1. Prepared by:
Mark Aldren M. Feliciano
Janine V. Samelo
Nevah Rizzah Sevilla
Bschem2

2. Introduction to PhilMech
 The Philippine Center for
Postharvest Development and
Mechanization (PHilMech)
formerly Bereau of Postharvest
Research and Extension being
the lead agency of the
Department of Agriculture
engaged in agricultural
mechanization, postharvest
research, development and
extension activities is
continuously developing
appropriate technologies to
empower the agriculture, fishery
and livestock sectors

3. Goals and Vision
 commercialization and utilization of improved
designs of postharvest equipment in the country
 aims to institutionalize a uniform procedure for
technology transfer and/or licensing that equally
serves the ends of intellectual property protection
and technology promotion

4. PHilMech’s Mandate
Generate, extend and commercialize appropriate and
problem-oriented postproduction technologies and
practices to reduce losses, improve food and feed quality
and maximize the benefits to various stakeholders
PHilMech’s Vision
Globally competitive farming and fishing communities
characterized by dynamic and responsive postharvest
technologies
PHilMech’s Mission
To empower farmers and fisherfolks by preserving and
adding value to their produce through dynamic
orchestration, research, technology promotion and policy
advocacy

5. Chemistry Laboratory Services
 The agency as a center for agricultural researches
makes a lot of analyses to insure the safety and
quality of the agricultural goods harvested from a
variety of different crops like corn, mango and banana.
These analyses were conducted at the laboratory
specified for that certain analysis
 The Laboratory Services Division, headed by Mr.
Alexander Joel G. Gibe, PhD, specializes in the
analysis of chlorophyll in leaf samples and
determination of aflatoxin B1 which are both needed to
aid the researches of the agency.

6.  Quantification of aflatoxin, a natural toxin produced
by the agricultural crops especially corn and peanut,
and analysis of chlorophyll in plants is one few of the
analyses that is conducted at the Chemistry
Laboratory.
 These analyses were done by persons who are
specifically trained to perform such analyses.
Specialization

7.  Currently, the agency has two trained personnel;
namely Mrs. Lyn Esteves, a graduate of Bachelor of
Chemistry in University of Sto. Tomas and Mr. Jester
Pangan, who are the only authorized persons to
conduct these analyses.
 Analysis of chlorophyll is conducted by UV-Vis
Spectrophotometry
 determination of aflatoxin utilizes High Performance
Liquid Chromatographic Method
 methods used for each analysis is based from AOAC
Official Methods

8. Duration of an Analysis
 A typical analysis of chlorophyll takes only an hour
to be completed
 aflatoxin takes two days for completion
day 1: preparation extraction of
samples
day 2: analysis and quantification of
aflatoxin

9. Analysis of Chlorophyll in Plants by UV-
Vis Spectrophotometry
 This analysis determines the total chlorophyll, chlorophyll
a, and chlorophyll b in leaf samples and other plant food
samples. In the analysis, chlorophyll is first extracted
using acetone that can take up water. After the
extraction, the solution is clarified and diluted to an
appropriate volume to measure chlorophyll content by
UV-Vis spectroscopy.
 Chlorophyll determinations are carried out in dim light
immediately after preparing the pigment extract solution
because absorption in the red and blue maxima is
highest in freshly isolated chlorophyll and then decreases
with time due to formation of allomeric chlorophyll forms
and possibly destruction of chlorophyll.

10.  During the analysis, aach sample is prepared with three
to five replicates. The appropriate sample sizes for
broccoli, mango, and tomato are 2g, 2g, and 10g
respectively. The extraction is done by adding 100 mg of
CaCO3 to the sample to neutralize plant acids. 30 mL of
100% acetone is then added and the solution is blended
for 3 minutes. The solution is then transferred to a 50 mL
graduated cylinder. The blender is rinsed with another 5
mL, which is also added to the graduated cylinder and
diluted to 30 mL with the solvent. The resulting solution is
transferred in a 50 mL centrifuge tube to be centrifuged
for 5 minutes at 3000 rpm at room temperature.

11.  The clear extract from the previous steps is aliquoted to
a 1 cm cuvette with a pipet to undergo an absorbance
reading against a 100% acetone in a UV –Vis at four
wavelengths.
750 nm (A750=0 for clear extract)
662 nm (chlorophyll a maximum using 100%
acetone)
645 nm (chlorophyll b maximum using 100%
acetone)
520 nm (for extracts from green plant tissues,
A250 should be < 10% of A662)

12.  The following equations are used in determining the
concentrations of chlorophyll a, chlorophyll b, and total
chlorophyll (µg/mL extract solution)
Chlorophyll a (µg/mL) = 11.24 A662 - 2.04 A645
Chlorophyll b (µg/mL) = 20.13 A645 - 4.19 A662
Total Chlorophyll (µg/mL) = 7.05 A662 + 18.09 A645
 The obtained value is multiplied by 30 mL to obtain the
total amount of chlorophyll a and b in the 30 mL extract.

13.  Determination of Aflatoxin B1 in Corn and
Peanut Butter by Immunoaffinity Column-
Liquid Chromatographic Method ( IAC-LC)
with Pre-Column Derivatization
This method is applicable to the determination of
aflatoxins B1 in corn and peanut butter. The aflatoxin is
extracted with methanol-water, purified using an antiboby
specific to aflatoxin, derivatized with trifluoroacetic acid,
separated by reversed-phase liquid chromatography and
detected by fluorescence.
In this analysis, samples are stored in a plastic bag or
container that are analyzed as soon as possible. If the analysis
cannot be done immediately, the samples are placed in the
freezer.

14.  A 25g test portion of corn sample is weighed into a flask.
Five grams of NaCl and 125 mL methanol:water (7+3) is
then added and blended for two minutes in explosion-
proof blender at high speed. Otherwise, shaking for 30
minutes in a wrist-action shaker or similar can can also be
done. Immediately after, the mixture is filtered through a
folded qualitative filter paper then a 15-mL portion of the
filtrate is pipette into 125-mL glass-stopper Erlenmeyer
flask. A 30 mL water is then added and the stopper is
placed then the solution is mixed. Using a glass
microfiber paper, the diluted extract is filtered at most 30
minutes before affinity column chromatography. In this
process, the filtrate should be clear. If not, refiltration
should be done. Immunoaffinity column chromatography
is done immediately

15.  In this process, the immunoaffinity should be let to come to room
temperature before removing the top cap from the column. The tip is
then cut and the column and reservoir is connected in a vacuum
manifold and a 25-mL reservoir is attached.
 A 15-mL second filtrate (equivalent to 1 g test portion) is pipetted into
the reservoir and is let to pass through the column at flow rate of
about 2 drops/second (6 mL/min). An air of 2-3 mL is passed through
the column for about 30 seconds. The column is then washed with
10 mL of water at a flow rate of 6 mL/min twice. Afterwards, a 2 to 3-
mL air is applying through the column to dry it. A milliliter of LC grade
methanol is applied to the reservoir and the solvent is allowed to
permeate the gel before elution for three minutes. Elution of the toxin
from the column into a 2-mL volumetric flask at a flow rate of 2 to 3
mL/min is then done. An additional 2 to 3 mL air is passed through
the column for 30 seconds. Then, the eluate is evaporated to
dryness in block heater set at about 40-45 °C using a gentle stream
of nitrogen. Afterwards, trifluoroacetic acid (TFA) derivatization is
done to increase the signal of the analyte.

16.  TFA derivatization is done by adding 200 µL hexane
and 50 µL TFA to the residue using a calibrated
pipettor, it was vort ex mixed for exactly 30 seconds to
ensure consistent reaction yields. After five minutes,
1.950 mL water:acetonitrile (9+1) is added and is vortex
mixed for 30 seconds. Separation of layer is then
waited for 10 minutes. Subsequently, 20 µL of
derivatized standard solution is injected into the LC
system.

17.  The following conditions should be observed for LC:
Mobile Phase: water:acetonitrile:methanol
(700+170+170)
Flow rate: 1 mL/min
Excitation wavelength: 360 nm
Emission wavelength: 440 nm
Column temp: 30°C

18.  The results were then evaluated by determining the
relative percent difference or RPD (for two values,
standard deviation for 3 or more values) for individual
test sample. The analysis of the test sample is rejected
if the RPD is more than 20% for concentrations equal
or more than 10 ppb and 30% for concentrations equal
of=r greater than 1 ppb but less than 10 ppb. These
values were adopted from the AOAC Peer-Verified
Methods Program Estimated Precision Data as a
Function of Analyte limit of the method (2 ppb) but less
than 10 ppb, an RPD of 30% is acceptable. For
concentrations below the detection limit (2ng/g), RPD
higher than 30% is acceptable. Test samples that do
not satisfy the required RPD are re-analyzed unless
there is a valid reason not to do so.

19. PhilMech’s Instruments
Shimadzu UV-2400 PC UV-Vis
Spectrophotometer
 Connected to a computer with
a preset database
management system that
stores the data of the analysis

20. Sample holders of UV-Vis
 It contains two
samples holders- one for
the sample and one for
the reference material

21.  Automated
printout of the
results

22.  AFB1 IAC-LC
HPLC for aflatoxin
 Uses an autosampler and
manual injection port
 Uses UV-Vis and
Fluorescence detector
 Connected to a computer
and automated readout
printer

23. . AFB1 IAC-LC autosampler

24. Lichrospher 100 column used in the unit.

25. Report sheet of a Determination of Aflatoxin B1 in Corn and Peanut Butter by
Immunoaffinity Column-Liquid Chromatographic Method ( IAC-LC) with Pre-
Column Derivatization

26. Quality Control for the Instruments
 Degassing of HPLC
 Careful handling of instruments to avoid
chemical spills
 Maintained cleanliness in the lab
 Instruments stored in air-conditioned rooms to
avoid dust particles won’t penetrate
 Only trained and authorized personnel are
allowed to enter the laboratory

27. Acquisition of Instruments
1) Approval of request letter
2) Bidding
-Php 500, 000.00 +, public bidding is being made
-less than 500, 000.00, sealed bidding
3) Delivery
4) Inspection, calibration, and instructions for the
authorized users