A Simple Method for Isolation of Citral Using Column

Chromatography

Adlina Savira,

Achmad Syahrani,

Marcellino Rudyanto

1,2

Department of Pharmaceutical Chemistry,

Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia.

Institute of Tropical Disease, Airlangga University, Surabaya, Indonesia.

Keywords Citral, Cymbopogon citratus, chromatograhy, separation.

Background Citral is the main component of lemongrass (Cymbopogon citratus) oil. This compound has biological

activities such as antibacterial, antifungi, analgesic, and antiinflammation. Citral also has importance for its

use as starting material for the synthesis of Vitamin A. Due to the broad utilisation of citral, it is important

to develop method of isolation which is relatively simple, low cost, but able to give pure citral in high yield.

Materials and method. Citral was isolated from commercially available lemongrass oil by simple column

chromatography using silica gel as stationary phase. Elution was carried out in isoctaric mode. Mobile phase

was chosen among hexane – diethyl ether, hexane – ethyl acetate, and hexane – ethanol based on separation

factor and Rf value on thin layer chromatography. Optimum ratio of sample and stationary phase was also

optimized based on isolation yield. Isolated citral was analyzed by gas chromatography – mass

spectroscopy. Results. The best separation factor on TLC was obtained from hexane – ethyl acetate (97:3)

as eluent. The best yield (49,61 ± 2,59 %) was obtained when stationary phase was used at ratio 20:1 to

sample.

1 INTRODUCTION

Lemongrass (Cymbopogon citratus) is a fast-

growing aromatic grass, growing to about 1 meter (3

feet) high with long, thin leaves.

It is native to Sri

Lanka and South India and is now widely cultivated

in the tropical areas of America and Asia, including

Indonesia.

Lemongrass is also one of the main

essential oil producing plants in Indonesia.

Indonesia, it is commonly known as sereh dapur and

its stem is used as a spice because of its distinctive

lemon-like aroma. This lemony odor is due to its

high content of the aldehyde citral, which ranges

from 65% to 85%.

Citral is a component of essential oils that can be

found in a variety of plants of the genus Citrus. It is

volatile, has a lemon-like odor and a form of light

yellowish oil.

Citral is a mixture of two compounds,

namely geranial (Citral-a or trans-citral) and neral

(Citral-b or cis-citral).

Besides in plants of the

genus Citrus, citral is also contained in essential oils

of lemon myrtle (90-98%), Litsea citrate (90%),

Litsea cubeba (70-85%), and Cymbopogon citratus

(65-85%).

This compound has various benefits,

such as antibacterial, antifungal, antiprotozoal,

ascaricidal, analgesic, antiinflammatory, and

antioxidant effects as well as hypoglycemic,

hypolipidemic and hypochesterolamic effects.

Citral is also an important starting compound for the

synthesis of vitamin A (retinol).

Due to the broad

utilisation of citral, it is important to develop method

of its isolation which is relatively simple, low cost,

but able to give pure citral in high yield.

One of the oldest isolation methods of citral is by

the reaction of adducting with sodium bisulfite.

Unfortunately, it is not the purest method because

sodium bisulfite can adduct other aldehydes and

methyl ketones available in lemongrass oil.

Therefore, other compounds which are also present

in lemongrass oil such as aldehyde compounds (e.g.

citranelal), or methyl ketone compounds (e.g. methyl

heptenone), can also be adducted together with

citral.

Steam distillation as well as partial fraction

distillation methods have also been used. In these

methods, isolation occurs successfully and produces

high-purity citral.

1,5

However, these methods have

their shortcomings. Separation of components such

as geraniol and nerol from citral is difficult because

they have a boiling point which differ only a few

Savira, A., Syahrani, A. and Rudyanto, M.

A Simple Method for Isolation of Citral Using Column Chromatography.

DOI: 10.5220/0009841500002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

degrees Celsius from citral. Furthermore, citral is

labile to high temperature, hence overheating can

lead to rearrangement, polymerization, and even

destruction of the citral.

Other methods already used include preparative

thin layer chromatography and column

chromatography.

5,8

Preparative thin layer

chromatography is only able to produce isolates in

very small amounts.

4,8

On the contrary, column

chromatography can be used on a large scale.

Column chromatography is very important in

industrial use because its methods can easily be

adopted from the laboratory scale to the production

scale.

Therefore, in this study, column chromatography

method was selected to isolate citral from

lemongrass oil. For the optimization of mobile phase

to be used for the isolation method, different

mixtures of mobile phase used in other experiments

were compared, such as mixtures of hexane-ether

based on Pushpakumari and Vatakencherry (1986),

mixtures of hexane-ethyl acetate based on Scott et

al. (1989) , and mixtures of hexane-ethanol based on

Purnamasari et al. (2016) of different ratios. The

goal was to determine which mobile phase mixture

at which ratio can produce the best resolution to

isolate citral. In addition, silica gel as stationary

phase and different sample-to-silica ratios were

compared to determine which ratio is the most

efficient for sample-loading based on the isolate’s

yield percentage obtained from each ratio. The

isolation results were further tested qualitatively and

quantitatively using gas chromatography -mass

spectrometry (GC-MS) and compared with

commercial citral as a standard.

2 MATERIALS AND METHODS

2.1 Materials

Lemongrass (C. citratus) oil was obtained from CV

M & H Farm. Commercial citral was obtained from

Aldrich. Hexane p.a., ethanol p.a., ethyl acetate p.a.

as well as silica gel 60 (0.063-0,200 mm) were

obtained from Merck. Ether p.a. were obtained from

Riedel-de Haen AG. The anisaldehyde reagents were

obtained from Merck. Iodine used to stain the spot

on TLC plate was obtained from Kimia Farma.

Filter paper, thin layer chromatography (TLC)

chamber, TLC silica gel 60 F254 were obtained

from Merck. To apply the lemongrass oil and the

fraction onto the TLC plate for the determination of

mobile phase mixture to be used for column

chromatography, 2 μl capillary pipes were used.

Chromatography was then performed in a 2,5 x 50

cm column and the fractionation results were

collected in 10 ml vials. The products were

subjected to gas chromatography (GC). GC was

performed on an Agilent Model 6890N, equiped

with mass spectrometer (MS) Agilent 5973 with

inert mass spectrum detector (MSD) and Head Space

Sampler Model 7697A HSS

2.2 Methods

2.2.1 Determination of Mobile Phase

Mixture for Column Chromatography

The solvent mixtures to be compared were hexane -

ether (97:3, v/v), hexane - ethyl acetate (97:3, v/v),

and hexane - ethanol (97:3, 98:2, 99:1, v/v).

Both the essential oil of lemongrass and the

commercial citral were applied on to the TLC plate.

The plate was then inserted into the saturated

chamber to be eluated with 5 ml of each solvent

mixture. Spots from the eluation process were

observed under UV lamps of λ 254 nm and also

stained by spraying anisaldehyde reagents or by

putting the TLC plate into chamber containing

iodine.

The best eluent for column chromatography was

chosen by taking Rf value and the separation factor

(α) of the standard spot (Rf 1) against the nearest

spot (Rf 2) into account. The separation factor (α) is

calculated by the formula:

  

1

2



12

11

2.2.2 Preparation of the Column for Column

Chromatography

The amount of silica gel needed for fractionation

depends on the results of the solvent mixture

optimization. The sample- to - silica gel ratios were

selected according to the guideline from Reichsstein

et al., (1960), as follows: 1:20, 1:35 and 1:50 if the

obtained separation factor (α) ≥1,5 ; 1:65 and 1:80 if

the obtained α ≤1,5.

2.2.3 Fractionation of Citral from

Lemongrass Oil Using Column

Chromatography and the

Determination of the Optimum

Sample-to-Adsorbent Ratio

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

Selected eluent was prepared for column

chromatography and 1 gram of lemongrass oil was

weighed before the fractionation. The flow rate was

set to approximately 1 drop per second. The droplets

of the eluent from the column were collected in

vials, each would contain 10 mL of droplets.

The TLC test was performed on every fifth vial.

The TLC test used the mobile phase of the column

chromatography. Spots from the elution were

observed under UV light 254 nm and stained by

anisaldehyde reagent or iodine vapor.

Fractions having the same Rf as the standard

were compared. This citral fraction was evaporated

in the rotary evaporator, until a constant weight was

obtained. Then the yield percentage of citral

obtained from fractionation was calculated with the

formula:

%  





100

The optimal sample-to-adsorbent ratio for

column chromatography was determined based on

the yield percentage of the obtained citral.

2.2.4 Identification of Obtained Citral Using

Gas Chromatography-Mass

Spectrometry

Citral obtained from fractionation with column

chromatography was injected into GC-MS. The gas

chromatography was equipped with a 30 m 0.25

mm, 0.25 mm film thickness column. Helium was

used as mobile phase with an average flow of 1.0

ml/min. The condition of the GC-MS was according

to the study from Bayala et al. (2018): Oven

temperature program was from 50° C (3.2 min) to

300° C at 8° C/min, 5 min post run at 300° C.

Sample was injected in split mode, injector and

detector temperature being at 250° C and 280° C

respectively. The peaks generated in the total ion

chromatogram are identified by comparing the mass

spectra obtained with the mass spectra found in the

GC-MS libraries.

3 RESULTS

3.1 The Optimum Mobile Phase

Mixture for Column

Chromatography

The data obtained from the TLC test with various

mixture of mobile phase (as described beforehand)

are listed in Table 1 below.

The best eluent for column chromatography was

chosen by taking Rf value and the separation factor

(α) of the spots on the TLC plate into account.

The optimal Rf value is 0.15-0.30.

The selected

solvent mixture is therefore hexane-ethyl acetate

(97: 3, v/v), due to its separation factor of ≥ 2.00

meaning that the separation between two compound

occured easily and its Rf value of 0.24 which lied

within the accepted range.

Table 1: Rf Values of lemongrass oil and comercial citral on TLC using various mobile phases

Mobile phase mixture and

its ratio (v/v)

Rf value of essential oil

Rf value of citral

standard

Hexane - ether (97:3)

0,06

0,18

0,06

0,18

3,44

Hexane - ethyl acetate (97:3)

0,03

0,24

0,39

0,03

0,24

2,02

Hexane - ethanol (97:3)

0,09

0,79

0,88

0,09

0,79

1,94

Hexane - ethanol (98:2)

0,05

0,49

0,68

0,05

0,49

2,21

Hexane - ethanol (99:1)

0,17

3.2 Fractionation of Citral from

Lemongrass Oil Using Column

A Simple Method for Isolation of Citral Using Column Chromatography

Chromatography and the

Determination of the Optimal

Sample-to-Adsorbent Ratio

After fractionation of citral from the lemongrass oil

as described before, the results obtained are listed in

Table 3.

Table 2. Fractionation results of citral from lemongrass oil

Sample-to-

Adsorbent

Ratio

Volume of

the eluent

(ml)

Volume of the

collected fraction

(ml)

1:20

400

200,00 ± 10,00

1:35

600

286,67 ± 5,77

1:50

700

326,67 ± 20,82

3.3 Organoleptical Analysis of the

Obtained Citral

The results from the organoleptical analysis were

compared with the data obtained from literature. The

organoleptical characteristics of the citral obtained

from the isolation are as follow:

Table 3. The organoleptics of the citral obtained from

fractionation compared with data from literature.

Data from literature

(Marques et al., 2013)

Obtained citral

Color

Pale yellow

Bright yellow

Form

Thick oil

Thick, oily

Odor

Lemony odor

Lemony/orange-

like odor

Figure 1: Pure citral obtained from the fractionation

3.4 Analysis of Weight and the Yield of

the Obtained Citral.

After the isolation of citral with different sample-to-

adsorbent ratios, the weight of pure citral and the

percentage of citral yield have been obtained as

indicated in Table 5. It was obtained from three

replications that the sample-to-adsorbent ratio of

1:20 can produce the highest yield percentage when

compared to the other ratios.

Table 4. Weight and the yield percentage of the obtained

citral

Weight and the Yield

Percentage of the Obtained

Citral

Sample – to

Adsorbent

ratio

1:20

1:35

1:50

Mean yield

percentage

49,61 ±

2,59 %

45,66 ±

2,84 %

38,30 ±

1,72 %

3.5 Identification of Obtained Citral

Using Gas Chromatography-Mass

Spectrometry GC-MS

Further qualitative and quantitative identification of

the obtained citral from was done using gas-

spectrometry mass chromatography (GC-MS) to

determine the purity of the isolate. The results of the

normalization percentage of neral (1) was 11,50%

and geranial (2) compound was 77,95%. Thus, the

obtained citral from the fractionation using 1:20

(w/w) sample – to - adsorbent ratio had the purity of

89.45%.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

DISCUSSION

In this research, the column chromatography method

of citral isolation from lemongrass oil (Cymbopogon

citratus) was optimized. The goal was to determine

which mobile phase mixture at which ratio can

produce the best resolution to isolate citral and

which sample-to-adsorbent ratio was the most

efficient for sample-loading. Mobile phase affects

the separation factor, while sample-to-adsorbent

ratio affects the effective theoretical plate number

of the chromatographic system.

Both separation

factor and the number of effective theoretical plates

are important parameters affecting the resolution of

a chromatographic system.

10,11

At the beginning of

the study, eluent mixture was selected using thin

layer chromatography (TLC) based on Rf value and

the separation factor (α) of the spots. The eluent

mixtures tested were hexane ether (95: 5, v/v),

hexane - ethyl acetate (97: 3, v/v), hexane - ethanol

(97: 3; 98: 2; and 99: 1, v/v). The optimal Rf value is

0.15-0.30, while the price of the optimal separation

factor is ≥ 1.5, meaning the separation between

compounds in essential oils was relatively easy.

Hence, a hexane-ethyl acetate solvent was selected

(97: 3, v/v) since the separation factor ≥ 2.00

classified as “easy separation” and the Rf value

within the range was 0.24.

After determining the

solvent mixture used as the mobile phase for

fractionation, fractionation process was performed

by column chromatography. In this study, an

isocratic chromatography method was used. The

chosen stationary phase was silica gel. To separate

polar compounds such as aldehydes (e.g., citral), a

polar stationary phase such as silica gel was required

since the surface comprises a highly polar hydroxyl

group and interacts with the dipole of a polar

solute,

hence silica gel was selected. At this stage,

the three sample-to-adsorbent ratios were compared

based on the yield percentage of the isolated citral.

The three sample-adsorbent ratios were 1:20, 1:35,

and 1:50 (w/w), respectively, chosen based on

guidelines from Reichstein et al. (1960).

With

each ratio, fractionation was performed with three

replication. The fractionation results were

evaporated until the weight stayed constant and the

yield percentage was calculated.

The results of the isolates obtained were yellow

liquid compounds smelling like lemon / orange. The

results of this organoleptic observation were

consistent with the data from the literature, which

states that the citral is a pale yellow liquid oil-like

compound smelling like lemon

4,8

A Simple Method for Isolation of Citral Using Column Chromatography

After three replication, the largest yield

percentage of 49,61 ± 2,59 %. was obtained by the

1:20 (w/w) sample – to - adsorbent ratio. This may

occur because the larger sample-to-adsorbent ratio,

more citral was retained on the surface of the silica

gel. The more the amount of silica, the greater the

surface area of the stationary phase and the greater

number of analyte interacting with the stationary

phase.

Therefore, it can be assumed that more

citral interacts with the polar hydroxyl group of

silica gel so that in larger amounts of silica the more

citral is left in the stationary phase. The variation in

the yield percentage of citral in one replication

compared to another could be caused by the drying

process, where the citral coalesced along with the

solvent.

Further qualitative and quantitative identification

of the obtained citral was done using gas-

spectrometry mass chromatography (GC-MS) to

determine the purity of the isolates. In

chromatogram the peak of neral can be seen at

retention time of 10.49 min and peak of geranial at

retention time of 11.45 min. Citral is a mixture of

cis-citral compounds (also called neral) and trans-

citral compounds (also called geranial).

The

difference in retention time of both compounds

could be caused by the interaction of the compound

with the stationary phase in the gas chromatographic

system. The column used was nonpolar, therefore

the polar compound came out first while the more

nonpolar compounds would be retained longer in the

column. It can be concluded that trans-citral

compounds (neral) are more polar than cis-citral

(geranial) compounds.

The normalization

percentage of neral was 11,50% and from geranial

77,95%. Thus, if added, the isolate obtained by a

1:20 (w/w) sample – to - adsorbent ratio had the

purity of 89.45%.

All in all, the method of fractionation using

column chromatography optimized in this study has

provided pure isolate with high yield, despite using

an isocratic elution. Hence, the optimized method

has the advantages of an isocratic elution such as the

relatively fewer solvents needed if compared to

gradient elution. Another advantage of the optimized

method would be its suitability for preparative use in

large amounts. Yet further studies need to be done in

order to adapt this optimized method to larger scale.

CONCLUSION

The optimal mobile phase mixture to isolate the

citral from lemongrass oil (Cymbopogon citratus)

using column chromatography method is hexane-

ethyl acetate with a ratio of 97: 3 (v/v), and the

optimal sample-to-adsorbent ratio to isolate citral

from the lemongrass oil (Cymbopogon citratus)

using column chromatography method is 1:20

(w/w).

REFERENCES

Bayala, B., Bassole, I.H., Maqdasy, S., Baron, S.,

Simpore, J. and Lobaccaro, J.M.A., 2018.

Cymbopogon citratus and Cymbopogon giganteus

essential oils have cytotoxic effects on tumor cell

cultures. Identification of citral as a new putative anti-

proliferative molecule. Biochimie. p. 1-9

Bidlingmeyer, B. A. (ed.)., 1989. Preparative liquid

chromatography. Journal of Chromatography Library

38. Amsterdam: Elsevier

Carbajal D., Casaco A., Arruzazabala L., Gonzalez, Tolon

Z., 1989. Pharmacological study of Cymbopogon

citratus leaves. JEthnopharmacol 25(1), p. 103-107.

Ella, M.U.E., Sumiartha, K.S., Suniti, N.W., Sudiarta, I.P.

and Antara, N.S., 2013. Uji Efektivitas Konsentrasi

Minyak Atsiri Sereh Dapur (Cymbopogon citratus

(DC.) Stapf) terhadap Pertumbuhan Jamur Aspergillus

Sp. Secara in vitro. E-Jurnal Agroekoteknologi

Tropika (Journal of Tropical Agroecotechnology),

2(1), p. 40-48.

Joga Rao, H., Kalyani, G., King, P., 2015. Isolation of

Citral from Lemongrass Oil Using Steam Distillation:

Statistical Optimization by Response Surface

Methodology. Int. J. Chem. Sci.: 13(3), p. 1305-1314

Marques, A.M. and Kaplan, M.A.C., 2013. Preparative

isolation and characterization of monoterpene isomers

present in the citral-rich essential oil of Pectis

brevipedunculata. Journal of Essential Oil Research,

25(3), p.210-215.

Oxtoby, D.W., Gillis, H.P., Nachtrieb, N.H., 2008.

Principle of Modern Chemistry. Sixth Edition.

Belmont: Thomson Brooks/Cole.

Purnamasari, P., Nashrianto, H. and Rusman, M.S., 2016.

Isolasi dan Identifikasi Senyawa Citral dalam Sereh

Dapur (Cymbopogon citratus) Menggunakan

Kromatografi Lapis Tipis Preparatif (KLTP) Dan

GC-MS. Skripsi, Fakultas Matematika dan Ilmu

Pengetahuan Alam Universitas Pakuan, Bogor, p. 1-9

Purwanto, D. A., Rudyanto, M., Annuryanti, F. 2016.

Produksi Vitamin A untuk Fortifikasi Minyak

Goreng Sawit dengan Bahan Baku Minyak Sereh

Dapur (Cymbopogon citratus) Mengacu

Permenperin No.87/M-IND/PER/12/2013. Laporan

Akhir Penelitian Prioritas Nasional Masterplan

Percepatan dan Perluasan Pembangunan Ekonomi

Indonesia 2011-2025 (PENPRINAS MP3EI 2011-

2025), Surabaya: Universitas Airlangga.

Pushpakumari, K.N. and Vatakencherry, P.A., 1985. A

new method of estimation of citral in lemon grass oil

by physical separation of citral. V International

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

Symposium on Medicinal, Aromatic dan Spice Plants

188, p. 241-246.

Ravinder, K., Pawan, K., Gaurav, S., Paramjot, K., Gagan,

S. and Appramdeep, K., 2010. Pharmacognostical

investigation of Cymbopogon citratus (DC) Stapf.

Scholars Research Library, 2, p.181-189.

Scott, R. P. W., 1989. Journals of Chromatography,

Amsterdam: Elsevier

Wilson, I.D (ed)., 2001. Encyclopedia of separation

science. Surrey: Academic Press.

A Simple Method for Isolation of Citral Using Column Chromatography