INTRODUCTION OF CHROMATOGRAPHY

Chromatography is a crucial biophysical method that makes it possible to identify, separate, and purify a mixture's
constituent parts for both qualitative and quantitative examination. Proteins can be separated according to features such as
size and shape, total charge, the number of hydrophobic groups on their surface, and their ability to bind to the stationary
phase[23].

Based on the nature of the interaction and the properties of the molecules, four separation techniques four methods of
separation—surface adsorption, partition, and exclusion of the size—are employed, depending on the molecular properties
and type of contact[24].
The two phases used in this approach are the mobile phase and the stationary phase. Depending on the sample's
composition, one phase of the process may be polar and the other non-polar [65].

Analytical methods generally differ significantly based on the sample type, minimum detection limit, apparatus, and
chromatographic process. Chromatographic separation, followed by identification and detection, are examples of specific
uses. mination of individual constituents, assessment of the distribution of the boiling range, and identification of the
various hydrocarbon group types[40].

Chromatography types :-
Chromatography types include column and ion-exchange chromatography,Gel-permeation (molecular sieve)
chromatography, affinity, gas, paper, thin-layer, dye- ligand, hydrophobic interaction, pseudo affinity, and high-pressure
liquid chromatography (HPLC) are among the chromatographic techniques that can be used.

Column chromatography- Chromatography in columns Given that proteins differ in terms of their size, structure, net charge,
stationary phase utilized, and binding capacity, chromatographic techniques can be used to purify each of these
distinguishing elements[23].
Today, HPLC is widely applied for separations and purifications in a variety of areas, including pharmaceuticals,
biotechnology, environmental, polymer, and food industries. It is accomplished by injection of a small amount of liquid
sample into a moving stream of liquid (called the mobile phase) that passes through a column packed with particles of the
stationary phase. The separation of a mixture into its components depends on the different degrees of retention of each [31].

Gas chromatography : An analytical method that is frequently used to separate and examine volatile and gaseous substances
is gas chromatography.To separate the analytes in gas chromatography, the sample is dissolved in a solvent and then
vaporized[53].

Thin layer chromatograph y : A thin glass plate coated in silica gel or aluminum oxide serves as the solid phase in thin-layer
chromatography.A solvent is selected as the mobile phase based on the characteristics of the mixture's constituent parts[39].

Paper chromatography: The partition concept, which divides different components between the liquid phases, is used in
paper chromatography.The aqueous solvent is contained in the stationary phase of this, which is the filter paper.The paper is
moved by the mobile phase. The capillary action of the pores allows for the separating process[82].

Advances in chromatography
One technique for distinguishing organic and inorganic molecules is chromatography. The definition of "color writing" is
chromatography. The foundation of chromatography is differential migration. The stationary phase, or column, is traversed
by the mobile phase, which collects the chemicals for testing. Taken with the compounds, the mobile phase proceeds
through the stationary phase. A glass tube with a diameter of 5 mm to 50 mm and a height of 5 cm to 1 m is the traditional
preparative chromatography column. For a 1 cm-diameter column, 25 g of adsorbent are needed in order to achieve a
greater separation than in a 2 cm-diameter column[79].

In order to solve more complex analytical problems where at least one of the techniques is a separation technique and the
other is a spectroscopic detection technique, hyphenated techniques have developed as a tool to find complete biological
shapes of herbal medicine arrangements or quotations. In contrast to a method that relies solely on logic, the coupling aims
to extract the material from a complicated mixture for identification and quantification. Capillary electrophoresis (CE), gas
chromatography (GC), liquid chromatography (LC), and high-performance liquid chromatography (HPLC) are examples of
separation procedures [58].
Without a doubt, thin-layer chromatography (TLC) is important. It is the only chromatographic technique that allows the
result to be shown as an image. Moreover, TLC is the only method where the chromatogram contains every component of
the sample. In contrast, not every component in the sample is displayed by HPLC and GC due to their selectivity. Stahl's
 work, which established the use of calcium sulfate as a binder and standardized layer thickness and chromatographic
development, was responsible for the actual breakthrough in TLC (Stahl, 1958), [34].
A sophisticated chromatographic method called two-dimensional liquid chromatography (2D-LC) combines two separate
chromatographic separations to increase peak capacity and resolution. It is intended to solve the drawbacks of conventional
one-dimensional chromatography, including the co-elution of analytes and a finite peak capacity[83].

Isolation of phytoconstituents by using chromatography

Isolation of Flavanoids
The leaves that had been shade-dried were ground into a powder (250 g), extracted using 70% (v/v) ethanol, and then
vacuum-concentrated at 60° ± 1 °C to remove all
moisture. It was kept in an airtight container in the refrigerator at 5 °C after drying in a hot air oven (40–45 °C). The residue
was named E. neriifolia hydro-ethanolic extract (HEEN).

To obtain the appropriate extracts, 250 g of dried E. neriifolia leaves were extracted in turn using pet-ether, benzene,
chloroform, ethyl acetate, and ethanol. The leaves were then macerated with distilled water (non-polar to polar). Using
quercetin as a standard, the flavonoid contents and their existence were ascertained using the Harborne (1998) method. TLC
was used to analyze the extracts[89].
Isolation of Glycoside
The N-Hexane/Ethyl Acetate/Methanol/Water solvent solution was degassed in about 2000 milliliters using a 20-minute
ultrasonic bath. For HSCCC separation, the column was completely filled with the upper phase (stationary phase) before the
lower phase (mobile phase) was added at a flow rate of 2.0 mL/min (head to tail). The device's speed was 860 rpm when
turned backward. At the moment of separation, it was 25 ◦C. The detection wavelength employed was 227 nm. The
powdered crude extract (about 100 mg) was dissolved in 10 mL of the solvent system prior to being injected onto the
column. After separation, two fractions were collected, referred to as I and II. Vacuum-formed fractions condensed at 50 °C
[91].
Isolation of saponine

Extraction Methods: Three times, 0.9 kg of dried Panax ginseng roots were ground into a coarse powder and steeped in 7.2
L of 90% aqueous ethanol for 45 minutes. The extract solution was first filtered, and then concentrated. The concentrated
solution was diluted with deionized water and separated using a 0.6 L macroporous resin column. Seven bed volumes of
deionized water were pushed through the column in order to remove impurities. Ginsenosides were then isocratically eluted
using four bed volumes of 70% aq. EtOH at a flow rate of 10 mL/min. At 60°C, the fractions were vacuum-concentrated to
get a crude saponin sample[80].

Medicinal Family Chemical Chromatogr Mobile Phase Reference

Plant Constituent aphic
Technique
Hibiscus Malvaceae Delphinidin 3- Chlorofarm : []
0-glucoside HPTLC Methanol(97:3 v/v)
(Anthocynin)
Ceiba Malvaceae Oxalate HPTLC
A] n-hexane : ethyl [19]
Pentandra(Ka flavonoids, acetate
pak tree) triterpenes (95 : 5 v/v)
B] Ethyl acetate :
methanol: water (100
: 1 v/v)
Urena Lobata Malvaceae Trans-tiliroside, Column CH2CL2 : MeOH [76]
B-sitosterol, Chromatogra (100 : 1 v/v)
quercetin phy
Pavonia Malvaceae 1-nonadecanol, Benzene : ethynol : [29]
Zeylanica bis(2- TLC Ammonia [18 : 2 :
ethylhexyl) 0.2] ethyl acelate :
Ester methanol : H20
 (10 : 1 : 0.5)

Grewia Malvaceae P-Coumaroyl Methanol : water [86]

asiatica Glycolic acid LC-MS (10 : 90 v/v) with
(Phalsa) 0.1% formic acid

Chorisia Malvacea Pyrocatechol, column Ethyl acetate: [60]

chodatii (silk e quercetin chromatogra Methanol (8:2)
floss tree) phy
Malvacea Isorhamnetin,m 0.1% formic acid in [20]
Malva e alvesterone,mal HPLC water and 0.1%
aegyptiaca L. valic acid formic acid in
(Mallow) methanol
Okra Malvacea Isoquercitrin, HPLC [13]
(Bhendi) e quercetin-3- A)1%acetic acid
gentiobioside glacial v/v
B) Acetonitrile

Lavatera Malvacea P- coumaric Column Ch2Cl2:MeoH [66]

trimestris e acid,kaempferol chromatogra
3-o-B-D- phy
lucoside
Blumea Malvacea Blumeatin,Dios HPLC Acetonitrile and [16]
balsamifera e metin,Chrysopl 0.2% aqueous acetic
(Balsa) enol acid

Shatavari Liliaceae Rutin, Gas [ 63]

Kaempferol, chromatogra
stigmasterol, phy-mass -
shatavarin IV spectrometry
Aloe vera Liliaceae Colletotrichun , TLC Ethyl acetate : [49]
Gloeosporiddes Methanol
, Fusarium
solani
Garlic Liliaceae Aliin , Albumin , HPLC Methanol : [90]
Volatile oils chloroform
Colchicum Liliaceae Luteolin , Methanol : [75]
Colchicine , GC-MS Chloroform
Colchifoline , 2-
demethylcolchi
cine
Garlic Liliaceae Alliin , Allicin HPLC Heptanslfonic acid : [73]
acetonitrile (1:1)
 Squill Liliaceae Scillaren A , High [77]
Scilliroside , performance
scillaren , gel -
proscillaridin chromatogra
phy

Onion Liliaceae Quercetin -3,4’- HPLC [ 64]

o-diglucoside
(3,4-Qdg);
quercetin -4’-o- -
glucoside(4’-
Qmg).

] Asparagus Liliaceae Shatavrin , HPLC Water and [59]

Stigmasterol acetonitrile
-Hexane :
ethylacetate (9:1)
 Urgenia Liliaceae Tannins ,phenol HPLC Acetonitryl : ethanol [45]
indica s , alkaloids , (85:15)
( Kunth) flavonoids
Lily Liliaceae Steroidal High [87]
saponins , performance -
sterols , gel
polysaccharides chromatogra
, alkaloids , phy
flavonoids
Shallot Liliaceae Flavonoids , Acidified water 1% [61]
onion(Black amino acid and - formic acid and .
onion) organosulfur acetonitrite
compound
Fritillaria Liliaceae Isosteroidal For TLC -ethyl [51]
alkaloids TLC ,GC ,HPL acetate -methanol -
C ammonium
hydroxide
(17:2:1)
Smilax Liliaceae Stilbenes and HPLC Acetonitrile and [41]
Flavonoids water containing
0.02% phosphoric
acid
Crocus sativus Liliaceae Safranal in HPLC 76% acetonitrile in [30]
stigmas of water ,1.0 ml/ min
Crocus sativus
Ruscus Liliaceae Steroidal Liquid Methanol : water [35]
glycoside chromatogra 50 :50
phy
Leeks Liliaceae Carbohydrates , Gas [68]
protein ,vitamin chromatogra -
, cellulose , phy
Minerals
Lilium Liliaceae Kaempferol LC 0.3ml phosphoric [15]
candidum acid in 500 ml
deionized water and
Acetonitril
50% :50% (v/v)
Gloriosa Liliaceae colchicine Over Chloroform : [81]
superba pressured acetone :
layered diethylamine
chromatogra 5:2:1
phy
Allium cepa Liliaceae Quercetin HPLC A)0.1% formic acid in [47]
glycoside ,3,4’- water and B) 0.1%
diglucoside and formic acid in
quercetin 4’ - methanol
monoglucoside
Tulip Liliaceae Abscisic acid Gas liquid Isopropyl alcohol : [74]
 chromatogra ammonium
phy hydroxide : distilled
water
10:1:1(v/v/v)
Aloe vera Liliaceae Aloin A and HPLC Water: Acetic acid [67]
Aloin B (98:2)
Angelica Umbellifer Polysaccharides TLC Toluene: ethyl [28]
(Angelica ae , acetate :acetic acid
sinensis) /apiaceae Ferulic acid, (90:10:2)
ligustilide

Asafoetida Umbellifer Ferulic acid TLC Toluene: ethyl [62]

(Hing) ae acetate :formic acid
(Ferula /apiaceae (60:40:10)
Asafoetida)
Anise Umbellifer Flavonoids HPLC Water:acetonitrile [33]
(Pimpinella ae (95%:5%)
anisum) /apiaceae
Azwain Umbellifer Thymol,P- HPTLC Hexane:diethyl ether [54]
(Trachysperm ae cymene, (8:2 v/v)
um ammi ) /apiaceae Gama terpinine
Carrot Umbellifer Alpha-carotene TLC n-hexane [72]
(Dacus ae Beta-carotene :benxotrifluoride
carota) /apiaceae :acetonitrile
(10:3.5:6.5)
Chervil Umbellifer Deoxypodophyll HSCCC n-hexane [52]
(Anthriscus ae otoxin (High speed :ethyl acetate
Sylvestris) /Apiaceae counter :metanol:water
current (7:3:5:5 v/v)
Chromatogra
phy)
Caraway Umbellifer Carvone, GS-MS 0.1%formic acid in [38]
(Carum carvi) ae Limonene water and
/Apiaceae 0.1%formic acid in
acetonitrile
Cumin Umbellifer Cuminaldehyde, HPTLC Hexane:diethyl ether [54]
(Cuminum ae Limonene, (8:2 v/v)
cyminum ) /Apiaceae Alpha and beta
pinene
Cow porshnip Umbellifer Coumarin, TLC n-hexane:water [48]
(Genus) ae sphondin, ( 100:0 to 0:100 )
(Heracleum /Apiaceae Xanthotoxin,
Lasiopetalum) pimpinellin,
Candinoside A-
D
Dill Umbellifer Umbelliprenin, Column C6H6 : Ethanol (9:1) [32]
(Anethum ae 7- chromatogra
graveolens) /Apiaceae isopentenyloxy phy
Coumarin
Umbelliferone

Funnel Umbellifer Flavonoids- TLC Ethyl acetate:acetic [57]

(Foeniculum ae rutin, acid: formic
vulgare mill) /Apiaceae Anethole acid:water
(100:11:11:26 )
Hogweed Umbellifer Coumarins, HPLC 1% formic acid in [43]
(Heracleum ae Dihydrocoumari water
Dissectum) /Apiaceae n, And 1% formic acid
Appearing in acetonitrile

Parsley Umbellifer Apiin apigenin HPLC Acetonitrile: water [44]

(Petroselinum ae formic acid
Crispum) /apiaceae (99.9: 0.1)
Lovage Umbellifer
Polyacetylene, HPLC Acetic acid [37]
(Levisticum ae Coumarin, : Acetonitrile
Officinale) /ApiaceaeCurcuminoids, ( 4.5% : 50%)
Lignans
Wild celery Umbellifer Flavonoids, TLC Chloroform [46]
( Angelic ae Saponine : methanol
Archangelica) /Apiaceae (80 : 20 )
Aswagandha Solanceae Withanolide, HPLC Acetonitril and [71]
withaferin A methanol ortho
phosphoric acid
Belladona Solanacea Tropane HPLC Water , acetonitrile [70]
e alkaloid 0.3 , trifluroacitic acid
hysocymine ,
atropine ,
scopolamine
solanum Solanacea Solancarpine , TLC Petroleum ether and [8]
e aseculin , chloroform and
cycloartanol; , methanol
aseculatin
Physalis solanceae Physalis , HPLC Hexane , ethyl [17]
flavonoids , acetate , methanopl,
caratinoids dichloro methane
Mandrgo solanceae Flavinoids ,alfa HPLC Hexane and acetone [21]
amylase ,kaemf
erol,luteoline
Stramonium Solanceae Hyoscine ,carbo TLC Acetone, wate,r [2]
hydrates ,atropi ammonia
ne
Jimsonweed Solanceae Spermine TLC CHCl3 and CH3OH [1]
Phenylalanine
atropine
Hysocymus solanceae Hysocymine TLC CHCL3 and CH3OH [6]
Alkaloid 0.5% and NH4OH
Fixed oil
Capsicum solanceae Capsanthin Solvant Ethanol and plant [22]
pepper B- carotene extraction material
Capsourubin
Lutin
Dubosia solanceae Scopolamine HPLC CH3OH- KH2PO4- [84]
Hysocymine D2O
Norhysosyamin
e
 Trigoidine
Valtropine
Angels solanceae Scopalamine Metabolic Methanol [27]
trumplet Hysocyamine Extraction Formic acid ( 1%)
9 gm of powder
Tridax Astereace Quercetin TLC Ethyl acetate: [88]
procumbens a Toluene: Formic acid
4:3.5:0.5
Calendula Asteracea Beta-sitosterol HPLC Methanol: water [42]
officinalis e Flavonoids 70:30
Phenols

Chicory Caffeic Acid HPLC Methanol : Formic [69]

Asteracea Chicoric Acid acid
e Chlorogenic
Acid
Daisy Asteracea Caffeic Acid TLC Chloroform:Methan [56]
e P-coumaric Acid ol
9:1

Zinnia Asteracea Anthocynins, TLC n-butanol:Acetic [55]

e Fatty Acids Acid:Water
Volatile 4:1:2
Constituents
Artichoke Asteracea Phenolic HPLC Ehylacetate:water:A [50]
e compounds, cetic Acid
Flavonoids 10:1:1
Yarrow Asteracea Flavonoids- HPLC TFA in water and in [78]
e Leuteolin-7-o- CH3CN(o.1%)
glycoside
Luteolin-3
Safflower Asteracea Yellow and Red TLC Water:Isobutanol:Et [12]
e Carthamin hanol:Formic Acid
4:7:4:4
Ageratum Asteracea Stigmasterol TLC Chloroform:Ethanol [36]
e Beta-sitosterol 9.8:0.2
Fatty Acid
Lettuce Asteracea 2-Nonynoic HPLC Hexane:Ethanol:Wat [85]
e Acid er
10:1:9
Marigold Asteracea Gallic Acid Toluene : Ethyl [25]
e Caffeic Acid HPTLC Acetate:Formic Acid
Quercetin 13:11:2
P-coumaric Acid
Wormwood Asteracea Polyphenols Acetone:Water: [18]
e Flavonoids HPLC 2:8
Condensed
Tannin
Acacia Legumino Arabin ,D- HPLC Acidified water(5% [7]
ceae Galactose , L acetic acid and
arabinose , L - acetonitril
rhamnose , D-
glucuronic acid
 Asoka Legumino Tannin and HPLC Acetonitril and [14]
ceae Crystalline Ethanol
Glycosides
Balsam of tolu Legumino Benzyle HPLC Journal Association
ceae benzoate ,Cinna Of analytical
mic Chemistry
acid ,Benzoic
acid , Resin
Bavchi Legumino Limonene ,Fixe HPLC Acetonitril and [3]
ceae d Oil ,Psoralen , Methanol
Isopsoralen ,Ba
vachin ,
Isoneobavachal
cone
Red tamarind Legumino Cyaniding -3- HPLC 0.01 % formic acid : [10]
ceae glucoside , 22.5 % methanol:
delphidin,, 50% acetonitril
pelargonidin
Guar gum Legumino Guaran ,Hydroly HPLC Orthophosphoric International
ceae sis galactose acid acetonitrile and Journal of molecular
and mannose glacial acetic acid Sciences ISSN 1433-
0067
2008 By MDPI.
Licorice Legumino Glycyrrhizin , HPLC Methanol and Water Nadine
ceae Glycyrrhetic pinder,Johannes
acid ,Liquiritin , B ,Torsten Hoppe
herniarin 7th Jan,2015.
Pea Legumino Saponin,coumar Column CAN and H2O MA.Sunil,V.S
ceae ins,flavonoids, chromatogra Sunitha ,A .ashita
phytosterols, phy 2nd july 2018.
phytoestrogen
Alfalfa( Medic Legumino Limonene ,Fixe Immunoaffini Acetonitril and [9]
ago sativa ) ceae d oil ,Psoralen, ty methanol
Isopsoralen,Bav chromatogra
achin,Isoneobav phy
achalcone.

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