Paper Chromatography: Whatmann no. 1 impregnated with formamide in propylene glycol.

Solvent system: Toluene: n-butanol saturated with water. (4:1) of (5:1) benzene or Chloroform.

Spray reagent: Raymonds reagent.

TLC: Partition chromatography is more suitable for cardiac glycoside as they are fairly polar in nature.

Adsorbent: Silica gel layer is used.

Solvent system:
- Methylene di chloride: pyridine: water
- Methylene di chloride: methanol : water (87:12:1)
- Ethyl acetate: pyridine: water (5:1:4)

Kedde's reagent (3,5, dinitro benzoic acid in methanol and 2N KOH) is quite selective and sensitive fives blue to violet color.
Anisaldehyde perchloric acid or chloramines tri chloro acetic acid are suitable.

ISOLATION: By Stass Otto Method

Since the drug is soluble in chloroform and water and also in alcohol.
• First macerate powder with aqueous alcohol for a week, filter.
• Filtrate + lead acetate to remove the impurities and filter.
• Filtrate passed hydrogen sulphide gas or sulphuric acid to remove excess of lead acetate.
• Filtrate is extracted with chloroform and concentrate chloroform layer to get total cardiac glycoside.

Cardenolides of cryptostegia madas caricasis F- Asclepediaceae.

The genins and glycosides are separated by column chromatography to get individual glycosides.

Silica gel column: Glycoside is treated with 1 gm celite 545 +4ml formamide, this mixture is stirred and heated at 50-60degrees for 20 minutes and then added to the column. Mobile Phase: Benzene: chloroform (3:1) this mixture is equilibrated with 20 ml of formamide and water.

Lintes column: (cellulose thread): This is used after washing with ethyl acetate, methanol, and water.

Mobile Phase: ethyl acetate is saturated with water.

Kiesulguhr column: Keiselguhr (0.5-0.6 mesh)

Mobile phase: ethyl acetate saturated with water.

1. By colorimetric method: which depends on three types of reactions.
1. keller killani acid ferric chloride (deoxy sugars)
2. alkaline sod picrate (lactose group)
3. m-dinitro benzene (lactone group)

• Standard is dissolved in 95% ethanol.
• Sample obtained from silica gel column with benzene and chloroform solvent system is collected.
• Aliquot of the eluate is transferred to flasks and solvent is evaporated.
• Add 3 ml of sod. Picrate (bal jet) with stirring and maintained at temperature 25 +3 degree C.
• Protect the mixture from intense light.
• After 16 minutes extinction is measured at 495nm.

Digoxin: By m-nitro benzene method:
• Pipette our 5ml of standard solution and 10ml of assay solution into conical flask.
• Evaporate the solvents on water bath and cool.
• Add 5ml of freshly prepared alkaline dinitrobenzene reagent and allow to stand for 5 min at the temperature not exceeding 30 degrees with frequent shaking.
• Determine the extinction at 620nm.
• Measurement is repeated at 1 minute interval until a maximum reading is obtained and compared with that of standard.

2. Modified Keller Killani method:
5ml of sample is treated with acid ferric chloride reagent (60mlglacial acetic acid +5ml conc sulphuric acid +1ml of 9% of ferric chloride solution) stir and allow to stand for 10 minutes, protected from light at a temperature not exceeding 30 degrees. Extinction is measured at 590 nm. Repeat the measurements at 2 min intervals until a maximum reading is obtained.

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