9. Ijmps -Chromatography a Competent Tool for Phytochemical Profiling

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CHROMATOGRAPHY A COMPETENT TOOL FOR PHYTOCHEMICAL P ROFILING

OF MEDICINAL PLANTS – A REVIEW

SONIA, N. S.1 & JESSYKUTTY, P. C.2 1Department of Plantation Crops and Spices, College of Agriculture, Vellayani,

Kerala Agricultural University, Thrissur, Kerala, India 2Associate Professor, Department of Plantation Crops and Spices, College of Agriculture Vellayani

Kerala Agricultural University, Thrissur, Kerala, India

ABSTRACT

Chromatography is a physical method of separation of components in a mixture by distributing it between a

stationary phase using a mobile phase. Based on geometry of technique there are planar chromatography and column

chromatography. In planar chromatography the stationary phase is present as a plane or on a plane and in column

chromatography it is embedded with in a tube. Planar chromatography includes paper chromatography, Thin Layer

Chromatography (TLC) and High Performance Thin Layer Chromatography (HPTLC). Column chromatography

includes Liquid Chromatography (LC), Gas Chromatography (GC) and Supercritical Fluid Chromatography (SFC). High

Performance Liquid Chromatography (HPLC) is a faster separation system with different modes of elution, mode of

functioning as well as separation techniques suited for heat sensitive compounds whereas GC can separate components

present in volatile oils only. SFC uses a supercritical fluid usually carbon dioxide for the separation of components which

are difficult to be handled by both LC and GC. Chromatographic systems are hyphenated with sophisticated detectors like

mass spectrometers (MS) or tandem mass spectrometers (MS – MS) for precise identification as well as quantification of

molecules.

KEYWORDS: Stationary, Mobile, Planar, Column, Mass Spectrometry

Received: Mar 02, 2016; Accepted: Mar 14, 2016; Published: Mar 21, 2016; Paper Id.: IJMPSAPR201609

INTRODUCTION

World Health Organisation (2008) has defined medicinal plants as plants that contain properties or

compounds that can be used for therapeutic purposes or those that synthesise metabolites to produce useful drugs.

It’s the aim of a drug researcher is to discover a new, safe and active chemical entity from the crude drug which has

therapeutic value for any disease or disorder.

For developing a drug, the crude drugs have to be dried, extracted and isolate the active components using

different analytical techniques. The isolates have to be further purified and standardized using pharmacological,

metabolic and pharmacokinetic studies. Once the compound was standardised/optimized for its therapeutic potential

the drug or dosage forms can be developed (Patil et al., 2013).

In all these stages the authenticity of both the crude drugs and product have to be ensured. When we get a

crude drug or a drug formulation, we have to assure the identity of its active ingredient as well as the impurity

profile of the drug for establishing its therapeutic safety. Since, misidentification of plants, contamination,

Original A

rticle

International Journal of Medicine and Pharmaceutical Sciences (IJMPS) ISSN(P): 2250-0049; ISSN(E): 2321-0095 Vol. 6, Issue 2, Apr 2016, 73-86 © TJPRC Pvt. Ltd.

74 Sonia, N. S. & Jessykutty, P. C.

Impact Factor (JCC): 5.4638 NAAS Rating: 3.54

substitution and adulteration of plants, failure of good manufacturing practice and incorrect preparations and/or dosages are

problems in drug industry (Calixo, 2000). Even after drug development, its quality (purity, strength, presence of any

degradation products etc.) has to be assured (Gillard and Ritter, 1997). Also, the quality of a drug or a drug formulation

varies with time under the influence of temperature, humidity, light etc. So, to establish a shelf life for the drug product by

recommending ideal storage conditions a stability testing is essential (Huynh-Ba, 2009).

For all these studies, high sensitivity and selectivity is required since, the concept of quality is a paramount factor

which must get good attention. WHO has emphasized the need of ensuring quality of medicinal plant products by using

modern techniques like ultraviolet/visible spectrophotometry, fluorimetry, titrimetry, electroanalytical techniques,

chromatographic methods, capillary electrophoresis and vibrational spectroscopies etc. by applying suitable parameters and

standards (Mehta et al., 2011). Of these, chromatography is a multi-disciplinary tool with higher degree of precision and

accuracy (Srivastava, 2011).

CHROMATOGRAPHY

Chromatography is a physical method of separation in which the components to be separated are distributed

between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves in a definite

direction (IUPAC, 2006). The word ‘chromatography’ is derived form two Greek words Chroma (colour) + Graphein

(writing) together means ‘colour writing’. Mikhail Semenovich Tswett invented chromatography in 1903 during his

research on plant pigments. He used the technique to separate various plant pigments such as chlorophylls, xanthophylls

and carotenoids in a plant extract. He had taken a glass column and filled with powdered limestone (CaCO3) (LC-GC

Europe, 2003).

In chromatography, the sample is dissolved in a mobile phase (may be liquid, gas or supercritical fluid) and is

allowed to run over the stationary phase (fixed to a solid surface). These two phases are selected in such a way that the

components of sample distribute themselves between mobile and stationary phases to varying degrees. Those components

strongly retained by the stationary phase move slowly with the flow of mobile phase and those which are weakly held will

travel rapidly through the column and gets eluted first. This difference in migration rates, make the sample components to

be separated into distinct (discrete) zones which can be used for quantitative or qualitative analysis (Miller, 2005).

Chromatographic fingerprint of a herb is a chromatographic pattern of the extract of some common chemical

components of pharmacologically active and/ or chemical characteristics (Patil and Shettigar, 2010). In the finger print we

have to look for its integrity and fuzziness or sameness and differences to chemically represent that herbal medicine.

Chromatography restricts not to analytical separations, it is used in separation of pure substances (purification), study about

kinetics of reactions, structural investigations on molecular scale also, in determination of physiochemical constants like

enthalpy, entropy, free energy etc.

TYPES OF CHROMATOGRAPHY

Among these techniques, for phytochemical profiling, choose a suitable one depending on the chemical

composition, chemical nature, physical and chemical properties and physical state of that test substance. Since, different

types of functional groups, acidic, basic or neutral substances, substances with different polarity, molecular mass,

refractive index, density, viscosity, also the different states of matter like solid liquid or gas. As shown here (Table 1.)

chromatography is classified based on various parameters (Chavan et al., 2013) of which geometry of the technique is

Chromatography a Competent Tool for Phytochemical Profiling of Medicinal Plants – A Review 75


found ideal to discuss all other types.

Table 1: Classification of Chromatography

Parameters Principle of Separation

Mobile Phase Used

Geometry of Technique

Scale of Operation

Mode of Technique

Elution Method

Types Partition GC Planar – Paper, TLC, HPTLC

Preparative Normal phase

Gradient Adsorption LC

Size exclusion SCF chromatography

Column – GC, HPLC, SFC, UPLC

Analytical Reverse phase

Isocratic Affinity Ion exchange

Based on the geometry of chromatographic technique there are planar and column chromatography. In planar

chromatography the stationary phase is present as a plane or on a plane and in column chromatography it is embedded with

in a tube (IUPAC, 2006).

Planar Chromatography

Planar chromatography includes Paper Chromatography, Thin Layer Chromatography (TLC) and High

Performance Thin Layer Chromatography (HPTL

Paper Chromatography

Paper chromatography works by the partition of solutes between water in the paper fibers (stationary phase) and the

solvent (mobile phase) (The Royal Society of Chemistry, 1997). This technique can be used in herbal studies for

prliminary screening or to know the presence of any particular compound. Devare et al. (2011) used this technique for

preliminary phytochemical screening of aminoacids in acetone, chloroform, ethanol and water extracts of Morinda

pubescence and reported that the leaves of Morinda is a rich source of different aminoacids also, water is a good solvent

for extracting it.

Paper chromatography can provide only a rough estimate of the compounds, separation takes place very slowly (10 to

20 hours) and also, the spots tended to be much more diffused. This technique is now obsolete, except perhaps as an

inexpensive technique for teaching chromatography in schools and colleges. But, its introduction must truly regarded as a

revolution since, it led ultimately to the award of Nobel Prize to Martin & Synge in 1952 (Wilson, 2000).

Thin Layer Chromatography (TLC)

TLC is a widely employed laboratory technique and is a modern version of paper chromatography. However,

instead of using a stationary phase of paper, it uses a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat,

inert substrate like glass, thick aluminum foil or plastic (Kumar et al., 2013).

The principle of TLC is the distribution of a compound between a solid fixed phase applied to a glass or plastic

plate and a liquid mobile phase, which is moving over the solid phase. A small amount of a compound or mixture is

applied to a starting point just above the bottom of TLC plate. The plate is then developed in the developing chamber that

has a shallow pool of solvent just below the level at which the sample was applied. As the solvent slowly travels up the

plate, the different components of the dye mixture travel at different rates and the mixture is separated into different

coloured spots. The mobile phase will carry the most soluble compounds the farthest up the TLC plate and less soluble

ones will stay behind (Singhal et al., 2009).

76

Impact Factor (JCC): 5.4638

The behavior of an individual compound in TLC is charact

and is expressed as a decimal fraction. The Rƒ is calculated by dividing the distance the compound travel

original position by the distance the solvent travelled from the original position (th

2007).

Figure 1:

Coloured substances can be seen directly

making them visible using spraying agents like iodine vapours, potassium permanganate, potassium dichromate etc. which

produces coloured areas in the region which they occupy. Also, some fluorescing materials can be added to the adsorbent

during preparation of TLC plate and these

Rƒ values can be used to aid in the identification of a subst

physical constant and comparison should be made only between spots on the same she

substances that have the same Rƒ value may be identical; those with different R

TLC is used for different purposes like identifying the presence of a particular compound in a crude drug as

reported by Kumar and Dhillon (2015) in

red data book for confirming the presence of

and tenoposide. TLC of methanolic extracts of its leaves and roots reported an

TLC helps in authentication of herbal drug for

Bilwadi churna, Gangadhar churna, and Pushyanug churna

(Bilva patra) by Singh and Kumar (2015). In the TLC plates, Bilva patra

Bilwadi churna showed three spots (0.89, 0.80 and 0.63) and Pushyanug churna showed two spots (0.89 and 0.66)

matching with two phytoconstituents of Bilva Patra whereas Gangadhar churna showed two spots of w

matching with Bilva Patra (0.63). These difference may be due to the difference in geographical distribution,

manufacturing process, stage of harvest etc. or it may be due to the intentional or unintentional adulteration.

Revathy et al. (2011) have tested various solvents systems in TLC for extraction of curcuminoids like curcumin

(C), demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) from the acetonic extract of

solvents systems were tried and among them d

C, DMC and BDMC were obtained for chloroform: methanol

compounds in HPLC.

Presence of antimicrobial compounds in

the technique is named as bioautography. Here, the test microbial cultures have to be poured over the resolved TLC plates

of particular medicinal plant. These plates on incubation for

presence of antimicrobial compounds which can be identified using the corresponding R

The behavior of an individual compound in TLC is characterized by a quantity known as Rƒ (Retention factor)

and is expressed as a decimal fraction. The Rƒ is calculated by dividing the distance the compound travel

original position by the distance the solvent travelled from the original position (the solvent front)

Figure 1: Determination of Retention Factor (Rf)

red substances can be seen directly in stationary phase, while colourless substances can be detected by

spraying agents like iodine vapours, potassium permanganate, potassium dichromate etc. which

red areas in the region which they occupy. Also, some fluorescing materials can be added to the adsorbent

during preparation of TLC plate and these can be viewed under the UV light (Archana et al., 2011).

values can be used to aid in the identification of a substance by comparison to standards.

and comparison should be made only between spots on the same sheet, run at the same time. Two

value may be identical; those with different Rƒ values are not identical.


Dhillon (2015) in Podophyllum hexandrum, an endangered species of medicinal plant listed under

he presence of Podophyllotoxin, a compound for the synthesis of anticancer drugs etoposide

and tenoposide. TLC of methanolic extracts of its leaves and roots reported an Rƒ value of 0.87 and 0.94 respectively

TLC helps in authentication of herbal drug formulations also. TLC finger prints of three polyherbal formulations

Bilwadi churna, Gangadhar churna, and Pushyanug churna were prepared for finding the presence of

(Bilva patra) by Singh and Kumar (2015). In the TLC plates, Bilva patra showed four spots at 0.89, 0.75, 0.65, and 0.52,


matching with two phytoconstituents of Bilva Patra whereas Gangadhar churna showed two spots of w

. These difference may be due to the difference in geographical distribution,


. (2011) have tested various solvents systems in TLC for extraction of curcuminoids like curcumin

(C), demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) from the acetonic extract of

solvents systems were tried and among them desired resolution of separation and Rf values matching with the standards of

tained for chloroform: methanol. So, it is selected as a suitable solvent for the separation of

Presence of antimicrobial compounds in medicinal plant samples can also be identified with the help of TLC and


of particular medicinal plant. These plates on incubation for a few days could yield some inhibition zones representing the

presence of antimicrobial compounds which can be identified using the corresponding Rf values.

Sonia, N. S. & Jessykutty, P. C.

NAAS Rating: 3.54

erized by a quantity known as Rƒ (Retention factor)

and is expressed as a decimal fraction. The Rƒ is calculated by dividing the distance the compound travelled from the

e solvent front) (Figure 1) (Skoog et al.,

rless substances can be detected by

spraying agents like iodine vapours, potassium permanganate, potassium dichromate etc. which

red areas in the region which they occupy. Also, some fluorescing materials can be added to the adsorbent

., 2011).

ance by comparison to standards. The Rƒ value is not a

et, run at the same time. Two

values are not identical.


, an endangered species of medicinal plant listed under

Podophyllotoxin, a compound for the synthesis of anticancer drugs etoposide

ƒ value of 0.87 and 0.94 respectively

. TLC finger prints of three polyherbal formulations

were prepared for finding the presence of Aegle marmelos Corr

showed four spots at 0.89, 0.75, 0.65, and 0.52,


matching with two phytoconstituents of Bilva Patra whereas Gangadhar churna showed two spots of which only one spot is

. These difference may be due to the difference in geographical distribution,


. (2011) have tested various solvents systems in TLC for extraction of curcuminoids like curcumin

(C), demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) from the acetonic extract of Curcuma longa. Three

esired resolution of separation and Rf values matching with the standards of

. So, it is selected as a suitable solvent for the separation of

medicinal plant samples can also be identified with the help of TLC and


a few days could yield some inhibition zones representing the

values. Saenego and Ndip (2012)



identified two compounds from Garcinia kola seeds, one with Rf of 0.53 which inhibits the growth of Streptococcus

pyogenes, Styphylococcus aureus and Salmonella typhimurium and another compound of Rf 0.63 which could inhibit

Salmonella typhimurium and Plesiomonads shigelloides.

TLC results are more reproducible, due to the use of much smaller silica particles as stationary phase that favours

a very efficient separation. The Rƒ is not a physical constant its value change with change in solvent system, adsorbent,

thickness of adsorbent and amount of the material spotted (Bilia et al., 2002).

High Performance Thin Layer Chromatography (HPTLC)

HPTLC is a sophisticated and automated form of thin – layer chromatography (TLC) with advanced separation

efficiency and detection limits. It is a powerful analytical technique equally suitable for qualitative and quantitative

analytical tasks (Sethi, 2013). Separation happens due to adsorption or partition or by both depending upon the nature of

adsorbents and the solvent system used. It is an instrument controlled by software (Kustrin and Hettiarachchi, 2014).

Here, sample is applied automatically and uniformily applied over the TLC plate specially designed for HPTLC

(silica particle size – 5 to 6 µm, layer thickness -100µm, sample volume – 0.1 to 0.5 µL, starting spot diameter – 1.0 to

1.5mm and seperated spot diamter – 2 to 5mm) using the sample applicator (Patel and Patel, 2008). The chromatogram is

developed inside a chamber filled with mobile phase and viewed inside a visualiser at different wavelenght of light and

finally documented. The software develops a peak corresponding to each compound and we can get the retention factor,

area of the height of the peak etc. from the result developed.

The use of modern apparatus such as video scanners, densitrometers and new chromatographic chambers and

more effective elution techniques, high resolution sorbents with selected particle size or chemically modified surface, the

possibility of combining with other instrumental methods and development of computer programs for method optimization

all make HPTLC an alternative to HPLC or GC. The major disadvantages include, bulky instrumentation, large space

requirement, expensive, requires dust free and stringent operation conditions and technically skilled persons to run the

system (Srivastava, 2011).

HPTLC can be used similar to TLC for the determination of bioactive component of herbal medicine, as a

chemotaxonomic tool in plant systematics and also for quality control and standardization of herbal drugs (Mahesh et al.,

2011).

Methanolic extract of leaves of Pala indigo tree/ Sweet indrajo (Wrightia tinctoria) [Family: Apocynaceae] popular

for its Psoriasis curing properties (Khyade and Vaikos, 2014) was analysed using HPTLC for the presence of compound

lupeol by Devi and Devakar (2012). Lupeol is an anti-inflammatory and anti-cancerous triterpenoid and it is reported to

contain 47.6mg of lupeol/ g of methanolic extract.

HPTLC analysis of Wrightia tinctoria (Pala indigo tree) and Wrightia tomentosa (Scarlet wrightia) by

Muruganandham et al. (2000) reported that indigotin, indigorubin, tryptanthrin, isatin, anthranallite and rutin were present

in Pala indigo tree whereas in Scarlet wrightia only anthranalite and rutin are present which can be a criteria for

distinguishing these species of medicinal plant. Another plant in Apocynaceae family is Holorrhena antidyssentrica

(Kudaka pala/ Kutja) containing an active principle conessine used for treating dysentery. Seeds and bark of this plant

resembles Wrightia tinctoria so, widely using as an adulterant in Wrightia tinctoria pharmaceutical preparations. Jirge

(2011) analysed two pharmaceutical formulations Kutja vati and Kutja capsules with Kutja bark using HPTLC and



reported a conessine content of 0.901 ± 0.71 % in Kutja bark and a lower value for both the formulations (Kutja vati –

0.725 ± 0.11 % and Kutja capsules - 0.692 ± 0.24 %)which may be due to adulteration.

Column Chromatography

Liquid Chromatography

A chromatography using a liquid as mobile phase is called liquid chromatography. The liquid used as the mobile

phase is called the “eluent” and here, stationary phase is usually a solid or a liquid. Sample solution is applied into a

porous stationary phase and mobile phase is delivered through the column at higher pressure so that separation occurs

based on the affinity of solute to the stationary phase. Need for higher degree of separation and faster analysis by refining

the packing material used as stationary phase to a size of 3 to 10 µm coupled with eluent delivery through a high pressure

pump facilitates the development of HPLC (High Performance Liquid Chromatography) (Charde et al., 2014). HPLC

instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column and a detector (Gupta and

Shanker, 2008).

Solvent delivery pump delivers the mobile solvent and the injector introduces it into the mobile phase or onto the

chromatographic bed. Most important component of the HPLC system is the column where the sample components get

separated when it passes through this. The column is 10 to 30 cm long stainless steel tube having a diameter of 3 to 5mm

filled with silica gel. The separated components in the column will go to a detector for quantification and got recorded in

the computer system. The system will provide the phytochemical components as a fingerprint containing peaks.

HPLC is a highly flexible system since, its mobile phase, stationary phase as well as elution method is modified

into different chromatography types for meeting wide range of analysis requirements (IUPAC, 2006). Normal phase and

reverse phase are the two types of HPLC based on mode of separation where, the stationary phase is more polar than the

mobile phase in normal phase and vice versa in reverse phase. Lipophilic substances like oils, fats and lipids are separated

by normal phase chromatography. Since, most of the plant extracts are polar compounds reverse phase chromatography is

widely using for phytochemical finger printing of medicinal plants. Sivakumar et al. (2014) have standardised HPLC

procedure for quantification of berberine an isoquinoline alkaloid, from Tinospora cordifolia (Guduchi) using acetonitrile:

water (60:40) as the solvent system. They had compared berberine content of wild and micropropagated Guduchi and

reported that methanolic extract of both samples contain berberine (1.2% berberine in micropropagated and only 0.2% in

the wild).

Silica particles using as stationary phase is now available in various modifications for specific applications in

chromatography like affinity chromatography, ion exchange chromatography and size exclusion chromatography. Of

which affinity chromatography is a technique based on selective non-covalent interaction between an affinity ligand bound

to the stationary phase and the analyte molecule. These affinity ligands like antibodies, enzyme inhibitors etc. selectively

and reversibly bind analyte molecules in the sample which could be eluted later by changing the elution parameters (Skoog

et al., 2007).

Utilising affinity chromatography a study was conducted by Paiva et al. (2003) to separate out trypsin inhibitors

present in Echinodorous paniculatus that could prevent the digestion of proteins. Here, a ligand obtained from another

plant (Carota mollis) which could bind trypsin inhibitor is first coated on to the chromatographic bed and they named it as

Cra Iso 1, 2, 3 – sepharose. When the extract was poured ligand binds the trypsin inhibitors and it is separated out from the

column using salt solution and it is quantified as 1mg protein contains 0.01mg trypsin inhibitor.



Another HPLC technique is ion exchange chromatography where the stationary phase is an ion exchange resin.

When the mobile phase passes through the resin, the electrostatically bound ions are released from resin and ions of the

like sign present in the extract got bounded preferentially (Sarzanini, 2002). This technique is most frequently used for the

separation and purification of proteins, polypeptides, nucleic acids, polynucleotides and other charged biomolecules

(Bonnerjera et al., 1986).

Isitua et al. (2015) had used Biochrom 30 an ion exchange chromatography column to separate different

aminoacids present in Moringa oleifera leaves collected from Africa. They had obtained 16 aminoacids with glycine is in

higher amounts (3.63%).

Size exclusion chromatography is yet another unique HPLC technique which uses a porous gel [stationary phase]

to separate the mixture through the column and separation happens according to the particle size. This is used to separate

high molecular mass products like proteins from low molecular mass species like amino acids and small peptides in

pharmaceutical drug preparations (Skoog et al., 2007).

From Solanum aculeatissimum using ion exchange chromatography a protease inhibitor was separated by

Krishnan et al. (2015) and they selected size exclusion chromatography using Sephadex G – 50 as the stationary phase to

purify this protease inhibitor into its components, trypsin inhibitor and chymotrypsin inhibitor. They could separate 285.7U

of trypsin and 270.9U of chymotrypsin from one mg protein (1U means the amount of substrate that can be catalyzed per

minute).

A HPLC system can be run in isocratic mode or in gradient mode. In isocratic elution process for the entire running

of HPLC system only a single mobile phase composition (a single solvent or solvent mixed in a particular ratio) is

delivered to the column using a single pump. In gradient elution, we can change the mobile phase composition using two

or more pumps according to a set programme as like the one which was used by Bhaskara Rao et al. (2015) for analysing

methanolic extract of Kaempferia galanga leaves. They used two solvents (Solvent A: 50mM sodiumphosphate in 10%

methanol and Solvent B: 70% methanol) in different proportions at specific time intervals and reported the presence of

genistein (Rt -778.85), coumaric acid (Rt - 23.82), ferulic acid (Rt - 25.39) and butein (Rt - 79.15).

In HPLC, a variety of separating techniques and column packings are available which make the system highly

efficient and flexible. HPLC can be used as a preparative method as well as a purification technique. This can be connected

with modern sophisticated detector or detectors in series for maximum precision in analysis. Most sample analysis is

carried out at room temperature (ICS – UNIDO, 2008).

The stationary phase of a liquid chromatography column is further refined to a particle size of less than or equal to

2.5µm and delivery of mobile phase at higher linear velocities under very high pressure of 103.5 MPa lead to the

development of an ultra – modern technique called UPLC (Ultra Performance Liquid Chromatography) (Roge et al., 2011).

Compared to HPLC compared to HPLC, resolution, sensitivity, speed of analysis and peak capacity (number of peaks

resolved per unit time in gradient separations) can be extended to new limits, so termed as UPLC (Kumar et al., 2014).

UPLC helps in faster analysis which ultimately result in early drug discovery as reported by Shang et al. (2015) that

they could analyse 17 hot water and methanolic extract of 17 different medicinal plants collected from a province of

Canada by using Acquity BEH C18 UPLC column (1.7µm, 2.1x100mm), 0.1% formic acid in water and 0.1% formic acid

in acetonitrile as mobile phase, at a flow rate of 0.5ml/minute and the sample volume used was only 1µL. They could get



the entire phytochemical data of these samples within hours whereas it takes days or even weeks in HPLC. More than 4000

metabolites were compared using the chromatograms and used further to fulfil their research objective.

Gas Chromatography

In gas chromatography (GC), a gas is used as mobile phase and stationary phase can be either a solid or a non-

volatile liquid coated on an inert supporting material. The stationary phase is held in a narrow column in an oven while

gaseous mobile phase travels through the column. Here, solutes present in the vapourised samples are separated when its

gets partitioned between mobile gaseous phase and stationary phase. Here, the mobile phase doesn’t interact with the

molecules of the analyte but only transport through the column. This is applicable to complex volatile biochemical entities

thermostable compounds (Adams, 2007).

Gas chromatography run by introducing the sample in liquid or gaseous form with the help of a GC syringe into

the injection port. It gets vaporized at injection port then passes through the column along with continuously flowing

mobile phase, usually H2 and get separated/detected at the detection port maintained by suitable temperature programming.

We can visualize the chromatogram in the computer system using the appropriate software.

Janicsak et al. (2003) have standardised a GC method for routene determination of oleanolic acid and ursolic acid

(triterpene compounds) in five medicinal plant species of Lamiaceae family like Hyssopus officinalis, Marrabium vulgare,

Melissa officinalis, Salvia officinalis and Satureja montana. Of these species, Salvia officinalis recorded maximum amount

of both the acids and Marrabium vulgare had the lowest.

The major advantages of gas chromatography are fast analysis, high resolution, reliable and simple technique

with, accurate quantification by coupling it with sophisticated detectors. But, the technique is suited only to volatile oils.

Since, the temperature of the columns are maintained above or equal to 3800C it is not suited to thermolabile compounds.

Sample preparation is also difficult since, it has to be dissolved in volatile solvents and moreover it’s an expensive process

(Eiceman, 2000).

Supercritical Fluid Chromatography (SFC)

Supercritical fluid chromatography (SFC) is a hybrid technique that combines the best features of gas and liquid

chromatography (Chavan et al., 2013). Here, the mobile phase is a fluid which is above and relatively close to its critical

temperature and pressure. The instrument is similar to a GC or LC which differs only in the mobile phase, a super critical

fluid usually carbon dioxide (critical temperature – 31.30C and critical pressure – 72.90atm) is using (Sairam et al., 2012).

SFC permits the separation and determination of a group of compounds which can’t be conveniently handled by either GC

or LC. Compounds that are nonvolatile/ thermally unstable can’t be handled by GC and those compounds with no

functional groups which failed to detect by spectrometry or electrochemical methods of LC can be detected by SFC (Skoog

et al., 2007). SFC has high throughput used for qualitative and quantitative analysis in drug industry.

Vitamin E a fat soluble vitamin was analysed using SFC in a better way than GC by Naegele (2015). Analysing

by SFC it is found that extra virgin olive oil contains α – tocopherol (184.8mg/kg) only and in virgin olive oil along with

tocopherols, its unsaturated derivatives like tocotrienols both in their α,β,γ and δ isomeric forms were present which

indicates the purity of extra virgin olive oil. Compared to LC and GC, SFC is 5 – 10 times faster and with better resolution

(5 times). Compounds with high molecular weight, thermolabile, non-volatile, polar and adsorptive in nature can be

analysed using SFC. Moreover the solvent is not at all harmful.

Chromatography a Competent Tool for Phytochemical Profiling o

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HYPHENATED CHROMATOGRAPHY TECHNIQUES

In all the modern chromatography techniques, separated fractions from the column are connected to detectors.

Today, several sophisticated detectors working on specific principles are available like, MS (Mas

(Diode Array Detector) and NMR (Nuclear Magnetic Resonance Detector) etc. which are possible to give additional

information on the separated components (Kamboj, 2012). Of these detectors, MS (Mass Spectrometry) is widely used for

phytochemical analysis and is usually fo

The separated components of the mixture from the chromatograph will enter into the MS through an interphase.

This is followed by ionization, mass analysis and detecti

the mass spectrometer (Van, 2005). The process of ionisation not only ionise the molecule but also break the molecule into

the fragments and detect these fragments with the help of electron

molecular ion of analyte form a finger print spectrum which is different from other analytes (Phalke and Kavade, 2013).

These will be compared with the online library of molecules or library maintained by

elemental composition etc. Thus, MS helps in quantifying the known compounds, identifying the unknown compounds and

also, elucidating the structure and chemical properties of the components.

A typical mass spectrum (Figure 2)

given ion (for singly charged ions this corresponds to the mass of the ion). Height of peak indicates the relative abundance

of a given ion (not reliable for quantitati

Liquid chromatography-mass spectrometry (LC

separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC

powerful technique used for many app

Steenkamp et al. (2004) reported that LC

was detected when the viscera samples of a dead body wa

conclude that the man died out of Datura poisoning, due to the consumption of

GC–MS was the first successful online combination of chromatography with mass spectrometry, an

used in the analysis of essential oil in herbal medicines. With the GC

the information related to qualitative and quantitative composition

establishing the relationship between different

today for the analysis of the volatile chemical compounds in herbal medicines (Gong

made between 2 varieties of Kacholam (

or Phytochemical Profiling of Medicinal Plants – A Review

HYPHENATED CHROMATOGRAPHY TECHNIQUES


Today, several sophisticated detectors working on specific principles are available like, MS (Mas



chemical analysis and is usually found attached with the LC as LC-MS, GC-MS, SFC-MS etc.


This is followed by ionization, mass analysis and detection of mass-to charge ratios of ions generated from each analyte by

The process of ionisation not only ionise the molecule but also break the molecule into

the fragments and detect these fragments with the help of electron impact ionisation and/or chemical ionization. The


These will be compared with the online library of molecules or library maintained by the system for getting its structure,


also, elucidating the structure and chemical properties of the components.

(Figure 2) is characterized by sharp, narrow peaks. X-axis indicates the m/z ratio of a


of a given ion (not reliable for quantitation). Peak intensity indicates the ion’s ability to desorb.

Figure 2: A Typical Mass Spectrum

mass spectrometry (LC-MS) is an analytical technique that combines the physical

separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC

powerful technique used for many applications which has very high sensitivity and selectivity (Wang

. (2004) reported that LC-MS analysis could help in forensic study also since, scopolamine and atropine

was detected when the viscera samples of a dead body was analysed using this technique

conclude that the man died out of Datura poisoning, due to the consumption of Datura stramonium

MS was the first successful online combination of chromatography with mass spectrometry, an

used in the analysis of essential oil in herbal medicines. With the GC–MS, not only a chromatographic fingerprint but also

the information related to qualitative and quantitative composition is made possible. This will be extremely useful for

different constituents and its pharmacology. Thus, GC–

for the analysis of the volatile chemical compounds in herbal medicines (Gong et al

rieties of Kacholam (Kaempferia galanga) for its volatile oil constituents using GC

81

[email protected]


Today, several sophisticated detectors working on specific principles are available like, MS (Mass Spectrometry), DAD



MS etc.


to charge ratios of ions generated from each analyte by

The process of ionisation not only ionise the molecule but also break the molecule into

impact ionisation and/or chemical ionization. The


the system for getting its structure,


axis indicates the m/z ratio of a


on). Peak intensity indicates the ion’s ability to desorb.

MS) is an analytical technique that combines the physical

separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a

lications which has very high sensitivity and selectivity (Wang et al., 2001).

MS analysis could help in forensic study also since, scopolamine and atropine

analysed using this technique. This aided the doctors to

Datura stramonium seeds.

MS was the first successful online combination of chromatography with mass spectrometry, and is widely

MS, not only a chromatographic fingerprint but also

. This will be extremely useful for

–MS is the most preferable tool

et al., 2001). A comparison was

) for its volatile oil constituents using GC – MS by Indrayan et



al. (2007) and reported that 58 compounds were identified from variety Kasthuri and 56 from Rajani. 45 compounds are

found common for both. 13 compound are peculiar to Kasthuri and is not present in Rajani. The major component

responsible for aroma in Kacholam, ethyltrans – p - methoxycinnamate is quantified to be 39% in Kasthuri and only 35%

in Rajani. So, Kasthuri is recommended for volatile oil production.

Chromatography is still competent by hyphenating it with another competent spectrometry called tandem mass

spectrometry which helps to analyze only selected specific ions of your interest by filtering out the untargeted ions.

Chromatography combined with MS – MS technique take the advantage of chromatography as a separation method and

MS - MS as a most precise identification method. MS - MS can provide on-line information for each individual peak in a

chromatogram. With the help of this hyphenation, in most cases, one could identify the chromatographic peaks directly on-

line by comparison with standard compounds, which made the MS–MS a powerful approach for the rapid identification of

phytochemical constituents. In phytochemical research, this is usually done to find out the presence of any trace elements

or heavy metals like mercury, lead etc. in the sample (Nair et al., 2010).

Here, two mass spectrometers are arranged one in front of the other (in tandem) separated by a collision cell. In

the first MS – ions of your interest, suppose mercury in your sample only will be filtered and allowed to enter the collision

cell where, it is collided with Argon or Nitrogen gas so that ions get fragmented. These ions are again filtered using the

second MS according to their m/z (mass/charge) ratio. Filtering can be done by several ways mainly according by

adjusting the frequency of oscillation of electric current according to the mass and charge of ions. Finally, the target ions

will be detected by a detector and quantified and a mass spectrum of specific ions only will be obtained (Prasain, 2012)

(Figure 3).

Figure 3: Principle of Tandem Mass Spectrometry

Paris polyphylla, a medicinal plant of Himalayas is rich in steroidal saponins like diosgenyl and pennogenyl

saponins which are proved to be successful against several human cancer cell lines. A study was done by Kumar et al.

(2014) to identify the presence of these two compounds in Paris polyphylla, for using it in a study against human lung

cancer cell lines. They developed HPLC chromatograms also, MS – MS spectra for each Rt values and reported the

presence of several pennogenyl and diosgenyl saponins in the Rt ranges 1.4 to 2.3 and 9.3 to 14.03 respectively.

CONCLUSIONS

Chromatography is a physical method of separation in which the components of a plant extract are separated

between a mobile phase and a stationary phase. It has a wide array of technical advancement which revolutionized

phytochemical research. It is a multidisciplinary tool which helps to identify the presence of some specific compounds

(qualitative analysis) which aid finding out the adulterants present in the pharmaceutical preparations, authentication of

herbal products as well as can sometimes act as a chemotaxonomical tool. Quantitative analysis can also be made with the

use of sophisticated techniques like HPTLC, HPLC, UPLC, GC, SFC etc. which are effective and reliable tools in the



entire chain of development of products like dietary supplements, nutraceuticals and various types of medicines. It is used

in quality control, purity check, clinical applications, metabolism studies, drug screening etc. Chromatography becomes

more competent when it is hyphenated with sophisticated detectors like mass spectrometry, tandem mass spectrometry etc.

which are capable of giving much more information about the components separated out using chromatography. The

molecular mass, structural elucidation of the compound etc. are made possible by using spectrometry whereas trace

compound analysis can be done using tandem mass spectrometry. Research is focused on development of new

chromatographic techniques and its hyphenations with more sophisticated detectors for enhancing the accuracy and

precision of phytochemical analysis.

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