Analysis of Flavonoids TLC Methods

Analysis of Flavonoids TLC Methods

Antiquity, paper chromatography is widely used for the analysis of flavonoids, but now more methods of analysis that is simple and inexpensive Thin Layer Chromatography (TLC / TLC). TLC's advantages are:

• The process of separation of compounds that are relatively short.
• Method detection reagents enough to spray.
• Can be analyzed in several samples at the same time.

TLC is suitable for initial orientation analysis of plant extracts before continuing to other analysis tools instruments such as HPLC, GC, etc..

Flavonoids have two benzene rings separated by propane and is a derivative of flavone. In general, water-soluble flavonoid compounds. The more brightly colored the conjugated compound. In the plants, flavonoid commonly found in the form of glycosides. The difference in the classification of flavonoids shown by the addition of oxygen, a heterocyclic ring and hydroxyl groups. These groups include catechins, leucoanthocyanidin, flavanones, flavanonol, flavones, anthocyanidins, flavonols, Chalcone, aurone and isoflavones.

There are many kinds of systems solvent / eluent used for the separation of flavonoids using TLC. One example of the results of the methylation or acetylation of flavones and flavonols require nonpolar solvents such as chloroform-methanol (15:1). Being such flavonoid aglycone apigenin, luteolin and quercetin can be separated with chloroform methanol (96:4) or with the same polarity. In general, the mobile phase TLC for flavonoid glycosides is ethyl acetate - formic acid - glacial acetic acid - water (100:11:11:26). If the addition of methyl ethyl ketone (ethyl acetate-methyl ethyl ketone-formic acid-glacial aseta acid - water (50:30:7:3:10), rutin and vitexin-2''-O-ramnosida be separated.

With regard to detection, flavonoids spot on TLC plates produce a yellow-brown spots white background when reacted with iodine vapor.

Flavonoids may appear as dark spots on a green background fluoresce when observed in UV light at 254 nm UV-plates containing fluorescent indicator (such as silica gel F254). If under 365 nm UV light, spot colors depending on the structure of flavonoids, can be yellow, green or blue fluorescent. It would be more clear and intense after being sprayed with the reagent.

Colors can be observed at 365 nm UV light are as follows:

• Quercetin, myricetin, and 3 & 7-O-glycosides: orange-yellow

• kaempferol, isorhamnetin, and 3 & 7-O-glycosides: yellow-green

• Luteolin and 7-O-glycosides: orange

• Apigenin and 7-O-glycosides: yellow-green

Further details regarding the use of reagents natural material products can be seen diartikel Brasseur and Angenot, 1986, p 351.

Ferric chloride in water or ethanol is generally apparition spot on analysis of phenolic compounds will provide a blue-black color on the detection of flavonoids. Similarly, Fast Blue Salt B form a blue or blue-purple.

Here is a list of TLC eluent for the separation of flavonoids on silica gel stationary phase:

Sampel	Eluen
Flavonoid aglycon	EtOAc–Isopropanol–H2O, 100:17:13 EtOAc– Chloroform, 60:40 Chloroform–MeOH, 96:4 Toluene– Chloroform –MeCOMe, 8:5:7 Toluene–HCOOEt–HCOOH, 5:4:1 Toluene–EtOAc–HCOOH, 10:4:1 Toluene–EtOAc–HCOOH, 58:33:9 Toluene–EtCOMe–HCOOH, 18:5:1 Toluene–dioxane–HOAc, 90:25:4
Flavonoid glycoside	n-BuOH–HOAc–H2O, 65:15:25 n-BuOH–HOAc–H2O, 3:1:1 EtOAc–MeOH–H2O, 50:3:10 EtOAc–MeOH–HCOOH–H2O, 50:2:3:6 EtOAc–EtOH–HCOOH–H2O, 100:11:11:26 EtOAc–HCOOH–H2O, 9:1:1 EtOAc–HCOOH–H2O, 6:1:1 EtOAc–HCOOH–H2O, 50:4:10 EtOAc–HCOOH–HOAc–H2O, 100:11:11:26 EtOAc–HCOOH–HOAc–H2O, 25:2:2:4 THF–toluene–HCOOH–H2O, 16:8:2:1 Chloroform –MeCOMe–HCOOH, 50:33:17 Chloroform –EtOAc–MeCOMe, 5:1:4 Chloroform –MeOH–H2O, 65:45:12 Chloroform –MeOH–H2O, 40:10:1 MeCOMe–butanone–HCOOH, 10:7:1 MeOH–butanone–H2O, 8:1:1
Flavonoid glucuronide	EtOAc–Et2O–dioxane–HCOOH–H2O, 30:50:15:3:2 EtOAc–EtCOMe–HCOOH–H2O, 60:35:3:2
Flavanone aglycone	CH2Cl2–HOAc–H2O, 2:1:1
Flavanone glycoside	Chloroform –HOAc, 100:4 Chloroform –MeOH–HOAc, 90:5:5 n-BuOH–HOAc–H2O, 4:1:5 (upper layer) Chalcones EtOAc–hexane, 1:1 Isoflavones CHCl3–MeOH, 92:8 Chloroform –MeOH, 3:1
Isoflavone glycoside	n-BuOH–HOAc–H2O, 4:1:5 (upper layer)
Dihydroflavonol	Chloroform –MeOH–HOAc, 7:1:1
Biflavonoid	Chloroform –MeCOMe–HCOOH, 75:16.5:8.5 Toluene–HCOOEt–HCOOH, 5:4:1
Anthocyanidin dan anthocyanin	EtOAc–HCOOH–2 M HCl, 85:6:9 n-BuOH–HOAc–H2O, 4:1:2 EtCOMe–HCOOEt–HCOOH–H2O, 4:3:1:2 EtOAc–butanone–HCOOH–H2O, 6:3:1:1
Proanthocyanidin	EtOAc–MeOH–H2O, 79:11:10 EtOAc–HCOOH–HOAc–H2O, 30:1.2:0.8:8