A REPORT ON THE INSTRUMENTAL ANALYSIS

MADE AT PHILIPPINE CENTER FOR POSTHARVEST DEVELOPMENT

AND MECHANIZATION (PHILMECH)

Prepared by:

Mark Aldren M. Feliciano

Janine V. Samelo

Nevah Rizzah Sevilla

Bschem2

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

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

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

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.

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

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

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

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.

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.

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)

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.

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.

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

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.

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.

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

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.

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

Sample holders of UV-Vis

It contains two samples holders- one for the sample and one for the reference material

Automated printout of the results

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

. AFB1 IAC-LC autosampler

Lichrospher 100 column used in the unit.

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

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

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