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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.
Chapter 6: TLC and HPLC
87
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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).
Chapter 6: TLC and HPLC
88
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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.
Colour of spot Rf value Standard Rf value
Colour of Flavonoids
Flavonoids
1 Orange 0.46 0.44 Orange Rutin
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).
Chapter 6: TLC and HPLC
89
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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
1 Orange 0.41 0.44 Orange Rutin
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
1 Orange 0.42 0.44 Orange Rutin
3 yellow 0.89 0.87 Yellow Quercetin
Chapter 6: TLC and HPLC
90
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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
2 Yellow 0.87 0.87 Yellow Quercetin
Spot No.
Colour of spot Rf value Standard Rf value
Colour of flavonoids
Flavonoids
1 Greenish orange 0.42 0.44 Orange Rutin
2 Yellowish brown 0.82 0.86 Yellow Quercetin
Chapter 6: TLC and HPLC
91
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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.
Colour of spot Rf value Standard Rf value
Colour of flavonoids
Flavonoids
1 Orange brown 0.46 0.44 Orange Rutin
2 Yellow 0.85 0.87 Yellow Quercetin
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.
Colour of spot Rf value Standard Rf value
Colour of Flavonoids
Flavonoids
1 Orange 0.48 0.44 Orange Rutin
2 Yellowish green 0.85 0.87 Yellow Quercetin
Table No 55: TLC of Cucumis setosus fruit
Chapter 6: TLC and HPLC
92
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Chapter 6: TLC and HPLC
93
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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.
Chapter 6: TLC and HPLC
94
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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
Chapter 6: TLC and HPLC
95
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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
(Spectral Graph No. 9), by comparing with standard retention time it is observed that the
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
Graph No. 11), by comparing with standard retention time it is observed that the presence
Chapter 6: TLC and HPLC
96
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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
(Spectral Graph No. 13), by comparing with standard retention time it is observed that the
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
Graph No. 15), by comparing with standard retention time it is observed that the presence
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
(Spectral Graph No. 17), by comparing with standard retention time it is observed that the
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
Graph No. 19), by comparing with standard retention time it is observed that the presence
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.
Chapter 6: TLC and HPLC
97
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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 = …. %
Chapter 6: TLC and HPLC
98
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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
Chapter 6: TLC and HPLC
99
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 2: HPLC chromatogram of standard Rutin and quercetin
VWD: Signal A, 298 nm Results
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
Chapter 6: TLC and HPLC
100
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No: 3 HPLC chromatogram of standard Rutin and Quercetin
VWD: Signal A, 298 nm Results
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
Chapter 6: TLC and HPLC
101
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Area % Report
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Cucumis
setosus fruit runs1st
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 4: HPLC chromatogram of Cucumis setosus fruit
VWD: Signal A, 298 nm Results
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
Chapter 6: TLC and HPLC
102
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Cucumis
setosus fruitruns 2nd
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
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
VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height %
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
Chapter 6: TLC and HPLC
103
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 6: HPLC of Lagenaria siceraria fruit VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height %
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
Chapter 6: TLC and HPLC
104
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Lagenaria
siscerariafruit runs2nd.
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 7: HPLC of Lagenaria siceraria fruit
Avg. of Sample area / avg. of std. area × std. conc. / sample conc. × 100
For Rutin: 16068580 /3083388943 × 1/100 × 100 = 0.0052 %
For quercetin: 0
VWD: Signal A, 298 nm Results
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
Chapter 6: TLC and HPLC
105
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Area % Report
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Tricosanthes
tricuspidata fruit run 1st
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
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
Chapter 6: TLC and HPLC
106
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Tricosanthes
tricuspidata fruit runs 2nd
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 9: HPLC of Trichosanthes tricuspidata fruit VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height % 1.353 19777509 46.15 868253 38.76 1.740 9222436 21.52 549187 24.52 2.083 5190338 12.11 449675 20.07 2.657 8665003 20.22 372940 16.65
Totals
42855286 100.00 2240055 100.00
Avg. of Sample area / avg. of std. area × std. conc. / sample conc. × 100
For Rutin: 9271614.5 /3083388943 × 1/100 × 100 = 0.0030 %
For quercetin: 0%
Area % Report
Chapter 6: TLC and HPLC
107
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Coccinia
grandis fruit runs 1st.
P4 - A.dat
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 10: HPLC of Coccinia grandis fruit
VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height % 1.713 14041096 8.35 610456 7.08 2.100 4350931 2.59 456357 5.29 2.637 43853342 26.08 3613888 41.93 4.247 44806216 26.65 498718 5.79 5.733 61100325 36.34 3440212 39.91
Totals
168151910 100.00 8619631 100.00
Chapter 6: TLC and HPLC
108
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Coccinia
grandis fruit runs 2nd.
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 11: HPLC of Coccinia grandis fruit
VWD: Signal A,
298 nm Results
Retention Time Area Area % Height Height %
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
Avg. of Sample area / avg. of std. area × std. conc. / sample conc. × 100
For Rutin: 37314647/3083388943 × 1/100 × 100 = 0.0121 %
For quercetin: 64206569.5/ 5110471667× 1/100 × 100 = 0.0125 %
Chapter 6: TLC and HPLC
109
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Area % Report
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Diplocyclos
palmatus fruit runs first time
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 12: HPLC of Diplocyclos palmatus fruit
VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height % 1.683 21166708 27.13 1025801 21.91 2.007 16138279 20.68 1124235 24.01 2.477 7277489 9.33 821866 17.55 2.627 19227856 24.64 1035082 22.11 5.750 14221666 18.23 675510 14.43
Totals
78031998 100.00 4682494 100.00
Chapter 6: TLC and HPLC
110
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Diplocyclos
palmatus fruit runs 2nd
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 13: HPLC of Diplocyclos palmatus fruit VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height % 1.700 25472319 28.69 1250691 27.85 2.027 21615529 24.35 1652661 36.80 2.520 30467710 34.32 1063355 23.68 5.770 11231801 12.65 524000 11.67
Totals
88787359 100.00 4490707 100.00
Avg. of Sample area / avg. of std. area × std. conc. / sample conc. × 100
For Rutin: 248477837/3083388943 × 1/100 × 100 = 0.0805 %
For quercetin: 12726734 / 5110471667× 1/100 × 100 = 0.0024 %
Chapter 6: TLC and HPLC
111
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Area % Report
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Cucumis
setosus leaf runs1st
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 14: HPLC of Cucumis setosus leaf VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height % 1.787 22007881 9.50 1685112 17.56 1.923 11489633 4.96 1649150 17.18 2.097 18306120 7.90 1799065 18.75 2.500 50878341 21.97 2909026 30.31 4.100 128916822 55.66 1554588 16.20
Totals
231598797 100.00 9596941 100.00
Chapter 6: TLC and HPLC
112
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Cucumis
setosus leaf runs 2nd.
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 15; HPLC of Cucumis setosus leaf
Avg. of Sample area / avg. of std. area × std. conc. / sample conc. × 100
For Rutin: 63711031.5 /3083388943 × 1/100 × 100 = 0.0206 %. For quercetin: 0
VWD: Signal A, 298 nm
Results
Retention Time Area Area % Height Height %
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
Chapter 6: TLC and HPLC
113
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Area % Report
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANETrichosanthes
tricuspidata leaf runs 1st
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 16: HPLC of Trichosanthes tricuspidata leaf
VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height %
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
Chapter 6: TLC and HPLC
114
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Trichosanthes
tricuspidata leaf runs 2nd
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 17: HPLC of Trichosanthes tricuspidata leaf
Avg. of Sample area / avg. of std. area × std. conc. / sample conc. × 100
For Rutin: 27972488 /3083388943 × 1/100 × 100 = 0.0090 % For quercetin: 0
VWD: Signal A, 298 nm Results
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
Chapter 6: TLC and HPLC
115
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Area % Report
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Coccinia
grandis leaf runs 1st
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 18: HPLC of Coccinia grandis leaf
VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height % 1.640 84155020 12.67 6583054 25.20 1.783 49628786 7.47 4154604 15.90 2.133 107400458 16.17 6206005 23.75 2.650 56283329 8.48 2595453 9.93 2.987 30263043 4.56 2292009 8.77 3.290 78273996 11.79 2244658 8.59 3.837 258028853 38.86 2052190 7.85
Totals
664033485 100.00 26127973 100.00
Chapter 6: TLC and HPLC
116
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Coccinia
grandis leaf runs 2nd time
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No19: HPLC of Coccinia grandis leaf
VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height % 1.583 167166698 21.99 8211671 33.92 2.073 136093344 17.90 7999390 33.04 2.570 45031107 5.92 2969822 12.27 3.227 87304409 11.48 2630285 10.86 3.763 324667190 42.70 2397970 9.91
Totals
760262748 100.00 24209138 100.00
Avg. of Sample area / avg. of std. area × std. conc. / sample conc. × 100
For Rutin: 50657218 /3083388943 × 1/100 × 100 = 0.0164 %
For quercetin: 0
Chapter 6: TLC and HPLC
117
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Area % Report
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Diplocyclos
palmatus leaf runs 1st time
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 20: HPLC of Diplocyclos palmatus leaf
VWD: Signal A, 298 nm Results
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
Chapter 6: TLC and HPLC
118
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Diplocyclos
palmatus leaf runs 2nd time
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
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
Avg. of Sample area / avg. of std. area × std. conc. / sample conc. × 100
For Rutin: 17255315.5 /3083388943 × 1/100 × 100 = 0.0055 %
For quercetin: 0
Chapter 6: TLC and HPLC
119
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Area % Report
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Lagenaria
siceraria leaf runs 1st time
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 22: HPLC of Lagenaria siceraria leaf
VWD: Signal A, 298 nm Results
Retention Time Area Area % Height Height % 1.620 73195298 18.75 3365084 26.38 2.100 72919419 18.68 4527518 35.49 2.600 14946128 3.83 1386496 10.87 2.820 36156324 9.26 1589194 12.46 3.280 166711976 42.71 1310667 10.27 7.087 5678145 1.45 299177 2.35 7.450 20709428 5.31 278689 2.18
Totals
390316718 100.00 12756825 100.00
Chapter 6: TLC and HPLC
120
Ph.D. Thesis: Miss Sandhya V. Rodge, 2015
Data File: E:\EzChrome\Enterprise\Projects\Default\Data\DAWANE\Lagenaria
siceraria leaf runs 2nd time
Method: E:\EzChrome\Enterprise\Projects\Default\Method\Routin.met
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
VWD: Signal A, 298 nmRetention Time
Spectral Graph No 23: HPLC of Lagenaria siceraria leaf
Avg. of Sample area / avg. of std. area × std. conc. / sample conc. × 100
For Rutin: 13457825.5/3083388943 × 1/100 × 100 = 0.0043 %. For quercetin: 0
VWD: Signal A, 298 nm Results
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
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