Validation Method of Determination Total

Phenol in Guava Leaf and Temu Hitam

Rhizome Extract

Anisa Maulida
(J3L213092)
Supervised by :
Harry Noviardi, MSi

Outline
Background

Summary
Material

and Method
Result and Discussion
Conclusion and Suggestion

Background
Free
Radicals

Validation
Method

Slowed
Determination
Total Phenol :
Folin-Ciocalteu
Method

Antioxidant

Phenol

Purpose
Validates the method of determination total phenol in guava leaf
(Psidium guajava L) and temu hitam rhizome (Curcuma aeroginosa)
by spectrophotometry uv-vis

Summary
Kingdom
Division
Sub division
Class
Ordo
Family
Genus
Spesies

Ph e n o l

Temu Hitam

Guava
: Plantae
: Spermatophyta
: Angiospermae
: Dicotyledonae
: Myrtales
: Myrtaceae
: Psidium
: Psidium guajava L

Kingdom
Divisi
Sub divisi
Kelas
Ordo
Famili
Genus
Spesies

: Plantae
: Magnoliophyta
: Commelinidae
: Liliopsida
: Zingiberales
: Zingiberaceae
: Curcuma
: Curcuma aeruginosa

Flow Chart of Spektrophotometry UV-Vis

Light
Source

Monochromarotor

Cuvette

Data Viewer

Detector

Amplifier

Summary
Validation Method
y

Linearity

An ability of analytical procedure that give

respond directly which are proportional to
the concentration of analyte in the sample

Accuracy
A measure to express the degree of clossness
between the reference value and the value
found.

Precision
A measure to express degree of scatter
(suitability) between a series of
measurement obtained from multiple
samples of the same homogenous sample.

LOD & LOQ

[x]?

LOD is lowest concentration of analyte in

sample that can be detected.
LOQ is lowest concentration of analyte in
sample that can be determined.

Materials and Method

Tools

Spectrophotometer UV-Vis

Glassware

Cuvette

Vortex

Micro Pipette &

Tips

Rotary
Evaporator

Materials

Extracti on

Simplisia guava leaf and

temu hitam rhizome

Sample simplicia : ethanol

technic 96% 1:3

Ethanol technical 96%,

Shake 3 hours

Methanol pro analyst,

Filtered

Folin Ciocalteu 7.5%,

Sodium Hydroxide 1 %,

Gallic Acid.

Residue + ethanol technic

Filtrate evaporated

Extract jelled

Determination of total phenol based Farmakope (2011)

Take 1 mL

Folin-C
7.5%
5 mL

Incubated
In dark room
8 min

NaOH 1%
4 mL

Incubated
In dark room
1 hour

Measure at
wavelength
730 nm

Material and Method

y

LINEARITY

Row standard gallic acid :

5, 15, 30, 50, 70 and 100
mg/L for linear regression
(6x repetition)
0.997.
y (b x)
a=
n
n x.y (x.y)
b=
2
nx (x)

r=

. .
2 ()2 2 ()2

PRECISION
Samples concentration
1000 mg/L
7 repetition
%RSD 2%.
SB
%RSD =
100%
x

Material and Method

LOD & LOQ

ACCURACY
Added standard gallic acid with
final concentration 5, 10, and
15 mg/L
7 repetition
98-102%
%recovery =
[Sample+Std][sample] (mg/L)
100%
[std] (mg/L)

Calculated by statistic
with linear regression
from 6 calibration curve
of standard gallic acid.

(yyi)

n2
3 Sy x
LOD =
b
10 Sy x
LOQ =
b

RESULT AND DISCUSSION

Method of determination total phenol is based on Folin-Ciocalteu
reagent ability to oxidize the hydroxyl group (-OH) in phenol
compound.
Phenolic
compounds
reduce
phosphomolibdatephosphotungstate in Folin-Ciocalteu reagent form a molybdenum
complex colored blue.

Sample + Folin-Ciocalteu

Blue complex

RESULT AND DISCUSSION

Absorbance

LINEARITY
Limit acceptance : 0.997

1.000
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000

y = 0.0007 + 0.009x
R = 0.9998

20

40

60

80

Concentration Gallic Acid (ppm)

100

120

Repetition

y= a + bx

1
2
3
4
5
6

y = 0.018 + 0.008x
y = 0.0007 + 0.009x
y = 0.0046 + 0.0104x
y = 0.0008 + 0.0087x
y = 0.004 + 0.009x
y = -0.001 + 0.007x

RESULT AND DISCUSSION

PRECISION
Limit acceptance : 2%

Sample

Content
(%w/w)

RSD (%)
Interday

Intraday

Guava

36.9

0.60

0.39

Temu
Hitam

5.32

0.97

1.5

RESULT AND DISCUSSION

ACCURACY
Limit acceptance : 98-102%

% Recovery
Sample
5 (mg/L) 10 (mg/L) 15 (mg/L)
Guava

99.81

99.32

99.71

Temu hitam

99.81

100.16

99.60

RESULT AND DISCUSSION

LOD and LOQ

LOD
(mg/L)

Sy

Slope (b)

0.0051
0.0090

1.7

LOQ
(mg/L)

5.7

Conclusion and Suggestion

Conclusion
Due to the result, validation method of determination total
phenol in guava leaf and temu hitam rhizome extract, with
parameter test linearity, precision, accuracy, LOD and LOQ,
have valid and can be used in daily analysis.
Suggestion
Parameter assay validation method can be tested other
parameters, like selectivity, robustness, ruggedness and
stability.

THANKYOU