SPDS SET 2: ASH-01

4.4.3) Reproducibility of results by analyzing same sample at various concentrations: The total withanolides contents were estimated using the same sample at different concentration. The contests were calculated by using only one standard concentration.

The results are tabulated.

4.4.4) Experiment

a) Standard preparation: Refer 4.2.4 – Dilution-3.

b) Sample preparation: The sample solution was prepared by weighing the quantity of the sample shown in the Table in a 250 ml beaker, extracted with 100ml of methanol, boiling on water bath for 10-15 minutes and repeated the procedure 3-4 times till the material is completely extracted or till the extracts turn colourless. Combined all the fractions, concentrated and made up the volume to 50ml with methanol. Filtered through 0.45 microns membrane filter paper.

Table-51: Concentration of the extract

Trial-1 (mg/100ml)

Trial-2 (mg/100ml)

Trial-3 (mg/100ml)

Trial-4 (mg/100ml)

Concentration-1 7.5mg/ml

751.1

753.6

747.4

755.3

Concentration-2 10.0mg/ml

1032.2

935.9

1072.5

1010.6

Concentration-3 15.0mg/ml

1659.6

1627.7

1503.2

1497.2

Concentration-4 20.0mg/ml

1959.2

2158.8

2100.3

2096.0

Concentration-5 25.0mg/ml

2762.4

2449.0

2509.0

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c) Procedure: 20 µ l of each of the standard solution, 20 µ l of each concentration of the

sample solutions were injected and Chromatograms were recorded. There are various

late eluting compounds and hence run time of the sample chromatograms were at least

for 60 minutes. From the peak area withanolides were calculated. RSD was

determined.

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3DJH RI