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Chapter 6: TLC and HPLC

86

Ph.D. Thesis: Miss Sandhya V. Rodge, 2015

THIN LAYER CHROMATOGRAPHY

Thin layer chromatography is the best known technique of plant biochemistry.

TLC is used for preliminary separation and determination of plant constituents. It is

helpful for proper identification, authentication of phytoconstituents. In present study the

TLC was performed to prepare qualitative TLC fingerprinting profile of methanol fruit

and leaf extracts of studied plant parts. Attempt was made to separate flavonoids in

studied plant parts.

TLC fingerprinting is of key importance for standardization of herbal drugs. It is

a powerful and relatively rapid technique to distinguish between chemical classes. A

simple chromatographic technique of quantitative microscopy is an important analytical

technique for powdered drugs, especially when chemical and other methods of evaluation

of crude drug fails to accurate measure of quality.

6.1.1 Preparation of thin layer plates

Thin layer glass plate (5cm×15cm) in size and 0.25 mm thickness was prepared

by spreading aqueous slurry of finely grind solid silica gel. The plates were activated in

an oven at 1100c -1200cfor 30 minutes before sample spotting.

6.1.2 Sample application and chromatogram development

The collected methanol extract was applied as a spot 1cm from edge of the plate

by using capillary tube. The plate was then placed in closed container saturated with

vapors of developing solvents i.e. Ethyl acetate: formic acid: acetic acid: water in volume

ratio 100:11:11:26 as a mobile phase and care was taken to avoid direct contact between

the sample and developer. It shows good separation of components. After developing the

plate up to two third of the length of plate it was removed from container and dried. After

dryness they were spray with flavonoids reagent (1% ethanolic-2-aminoethyl

diphenlyborinte solution) and examined in ultra violet chamber.


87


6.1.3 Observations

TLC chromatogram of in the methanol extracts the spots were characterized by

Rf values and colour obtained under UV light which are tabulated in Table No 46-57.

The methanol extract was selected as it contains flavonoids as a major phytoconstituents

and exhibits more fluorescence spots.

Solvent system: Ethyl acetate: formic acid: acetic acid: water in volume ratio

100:11:11:26. Chamber saturation: 30 minutes. Temperature: 29-33. Detection: UV at

256nm.

6.2.1 Thin layer chromatography of Flavonoids of Coccinia grandis leaf

The thin layer chromatography of methanol leaf extract of Coccinia grandis

shows separation of two flavonoids i.e. rutin and quercetin with distinctive colours. Two

spots were observed with different colour and Rf values determined by measuring the

distance traveled by solvent and distance traveled by the solute. The spot 1with orange

colour and spot 2 shows yellow colour (Fig No 48). The Rf values are 0.42 and 0.85

respectively. It was compared with standard Rf values for the confirmation of the rutin

and quercetin (Table No 46).

Spot No.

Colour of spot

Rf value Standard Rf value Colour of flavonoids

Flavonoids

1 Orange 0.42 0.44 Orange Rutin

2 Yellow 0.85 0.87 yellow Quercetin

Table No 46: TLC of Coccinia grandis leaf

6.2.2 Thin layer chromatography of Flavonoids of Coccinia grandis fruit

The thin layer chromatography of methanol fruit extract of Coccinia grandis

shows separation of two spots with different colours. The spot one with orange colour

(Fig No 48) and Rf value is 0.40 which confirmed as rutin and spot two with greenish

yellow colour and 0.84 Rf value confirmed as quercetin (Table No 47).


88


Spot No.

Colour of spot Rf value Standard Rf value

Colour of Flavonoids

Flavonoids

2 Orange yellowish 0.40 0.44 Orange Rutin

3 Greenish yellow 0.84 0.87 Yellow Quercetin

Table No 47: TLC of Coccinia grandis fruit

6.3.1 Thin layer chromatography Lagenaria siceraria leaf

Thin layer chromatography of methanol leaf extract of Lagenaria siceraria

exhibit the separation of two flavonoids with distinctive colours developed after spray of

1% ethanol 2-aminoethyl and diphenlyborinte solution. The spot 1 with orange colour

and its Rf value is 0.42 confirmed as rutin, spot second with yellowish green colour (Fig

No 49) and Rf value is 0.86 confirmed as quercetin (Table No 48).

Spot No.



Flavonoids


2 Yellowish green 0.86 0.87 Yellow green Quercetin

Table No 48: TLC of Lagenaria siceraria leaf

6.3.2 Thin layer chromatography Lagenaria siceraria fruit

The thin layer chromatography of Lagenaria siceraria fruit extract differentiated in

to two spots with distinct colours. By measuring Rf values it is identified as flavonoids.

The spot 1 with orange colour and Rf value is 0.42 which confirms the presence of rutin.

The spot 2 with yellow colour (Fig No 49) and Rf value is 0.89 confirms quercetin

(Table No 49).


89


Table No 49: TLC of Lagenaria siceraria fruit

6.4.1 Thin layer chromatography of flavonoids of Trichosanthes tricuspidata leaf

TLC of Trichosanthes tricuspidata leaf extract shows two spots with different colours

and Rf values, the spot no 1 indicates orange colour with Rf value 0.43 and spot no. 2

was yellow coloured (Fig No 50) and its Rf is values 0.88. On the basis of Rf values

and colour it is confirmed as rutin and quercetin (Table No 50).

Spot No.

Colour of spot

Rf value Standard Rf value Colour of flavonoids

Flavonoids


2 Yellow 0.88 0.87 Yellow Quercetin

Table No 50: TLC of Trichosanthes tricuspidata leaf

6.4.2 Thin layer chromatography of flavonoids of Trichosanthes tricuspidata fruit

The thin layer chromatography of methanol fruit extract of Trichosanthes tricuspidata

shows separation of two flavonoids with distinctive colour. The first spot with orange

brown colour and Rf value is 0.46 confirmed as rutin, spot no 2 with yellowish green

colour (Fig No 50) and Rf value is 0.85 confirmed quercetin (Table No 51).

Spot No. Colour of spot Rf value Standard Rf value Colour of flavonoids Flavonoids

2 Orange brown 0.47 0.44 Orange Rutin

3 Yellowish green 0.85 0.87 Yellow Quercetin

Table No 51: TLC of Trichosanthes tricuspidata fruit

Spot No.

Colour of spot

Rf value Standard Rf value

Colour of flavonoids Flavonoids


3 yellow 0.89 0.87 Yellow Quercetin


90


6.5.1 Thin layer chromatography of flavonoids of Diplocyclos palmatus leaf

Thin layer chromatography of extract of Diplocyclos palmatus leaf extract shows

separation of two flavonoids with distinctive colours developed after spray of 1% ethanol

2-Aminoethyl biphenyl borinate solution. The spot one with yellowish orange colour and

Rf value is 0.43 confirmed as rutin. The spot no 2 with yellow colour (Fig No 51) and Rf

value is 0.87 confirmed as quercetin (Table No 52).

Table No 52: TLC of Diplocyclos palmatus leaf

6.5.2 Thin layer chromatography of Diplocyclos palmatus fruit

The thin layer chromatography of Diplocyclos palmatus fruit extract exhibit two

spots with distinct colours, by measuring Rf values the identification of flavonoids were

confirmed. The spot no. 1 with greenish orange colour and Rf value is 0.42 confirms

flavonoids rutin, spot no.2 with yellowish brown colour (Fig No 51) and Rf value is

0.88confirms the flavonoids quercetin (Table No 53).

Table No 53: TLC of Diplocyclos palmatus fruit

Spot No.

Colour of spot Rf value

Standard Rf value

Colour of flavonoids

Flavonoids

1 Yellowish orange 0.43 0.44 Orange Rutin


Spot No.



Flavonoids

1 Greenish orange 0.42 0.44 Orange Rutin

2 Yellowish brown 0.82 0.86 Yellow Quercetin


91


6.6.1 Thin layer chromatography of Cucumis setosus leaf

The thin layer chromatography of Cucumis setosus leaf extract exhibit the presence of

two spots with different colours and Rf values. The spot no.1 with orange brown colour

and Rf value is 0.46 confirmed as rutin. The spot no. 2 with yellow colour (Fig No 52)

and Rf value 0.85 confirms as quercetin (Table No 54).

Spot No.



Flavonoids

1 Orange brown 0.46 0.44 Orange Rutin


Table No 54: TLC of Cucumis setosus leaf

6.6.2 Thin layer chromatography of Cucumis setosus fruit

The thin layer chromatography of Cucumis setosus fruit extract shows two spots

with distinct colours. The spot no. 1 with orange colour and Rf value is 0.48 confirms

flavonoids rutin, spot no. 2 with yellowish green colour (Fig No 52) and Rf value is 0.86

confirms flavonoids quercetin (Table No. 55).

Spot No.



Flavonoids


2 Yellowish green 0.85 0.87 Yellow Quercetin

Table No 55: TLC of Cucumis setosus fruit


92



93


Plant name Rutin Quercetin

Plant parts Leaf Fruit Leaf Fruit

Coccinia grandis 0.42 0.40 0.85 0.84

Lagenaria siceraria 0.46 0.42 0.86 0.89

Trichosanthes tricuspidata 0.41 0.47 0.88 0.85

Diplocyclos palmatus 0.43 0.42 0.87 0.82

Cucumis setosus 0.46 0.48 0.85 0.85

Table No 56: TLC of leaf and fruit extract of studied plants

The TLC of present study noticed that the Rf values of leaf are 0.46 and 0.46 which

concludes the presence of rutin was detected in Lagenaria siceraria and Cucumis setosus.

Whereas, in fruit extract the Rf values are 0.47 and 0.48 in Trichosanthes tricuspidata

and Cucumis setosus respectively.

The leaf extract shows 0.88 and 0.87 are the Rf values in Trichosanthes

tricuspidata and Diplocyclos palmatus respectively confirming the presence of quercetin.

While the fruit extract exhibit 0.89 Rf value in Lagenaria siceraria indicating presence of

quercetin (Table No 56).

The TLC of reveals presence of rutin and quercetin in leaf and fruit extract further

it was confirmed HPLC analysis.


94


6.7 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS

The determination of rutin and quercetin the HPLC analysis was performed at

Chemistry Research Laboratory, Deogiri College, Aurangabad.

a) Materials and reagents

Quercetin and rutin were purchased from Sigma-Aldrich, Bangalore, India. Methanol,

water, acetic acid and Acetonitrile were of HPLC grade from SD fine chemicals,

Mumbai, India. All the reagents and chemicals used were of analytical and HPLC grade.

Water (HPLC grade) was obtained from Milli Q RO system.

b) Plant Material

The dried powdered leaf and fruit drug of selected medicinal plants were used for

HPLC analysis.

c) Preparation of sample solution

About 0.10 g of dry plant powder was accurately weighed in a stopper tube, and then

in the stopper tube, 10.0 ml of aqueous methanol (40%) was added and incubated in for

about 24 hrs. After that the tube was sonicated in an ultrasonic bath (Model: USC 100,

Frequency: 50 Hz, Amkette industries Pvt. Ltd, India), for 15 min. The contents of the

tube were filtered through Whatman filter paper No 41 (Merck, India). The filtrate was

used for further analysis.

d) Preparation of standard solution

The standard stock solution (1 mg/ml) of quercetin and rutin were prepared by

dilution in methanol. These stock solutions were stored in light resistant containers and

used for calibration curve of quercetin and rutin.

e) Mobile Phase Preparation

The solutions of Methanol (HPLC Grade), acetonitrile (HPLC grade) and Water

(HPLC Grade) with 1 acetic acid in the ratio of 40:15:45 were prepared and mixed

thereafter it is sonicated.

f) Instrumentation and Chromatographic conditions

HPLC analysis was performed with Aglient technology HPLC system. The analysis was

performed by using Zorbax SB-C18, 250 X 4.6 mm internal diameter with 5 micron


95


particle size column and PDA detector set at 298 nm, in conjunction with a mobile phase

of Methanol, Acetonitrile and Water with 1% acetic acid in the ratio of 40:15:45%(v/v) at

a flow rate of 1.0 ml/min. The run time of samples was 10 minute and the operating

pressure: 131 bar. The separation was performed at ambient temperature. The injection

volume was 20μl.

The methanolic leaf and fruit extracts of selected plants are used for HPLC analysis

for the confirmation of flavonoids which are detected in TLC process. The HPLC

chromatographic pattern corresponding to the methanol extract of selected plants were

monitored at 298 nm is shown in Spectral Graph Nos 4-23.

In the present study, characterization of chemical compounds was performed based on

UV/visible spectrometry, as well as by the comparison of retention times with those of

semi synthesized reference compound Rutin and Quercetin. The retention time of

standard rutin and quercetin is 2.593 and 5.643 respectively. The compound was

confirmed by HPLC with UV spectrometry provided peak information and retention time

in minute.

The retention time of rutin in Cucumis setosus fruit found to be 2.037 (Spectral

Graph No. 5), by comparing with standard retention time it is observed that the presence

amount of rutin in Cucumis setosus fruit having 0.0078 percentage. The quercetin was

not detected. The retention time of rutin in Lagenaria siceraria fruit found to be 2.413

(Spectral Graph No. 7), by comparing with standard retention time it is observed that the

presence amount of rutin in L. siceraria fruit having 0.0052 percentage. The quercetin

was not detected.

The retention time of rutin in Trichosanthes tricuspidata fruit found to be 2.657


presence amount of rutin in T. tricuspidata fruit having 0.0030 percentage. The quercetin

was not detected.

The retention time of rutin in Coccinia grandis fruit found to be 2.503 (Spectral



96


amount of rutin in C. grandis fruit is 0.0078%. The retention time of quercetin is 5.613

(SpectralGraphNo10) showing 0.0125% amount of quercetin.

The retention time of rutin in Diplocyclos palmatus fruit found to be 2.520


presence 0.0805% amount of rutin. Whereas, the retention time of quercetin is 5.770

(Graph No 12). The amount of quercetin found in D. palmatus is about 0.0024%.

The retention time of rutin in Cucumis setosus leaf found to be 2.363 (Spectral


amount of rutin in Cucumis setosus leaf having a 0.0206 percentage. The quercetin was

not detected.

The retention time of rutin in Trichosanthes tricuspidata leaf found to be 2.443


presence amount of rutin in T. tricuspidata leaf having 0.0090 percentages. The quercetin

was not detected.

The retention time of rutin in Coccinia grandis leaf found to be 2.570 (Spectral


amount of rutin in C. grandis leaf has 0.0164 percentage but the quercetin was not found.

The retention time of rutin in Diplocyclos palmatus leaf is 2.607 (Spectral Graph

No. 21), by comparing with standard retention time it is observed that the presence

0.0055 amount of rutin in D. palmatus leaf. The quercetin is absent.

The retention time of rutin in Lagenaria siceraria leaf is 2.657 (Spectral Graph

No.23), by comparing with standard retention time it is observed that the presence of

0.0043 amount of rutin in L. siceraria leaf. The quercetin was absent.

In present study quantification of rutin in methanol extract it is observed that

maximum percentage of rutin is detected in Diplocyclos palmatus fruit and Cucumis

setosus i.e. about 0.0805% and 0.0206% respectively. The notable percentage of rutin

also detected in Coccinia grandis leaf and fruit i.e. 0.0164% and 0.0121% respectively.


97


Trichosanthes tricuspidata leaf and Cucumis setosus fruit has rutin content 0.0090% and

0.0078% respectively. The rutin also detected in Diplocyclos palmatus leaf, Lagenaria

siceraria fruit, L. siceraria leaf and Trichosanthes tricuspidata fruit i.e. 0.0055%,

0.0052%, 0.0043% and 0.0030% respectively. The Coccinia grandis fruit has maximum

percentage of quercetin i.e. 0.0125% and minimum percentage of quercetin 0.0024% is

detected in Diplocyclos palmatus. The flavonoids quercetin is not found in leaf and fruits

of Lagenaria siceraria, Trichosanthes tricuspidata and Cucumis setosus (Table No 57).

Sample Sample Name Flavonoid content in plant at 1mg/ml conc.

Rutin (%) Quercetin (%) 1 C. setosus fruit 0.0078 - 2 L. siceraria fruit 0.0052 - 3 T. tricuspidata fruit 0.0030 - 4 C. grandis fruit 0.0121 0.0125 5 D. palmatus fruit 0.0805 0.0024 6 C. setosus leaf 0.0206 - 7 T. tricuspidata leaf 0.0090 - 8 C. grandis leaf 0.0164 - 9 D. palmatus leaf 0.0055 - 10 L. siceraria leaf 0.0043 -

Table No 57: HPLC analysis of selected pants

STD: Rutin and quercetin: RQ std. p1, RQ std. P2, RQ std. P3

Std. Concentration: 1 mg/ml (dissolve in 100 % HPLC grade methanol)

Sample concentration: 100 mg/ml (dissolve in 100 % HPLC grade methanol)

Rutin and Quercetin present in plant extract calculated as: Avg. of Sample area / avg. of

std. area × std.conc. / sample conc. × 100 = …. %


98


Area % Report

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\standard Rutin

and Quercetin 1st .

Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met

Acquired: 7/31/2014 12:50:07 PM

Printed: 8/1/2014 2:25:36 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

1000

2000

Volts

0

1000

2000

2.42

3

3.01

3

5.44

3

VWD: Signal A, 298 nmRetention Time

Spectral Graph No 1: HPLC chromatogram of standard Rutin and Quercetin

VWD: Signal A, 298 nm Results

Retention Time Area Area % Height Height % 2.423 195526907 23.05 19014963 35.80 3.013 5317694 0.63 419008 0.79 5.443 647442198 76.32 33673089 63.41

Totals

848286799 100.00 53107060 100.00


99


Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\standard Rutin and Quercetin 2nd Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met

Acquired: 7/31/2014 1:03:45 PM Printed: 8/1/2014 2:27:47 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

1000

2000

Volts

0

1000

2000

2.36

0

2.94

7

5.39

3


Spectral Graph No 2: HPLC chromatogram of standard Rutin and quercetin


Retention Time Area Area % Height Height % 2.360 433325943 45.65 40303227 59.25 2.947 12812481 1.35 938404 1.38 5.393 503018933 53.00 26782471 39.37

Totals

949157357 100.00 68024102 100.00


100


Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\standrd Rutin and Quercetin3rd Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met

Acquired: 7/31/2014 1:16:18 PM Printed: 8/1/2014 2:28:26 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

500

1000

1500

Volts

0

500

1000

1500

1.66

0

2.12

7

2.59

3 3.18

0

5.64

3


Spectral Graph No: 3 HPLC chromatogram of standard Rutin and Quercetin


Retention Time Area Area % Height Height % 1.660 3359127 0.49 419115 0.85 2.127 1115550 0.16 125397 0.25 2.593 296163833 43.16 27996884 56.85 3.180 2850593 0.42 344868 0.70 5.643 382680369 55.77 20363553 41.35

Totals

686169472 100.00 49249817 100.00

Avg. of std. rutin area = 3083388943 and avg. of std. Quercetin area = 5110471667


101


Area % Report

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Cucumis

setosus fruit runs1st


Acquired: 7/30/2014 2:55:39 PM

Printed: 8/1/2014 1:34:56 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

20

40

Volts

0

20

40

1.34

3

2.03

72.

227


Spectral Graph No 4: HPLC chromatogram of Cucumis setosus fruit


Retention Time Area Area % Height Height %

1.343 25943834 37.66 844139 48.85

2.037 8008988 11.62 469249 27.15

2.227 34944984 50.72 414789 24.00

Totals

68897806 100.00 1728177 100.00


102



setosus fruitruns 2nd


Acquired: 7/30/2014 3:08:05 PM

Printed: 8/1/2014 1:45:50 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

25

50

75

Volts

0

25

50

75

1.77

3

2.50

3

3.12

0


Spectral Graph No 5: HPLC of Cucumis setosus fruit

Avg. of Sample area / avg. of std. area × std. conc. / sample conc. × 100

For Rutin: 24350097/3083388943 × 1/100 × 100 = 0.0078 % For quercetin: 0



1.773 31458762 40.97 1206449 58.30

2.503 16341109 21.28 511981 24.74

3.120 28985810 37.75 350899 16.96

Totals

76785681 100.00 2069329 100.00


103


Area % Report

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Lagenaria

sicerariafruit runs 1st

.Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met

Acquired: 7/30/2014 3:20:51 PM

Printed: 8/6/2014 2:06:55 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

50

100

150

Volts

0

50

100

150

1.51

31.

713

1.95

7

2.30

02.

620

2.84

0


Spectral Graph No 6: HPLC of Lagenaria siceraria fruit VWD: Signal A, 298 nm Results


1.513 34251977 14.03 2272020 25.18

1.713 15901567 6.51 1581060 17.52

1.957 32137625 13.16 1780333 19.73

2.300 23370690 9.57 1217595 13.49

2.620 7783822 3.19 980779 10.87

2.840 130724890 53.54 1192029 13.21

Totals

244170571 100.00 9023816 100.00


104



siscerariafruit runs2nd.


Acquired: 7/30/2014 3:34:05 PM

Printed: 8/1/2014 1:48:35 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

50

100

150

Volts

0

50

100

150

1.29

71.

503

1.74

3

2.09

02.

413

2.62

7

3.09

3


Spectral Graph No 7: HPLC of Lagenaria siceraria fruit


For Rutin: 16068580 /3083388943 × 1/100 × 100 = 0.0052 %

For quercetin: 0


Retention Time Area Area % Height Height % 1.297 31716118 11.29 2464805 22.30 1.503 23008948 8.19 1800986 16.29 1.743 38273099 13.63 2087417 18.88 2.090 24417802 8.69 1335990 12.09 2.413 8766470 3.12 1076681 9.74 2.627 37014822 13.18 1352446 12.23 3.093 117684455 41.90 935708 8.46

Totals

280881714 100.00 11054033 100.00


105


Area % Report

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Tricosanthes

tricuspidata fruit run 1st


Acquired: 7/30/2014 3:47:11 PM

Printed: 8/7/2014 12:25:45 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

25

50

75

Volts

0

25

50

75

1.18

3

1.59

0

1.93

0

2.48

3


Spectral Graph No 8: HPLC of Trichosanthes tricuspidata fruit VWD: Signal A, 298 nm Results

Retention Time Area Area % Height Height % 1.183 27836694 39.36 1229400 36.74 1.590 14911109 21.08 873401 26.10 1.930 18102337 25.59 657818 19.66 2.483 9878226 13.97 585827 17.51

Totals

70728366 100.00 3346446 100.00


106


Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Tricosanthes

tricuspidata fruit runs 2nd


Acquired: 7/30/2014 4:02:47 PM

Printed: 8/7/2014 12:35:56 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

20

40

Volts

0

20

40

1.35

3

1.74

0

2.08

3

2.65

7


Spectral Graph No 9: HPLC of Trichosanthes tricuspidata fruit VWD: Signal A, 298 nm Results


Totals

42855286 100.00 2240055 100.00


For Rutin: 9271614.5 /3083388943 × 1/100 × 100 = 0.0030 %

For quercetin: 0%

Area % Report


107


Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Coccinia

grandis fruit runs 1st.

P4 - A.dat


Acquired: 7/31/2014 1:28:26 PM

Printed: 8/7/2014 12:41:25 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

100

200

Volts

0

100

200

1.71

3

2.10

0

2.63

7

4.24

7

5.73

3


Spectral Graph No 10: HPLC of Coccinia grandis fruit



Totals

168151910 100.00 8619631 100.00


108



grandis fruit runs 2nd.


Acquired: 7/31/2014 1:41:05 PM

Printed: 8/7/2014 12:42:56 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

100

200

Volts

0

100

200

1.57

3

1.95

0

2.50

3

5.61

3


Spectral Graph No 11: HPLC of Coccinia grandis fruit

VWD: Signal A,

298 nm Results


1.573 11263453 9.77 495759 7.41

1.950 5982308 5.19 373799 5.59

2.503 30775952 26.68 2290210 34.25

5.613 67312814 58.36 3527731 52.75

Totals

115334527 100.00 6687499 100.00


For Rutin: 37314647/3083388943 × 1/100 × 100 = 0.0121 %

For quercetin: 64206569.5/ 5110471667× 1/100 × 100 = 0.0125 %


109


Area % Report

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Diplocyclos

palmatus fruit runs first time


Acquired: 7/31/2014 1:54:59 PM

Printed: 8/7/2014 2:19:20 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

20

40

60

Volts

0

20

40

60

1.68

32.

007

2.47

72.

627

5.75

0


Spectral Graph No 12: HPLC of Diplocyclos palmatus fruit



Totals

78031998 100.00 4682494 100.00


110



palmatus fruit runs 2nd


Acquired: 7/31/2014 2:07:26 PM

Printed: 8/7/2014 2:21:52 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

50

100

Volts

0

50

100

1.70

02.

027

2.52

0

5.77

0


Spectral Graph No 13: HPLC of Diplocyclos palmatus fruit VWD: Signal A, 298 nm Results


Totals

88787359 100.00 4490707 100.00


For Rutin: 248477837/3083388943 × 1/100 × 100 = 0.0805 %

For quercetin: 12726734 / 5110471667× 1/100 × 100 = 0.0024 %


111


Area % Report


setosus leaf runs1st


Acquired: 7/31/2014 2:51:31 PM

Printed: 8/7/2014 2:30:12 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

50

100

150

Volts

0

50

100

150

1.78

71.

923

2.09

7

2.50

0

4.10

0


Spectral Graph No 14: HPLC of Cucumis setosus leaf VWD: Signal A, 298 nm Results


Totals

231598797 100.00 9596941 100.00


112



setosus leaf runs 2nd.


Acquired: 7/31/2014 3:03:31 PM

Printed: 8/7/2014 2:34:09 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

100

200

300

Volts

0

100

200

300

1.64

71.

790

1.96

02.

183

2.36

3

2.82

72.

990

3.23

3

3.98

7

4.41

0

7.12

0


Spectral Graph No 15; HPLC of Cucumis setosus leaf


For Rutin: 63711031.5 /3083388943 × 1/100 × 100 = 0.0206 %. For quercetin: 0

VWD: Signal A, 298 nm

Results


1.647 33694179 4.78 2499826 8.26

1.790 14728686 2.09 2306442 7.62

1.960 30250221 4.29 2624901 8.67

2.183 44433300 6.31 3911486 12.93

2.363 76543722 10.86 4565752 15.09

2.827 66136573 9.39 3857577 12.75

2.990 38974944 5.53 3230675 10.68

3.233 94809727 13.46 2537728 8.39

3.987 61779962 8.77 2177838 7.20

4.410 195880400 27.80 1940676 6.41

7.120 47400556 6.73 609204 2.01

Totals 704632270

100.00 30262105


113


Area % Report

Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANETrichosanthes

tricuspidata leaf runs 1st


Acquired: 7/31/2014 3:16:27 PM

Printed: 8/1/2014 2:10:57 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

100

200Vo

lts

0

100

200

1.22

71.

370

1.51

01.

760

1.95

0

2.39

32.

570

2.81

7

3.44

7

6.71

0


Spectral Graph No 16: HPLC of Trichosanthes tricuspidata leaf



1.227 40100248 6.88 3247524 13.12

1.370 15423991 2.65 2368533 9.57

1.510 26852531 4.61 2398456 9.69

1.760 39329546 6.75 3386874 13.68

1.950 55628351 9.55 3482788 14.07

2.393 57250322 9.82 2992508 12.09

2.570 30906423 5.30 2531218 10.23

2.817 68272912 11.71 2048759 8.28

3.447 213152661 36.57 1787664 7.22

6.710 35875155 6.16 507967 2.05

Total 582792140 100.00 24752291 100.00


114


Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Trichosanthes

tricuspidata leaf runs 2nd


Acquired: 7/31/2014 3:33:28 PM

Printed: 8/1/2014 2:12:51 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

100

200

Volts

0

100

200

1.67

71.

823

2.20

32.

443

2.

727

3.01

33.

300

3.64

33.

877

5.39

3

7.17

3


Spectral Graph No 17: HPLC of Trichosanthes tricuspidata leaf


For Rutin: 27972488 /3083388943 × 1/100 × 100 = 0.0090 % For quercetin: 0


Retention Time Area Area % Height Height % 1.677 36955418 9.09 3160522 17.76 1.823 33893431 8.34 2058097 11.57 2.203 28378443 6.98 2192431 12.32 2.443 25038553 6.16 1863288 10.47 2.727 38631407 9.50 1953521 10.98 3.013 19587245 4.82 1492151 8.39 3.300 27701364 6.81 1346923 7.57 3.643 17604413 4.33 1297527 7.29 3.877 92675800 22.79 1294159 7.27 5.393 53843810 13.24 731329 4.11 7.173 32255321 7.93 404661 2.27

Totals

406565205 100.00 17794609 100.00


115


Area % Report


grandis leaf runs 1st


Acquired: 7/31/2014 3:47:27 PM

Printed: 8/1/2014 2:14:32 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

200

400

Volts

0

200

400

1.64

01.

783

2.13

3

2.65

02.

987

3.29

0

3.83

7


Spectral Graph No 18: HPLC of Coccinia grandis leaf



Totals

664033485 100.00 26127973 100.00


116



grandis leaf runs 2nd time


Acquired: 7/31/2014 4:00:59 PM

Printed: 8/7/2014 2:42:44 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

200

400

Volts

0

200

400

1.58

3

2.07

3

2.57

0

3.22

7

3.76

3


Spectral Graph No19: HPLC of Coccinia grandis leaf



Totals

760262748 100.00 24209138 100.00


For Rutin: 50657218 /3083388943 × 1/100 × 100 = 0.0164 %

For quercetin: 0


117


Area % Report


palmatus leaf runs 1st time


Acquired: 7/31/2014 4:12:57 PM

Printed: 8/7/2014 2:45:11 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

200

400

Volts

0

200

400

1.58

01.

720

2.06

3

2.56

3

3.24

7

3.76

3


Spectral Graph No 20: HPLC of Diplocyclos palmatus leaf


Retention Time Area Area % Height Height % 1.580 60828179 12.83 4499184 22.67 1.720 35647024 7.52 3261457 16.44 2.063 103610614 21.86 7077381 35.67 2.563 22046218 4.65 1817632 9.16 3.247 59849147 12.63 1674964 8.44 3.763 191994139 40.51 1511628 7.62

Totals

473975321 100.00 19842246 100.00


118



palmatus leaf runs 2nd time


Acquired: 7/31/2014 4:24:51 PM

Printed: 8/7/2014 2:46:24 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

200

400

Volts

0

200

400

1.62

3

2.11

0

2.60

7

3.29

3

3.82

0

7.44

3


Spectral Graph No 21: HPLC of Diplocyclos palmatus leaf VWD: Signal A, 298 nm Results

Retention Time Area Area % Height Height % 1.623 73572350 19.68 3402986 23.08 2.110 95915636 25.65 7180709 48.70 2.607 12464413 3.33 1406381 9.54 3.293 46345765 12.40 1296612 8.79 3.820 132685589 35.49 1174470 7.96 7.443 12891580 3.45 284608 1.93

Totals

373875333 100.00 14745766 100.00


For Rutin: 17255315.5 /3083388943 × 1/100 × 100 = 0.0055 %

For quercetin: 0


119


Area % Report


siceraria leaf runs 1st time


Acquired: 7/31/2014 4:35:17 PM

Printed: 8/1/2014 2:21:23 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

100

200

300

Volts

0

100

200

300

1.62

0

2.10

0

2.60

02.

820

3.28

0

7.08

7

7.45

0


Spectral Graph No 22: HPLC of Lagenaria siceraria leaf



Totals

390316718 100.00 12756825 100.00


120



siceraria leaf runs 2nd time


Acquired: 7/31/2014 4:47:07 PM

Printed: 8/1/2014 2:22:37 PM

Minutes0 1 2 3 4 5 6 7 8 9 10

Volts

0

50

100

150

Volts

0

50

100

150

1.68

31.

813

2.16

3

2.65

7 2.88

0

3.33

3

5.12

3

7.51

3


Spectral Graph No 23: HPLC of Lagenaria siceraria leaf


For Rutin: 13457825.5/3083388943 × 1/100 × 100 = 0.0043 %. For quercetin: 0


Retention Time Area Area % Height Height % 1.683 32300176 9.89 2605387 22.51 1.813 30160893 9.24 2229469 19.26 2.163 44154982 13.52 1845946 15.95 2.657 11969523 3.66 1195585 10.33 2.880 36092503 11.05 1633136 14.11 3.333 94342045 28.89 1154516 9.98 5.123 57813137 17.70 646200 5.58 7.513 19759679 6.05 262577 2.27

Totals

326592938 100.00 11572816 100.00

THIN LAYER CHROMATOGRAPHY - Shodhgangashodhganga.inflibnet.ac.in › bitstream › 10603 › 78504...

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