Determination of Tanninoids. Analytical Pharmacognosy
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1 QUIZ If the manager of a phytopharmaceutical industry wish to buy one chromatographic equipment, which one you recommend, HPLC or TLC densitometer. What are the reasons to support your recommendation.
2 Determination of Tanninoids Analytical Pharmacognosy
3 What is tannin Tannin is a natural product that can convert hide to leather by binding protein and make them resistant to proteolytic enzyme. Its reaction to living tissue named as astringent and is used as the basis for their use in medicine. Chemically tannins is a mixture of polyphenol that is difficult to be separated and crystallized, easily oxidized and polymerized, low solubility in solvent.
4 Principle of the measurement Gallic Acid as reference. Determination based on reaction with phosphotungstomolibdate in solution of Na 2 CO 3. Measurement at 760 nm.
5 Total Phenol Measurement is conducted as that of standard solution.
6 Non adsorbed phenol Sample solution + Casein. Filter the mixture Filtrate + reagent for phenol and measure.
7 Total Tannin Total Tannin= Total phenol Non adsorbed phenol
8 Alternative tannin Determination 1. Principle: Tannin reacts with hide (collagen) to form insoluble product that can be quantified by gravimetric method.
9 Method 1. Boils 2 g in water for 30 minutes. 2. Cool and ad to 250 ml. 3. Filter the solution. 4. First filtrate (50 ml) evaporated to dryness T1. 5. Second filtrate (80 ml) + hide powder (6g) and shake for 60 minutes. Filter (50 ml) and evaporate to dryness T2 6. Prepare control: 80 ml water + 6 g hide filter 50 ml evaporated to dryness (T0) 7. Tannin content (%): [T1 - (T2 -T0)] x 500 W W in gram
10 Titrimetric Method Tannin solution + indigosulphonate Titrate with potassium permanganate. 1 ml 0.1 N KMnO 4 ~ tannin.
11 Determination of Alkaloid What do you know about alkaloid. Please mention plant containing alkaloid and what are they for. How alkaloid is classified. How to detect alkaloid. How to fractionate alkaloid. How to measure the quantity of alkaloid
12 Determination of Alkaloid Fractionation Alkaloid rich fraction Back titration using acid-base titration. Titration under non-aqueous system. Spectrophotometric method.
13 Antraquinone What is anthraquinone Please mention plants containing anthraquinone and what are they for. Please describe the characteristic of anthraquinone.
14 Determination of Anthraquinone 0.1 g extract + 10 ml hot water, cool and extract with benzene (10 ml). Water fraction + 5% FeCl3 (10 ml) + HCl 5 ml reflux 10 minutes. Extract with benzene Evaporate the residue and dissolve in 5 ml KOH (5% in MeOH). Measure at 515 nm. Use anthraquinone glycoside as reference.
15 Determination of Anthraquinone Benzene is very poisonous, can it be replaced by other solvent? What the intention of refluxing the extract with FeCl3.
16 ALCOHOL Methanol Ethanol Propylene glycol Propanol Isopropanol Glicerol Sorbitol Mannitol
17 Determination of Ethanol Determination by GLC The separation at o C. Number of plate: > 700, calculated based on the peak of n-propanol. Resolution of ethanol-propanol > 2.
18 Alternative method HPLC very simple Spectrophotometry
19 Test for fats and fatty oils Relative density Refractive index Melting range Congealing point of fatty acid Acid value Saponification value Hydroxyl value Iodine value
20 Test for fats and fatty oils Peroxide value Heating test Foreign matter Water and volatile matter
21 Carbohydrate (Ref Chaplin and Kennedy) What is carbohydrate Can you give at least 10 examples of carbohydrate. How to classify carbohydrate What is the physical and chemical characteristic of carbohydrate What is their use in pharmacy Are they pharmacologically active compound?
22 Analysis How to identify the presence of carbohydrate How to separate carbohydrate How to measure the amount or quantity of carbohydrate compounds
23 Total sugar assay Sensitivity 0-20 μg in 0.2 ml, final volume 1.0 ml. Reagent: orcinol in concentrated sulfuric acid (2 g/l). 0.2 ml samples ml reagent. Heat 80 o C for 15 min and cool rapidly at room temp. Measure the absorbance at 420 nm.
24 Reducing sugar assays Tetrazolium blue assay Sensitivity: 0-10 μg in 0.1 ml, Final volume 1.0 ml. Reagent: 3 vol 0.3 M NaOH to 1 vol of tetrazolium blue (1% w/v) and stir to completely dissolved. Dilute with 5 vol. water and store at 5 o C. 0.1 ml samples ml reagent, heat 30 min at 100 oc, cool. Add 1 ml toluene and shake until no more colour extracted from the aqueous layer. Determine at 570 nm
25 Instrumental analysis TLC HPLC
26 Ester What is ester Can you give some examples of ester compound What is their use in pharmacy How to analyse them
27 Fats and fixed oils Waxes
28 Drug containing esters Benzoin (cinnamic, benzoic and their esters) Tolu balsam (resin alcohols combined with cinnamic and benzoic acids) Peru balsam (benzyl cinnamate, cinnamyl-cinnamate).
29 Flavonoid What do you know about flavonoid. Please mention plant containing flavonoid and what are they for. How flavonoid is classified. How to detect the presence of flavonoid. How can you fractionate flavonoid. How to measure the quantity of flavonoid.
30 Determination of flavonoid Principle: Flavonoid hydrolysis aglycone + AlCl 3 in the presence of Hexametilenetetramine.
31 Method Crude drug Hexamethylene tetramine Acetone HCl LLE with EtOAc AlCl3
32 Reference compound: hesperidin or rutin Maximum wave length? Total flavonoid: [(A o x 1.25) sample weight] %
33 Steroid What is steroid Please mentions plants containing steroid and their use in pharmacy. How to detect the presence of steroid. How to measure their quantity
34 Determination of steroid Stock solution: 1 mg sitosterol in ethanol to yield solution containing 5, 10 and 20 μg/ml. 1 g extract in 20 ml EtOH Add tetrazolium (50 mg in 10 ml MeOH). Add tetrametilammonium hydroxide Let react for 90 minutes. Measure absorbance at 525 nm.
35 CYANOGENIC GLYCOSIDE GLUCOSINOLATE Cassava manihotoxin Bitter almond amygdalin Lineseed linamarin Phaseolus lunatus phaseolunatin
36 Test for Cyanogenetic glycoside Well broken crude drug moisten Filter paper treated with picric acid (yellow) sodium isopurpurate (brick-red). Filter paper treated with guaiacum resin in ethanol, CuSO4 solution. blue colour with prissic acid.
37 Quantitative determination Crude drug + Tartaric acid distill Distilled hydrocyanate titrate with Silver nitrate. More sensitive GLC
38 Glucosinolate RC=N-O-SO2.OX S.C6H11O5
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