A., " A Simple 

method for extraction and 

partial purification of 

chlorophyll from plant 

material, using dioxane", 

J.Biochem. 76, No.4, 901-

904(1974).  

5- Shoaf, W.T. and B.W. Lium, " 
Improved extraction of 

chlorophyll a and b from algae 

using dimethyl sulfoxide", 

Limnol. Oceanogr. 21, 926- 

928 (1976).  

6- Hiscox, J.D. and G.F. 
Israaelstan. "A method for the  

extraction of chlorophyll from 

leaf tissue without maceration". 

Can. J. Bot. 57, 1332—1334 

(1979).  

7-  Johnson, A. B., "Chlorophyll 
extraction  techniques",  In: 

"Methods in  Plant 

Physiology", Editors: E. Will 

and F. Bill, Cambridge 

University  Press, Cambridge, 

25-30(1989).  

8- Agar, I.T., S. Cetiner, J.M. 
Garcia and J. Streif, "A method 

of  chlorophyll extraction  from 



Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
58 

 

 

fruit peduncles: Application to 

red currants",  Tr. J. of 

Agricultural and Forestry 18, 

209-212 (1994).  

9- Eigenberg, K.E., Croasmun, 
W.R., "Extraction of 

chlorophyll from chlorophyll-

proteins", J.Biochim. Biophys. 

Acta, 642, 3, 438(1981).    

10- Wood, L. W. ,  "Chloroform-
methanol Extraction of 

chlorophyll a",  Can. J. Fish. 

Aquat. Sci. 42,  38-43(1985).     

11- Judah, M.A., Burdick, E.M., 
and Robert, G.C., 

"Chlorophyll, by solvent 

extraction",  Ind& Eng. Chem. 

46, 11, 2262-2271(1954).  

12- Shearon, W., Gee, D.F., 
"Caroten and chlorophyll 

commercial chromatographic 

production",  Ind. & Eng. 

Chem. 41, 2,    218-226(1949). 

13-  Al-Jomaily, K.K., 
"Optimization of chlorophyll 

extraction from alfalfa meal", 

M.Sc. Thesis, University of 

Technology, Baghdad (2000).  

14-  Sungtan, C., Kweiliang, S., 
and Chungliou, "Liquid-solid 

mass transfer in extractor", 

Chem. Eng. 38, Dec., 17-

22(1988).  

15- Davies, J.T., and Rideal, E.K., 
"Interfacial phenomena", 

Academic Press, Inc., 2nd 

edition  (1961).  

16- Othmer, K., "Encyclopedia of 
chemical technology", 2nd 

edition, John Wiely and Sons, 

Inc., Vol.5, 339(1964).  

17- Harborne, J.B., "Photochemical 
Methods", Chapman and Hall, 

London, 1973. 

 

 

 

 



Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
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Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
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Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
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Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
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o
f 

 S
o

lv
e
n

t 
C

o
n

c
e
n

tr
a
ti

o
n

 o
n

  
C

h
lo

ro
p

h
y
ll

 E
x
tr

a
c
ti

o
n

 w
it

h
 H

e
x
a
n

e
-

T
o

lu
e
n

e
 B

le
n

d
 a

t 
A

 C
ir

c
u

la
ti

o
n

 R
a
te

 o
f 

0
.2

4
 c

u
.m

/h
.

0

1
0

2
0

3
0

4
0

5
0

6
0

7
0

8
0

9
0

1
0
0

0
2
0
0
0

4
0
0
0

6
0
0
0

8
0
0
0

1
0
0
0
0

1
2
0
0
0

1
4
0
0
0

E
x
tr

a
c
ti

o
in

 T
im

e
, 

s
e
c

Chlorophyll Extracted (wt%)

s
o
lv

e
n
t 

c
o
n
c
e
n
tr

a
ti
o
n
 =

  
1
0
0
%

 T
o
lu

e
n
e

s
o
lv

e
n
t 

c
o
n
c
e
n
tr

a
ti
o
n
 =

  
1
0
0
%

 H
e
x
a
n
e

s
o
lv

e
n
t 

c
o
n
c
e
n
tr

a
ti
o
n
 =

  
7
9
%

 H
e
x
a
n
e



Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
88 

 

 

 

 

 

 

F
ig

u
re

 (
7
) 

: 
E

ff
e
c
t 

o
f 

V
o

l.
 o

f 
S

o
lv

e
n

t/
W

t.
 o

f 
S

o
li

d
 R

a
ti

o
 o

n
 C

h
lo

ro
p

h
y
ll

 E
x
tr

a
c
ti

o
n

 

w
it

h
 H

e
x
a
n

e
-T

o
lu

e
n

e
 B

le
n

d
 (

7
9
%

 H
e
x
a
n

e
) 

a
t 

A
 C

ir
c
u

la
ti

o
n

 R
a
te

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f 

0
.2

4
 c

u
.m

/h
.

0

2
0

4
0

6
0

8
0

1
0
0

1
2
0

0
2
0
0
0

4
0
0
0

6
0
0
0

8
0
0
0

1
0
0
0
0

1
2
0
0
0

1
4
0
0
0

E
x
tr

a
c
ti

o
in

 T
im

e
, 

s
e
c

Chlorophyll Extracted (wt%)

v
o
l.
s
o
lv

e
n
t/

w
t.

s
o
li
d
 =

  
(8

/1
)

v
o
l.
s
o
lv

e
n
t/

w
t.

s
o
li
d
 =

  
(1

1
/1

)

v
o
l.
s
o
lv

e
n
t/

w
t.

s
o
li
d
 =

  
(1

4
/1

)



Dr.Ahmed Jawad /Al-khwarizmi Engineering Journal, Vol.2, No.1,PP 85-97  (2006) 
 

 
88 

 

 

 

وروفيل من نبات الجتأستخالص الكل  

 

 

 د.عصام كامل                      د.احمد جواد علي                   خالد خضير
قسم الهندسة الكيمياوية           قسم الهندسة الكيمياوية األحيائية                 قسم الهندسة الكيمياوية  

الجامعة التكنلوجية                    يكلية هندسة الخوارزم               الجامعة التكنلوجية  
 جامعة بغداد

  
 :الخالصة

تم في هذا البحث دراسة عملية أستخالص الكلوروفيل من الجت المجفف والمطحون بطريقة التغلغل. تم أستخدام نظامين منن 
تولوين . تم دراسة تنثثير مدندل التندويرر تركينم المنذي  -ماء  ومنظومة هكسان-المذيبات لألستخالص  وهي منظومة أيثانول
المستخدمة . في كال المنظومتين  تم التوصل الى أن تركينم المنذي  هنو الدنصنر  و نسبة حجم المذي  الى ومن المادة الصلبة

 األكثر تثثيرا.