FACULTY OF FOOD SCIENCE AND

NUTRITION

NT20703 FOOD ANALYSIS AND

INSTRUMENTATION

LABORATORY MANUAL

Name: _______________________________

Matrix No.: ____________________________

Content

Page Number

Lab Report Format .................................................................................................................... 3

Lab 1: Moisture and Ash Content Determination & pH Meter Calibration ....................................... 4

Lab 2: Protein Content Determination ......................................................................................... 6

Lab 3: Fat Content Determination............................................................................................... 8

Lab 4: Crude Fiber Content Determination ................................................................................ 10

Lab 5: Dietary Fiber Determination – Demonstration .................................................................. 11

Lab 6: Spectrophotometer ...................................................................................................... 13

Lab 7: Inductively Coupled Plasma Mass Spectrometry (ICPMS) – Demonstration ......................... 14

Lab 8a: High Performance Liquid Chromatography – Demonstration............................................ 15

Lab 8b: Ultra Performance Liquid Chromatography-Mass Spectroscopy (UPLC-MS) –Demonstration ...
............................................................................................................................................. 16

Lab 9: Gas Chromatography (GC) & Gas Chromatograph Mass Spectroscopy (GC-MS) –Demonstration
............................................................................................................................................. 17

Updated on 23 Sept 2019

2
Lab Report Format
A. Cover page

FACULTY OF FOOD SCIENCE AND NUTRITION

NT20703 FOOD ANALYSIS AND INSTRUMENTATION

TITLE OF LAB

GROUP NUMBER:

DATE (DAY) OF EXPERIMENT:

MEMBER’S NAME MATRIX NO. SIGNATURE

B. Report content:
Proximate analysis
1. Introduction
2. Objectives
3. Materials & Methods
4. Result & Discussion
5. Conclusion
6. Reference

Instrumentation
1. Introduction
-Principle of Instrument
-Components of Instrument
2. Standard Operating Procedure (SOP)
3. Precaution Step
4. Application
5. Reference

*Ensure all references are from a reliable source and according to following format:
Judprasonga, K, Jongjaithet, N, & Chavasit, V. 2016. Comparison of methods for iodine analysis in
foods. Food Chemistry 193, 12-17. [Journal]

Nielsen, S.S. 2010. Food Analysis. 4th Edition. New York: Springer. [Book]

FAO. 2003. Food energy - methods of analysis and conversion factors. [Online]. In
http://www.fao.org/docrep/006/Y5022E/y5022e00.htm#Contents [Website]

3
 Lab 1: Moisture and Ash Content Determination & pH Meter Calibration

Determination of Moisture Content (2 Methods)

First m ethod: Oven Method (Conventional Method - AOAC )

Apparatus:

Oven
Porcelain crucible
Analytical balance

Food Sample (2 – 5g)

Procedures:

1. Weigh blank crucible, a using analytical balance.

2. Place sample (2 – 5g) into the crucible and weigh together with the crucible, b.
3. Put crucible into the oven at 103°C for overnight.
4. Cool crucible in desiccators.
5. Weigh crucible again for the last reading, c.
6. Calculate moisture content using formula.

𝑏𝑏 − 𝑐𝑐
𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐, % = × 100
𝑏𝑏 − 𝑎𝑎

Second M ethod: Moisture Analyzer (Rapid Method)

Apparatus:

Moisture Analyzer
Aluminium plate (suitable with moisture analyzer)

Food Sample (As much as required by the moisture analyzer).

Procedures:

1. Moisture analyzer was turn on.

2. Place the blank aluminium plate onto the moisture analyzer and press TARE button.
3. After TARE, moisture analyzer will automatically open for sample analysis.
4. Place sample, and notice there is +/- sign to indicate whether samples are enough. Place
sample until the sign is in the middle between the + and – sign.
5. Press the start button and moisture analyzer will start to operate and will automatically stop.
Moisture content will be shown on the screen in %.

4
Determination of Ash Content

Apparatus:

Furnace
Bunsen burner
Crucible
Analytical Balance
Tongs

Food Sample (2 – 5g)

Procedures:
1. Weigh blank crucible as a.
2. Weigh food sample into the crucible (2 – 5g) as c.
3. Burn the sample on Bunsen burner until no white smoke is visible.
4. Transfer the crucible into the furnace at 550°C for overnight.
5. Cool the crucible in the desiccators prior to weighing.
6. Weigh the crucible for the final reading as b.
7. Calculate ash content using the formula:

𝑏𝑏 − 𝑎𝑎
𝐴𝐴𝐴𝐴ℎ 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐, % = × 100
𝑐𝑐

pH Meter Calibration

Apparatus:

pH meter
Beakers
Distilled water dispenser
Tissue paper

Food samples in liquid form (enough to immerse electrode), pH standard buffer pH7, 4 & 10.

Procedures:
1. Turn on the pH meter.
2. Open pH electrode cap and rinse with distilled water and dab clean with tissue paper.
3. Switch pH meter to calibration mode.
4. Start with buffer pH7, wait until complete calibration.
5. Complete calibration with pH 4 and/or pH10. Make sure the electrode is rinsed with distilled
water and dab clean with tissue paper before switching pH buffer.
6. Test food sample by switching pH meter to test mode and immerse electrode into the food
sample and wait until it’s READY. Record the reading.
7. Rinse and dab, and cover it back before switch off.

5
 Lab 2: Protein Content Determination

Determination of Crude Protein Content – Kjeldahl Method

Apparatus:

Digestion tube
Digester
Kjeltec 2300
Analytical Balance

Food Samples: 2g for normal protein content, and 0.5g – 1g for high protein content sample.

Chemical reagents:
Concentrated sulphuric acid, catalyst (2 selenium tablet or 10 parts of potassium sulphate and 1 part
copper sulphate).

Procedures:
Digestion process
1. Food samples are weighed accordingly and put into the digestion tube.
2. Add catalyst and sulphuric acid into the digestion tube and heat up using the digester at the
highest heat for 1h 30min or until the solution is clear.
Note: Selenium tablet will form yellowish clear solution while potassium sulphate and
copper sulphate catalyst will form a green-blue clear solution.

Distillation and Titration process.

1. Distillation and titration process will be carried out using instrument KJELTEC 2300. All
procedures and instruction before analysis must be carried out accordingly. Refer to the
following page for the SOP.
2. Note that the distillation process is using 40% NaOH solution, while for titration process 1%
boric acid mixed with indicator solution (methyl red and bromocresol green) will be used. The
entire chemicals reagent will be prepared and kept in the solution tank together with the
KJELTEC instrument.
3. The result will be shown in the KJELTEC in % protein.

6
 KJELTEC 2300 PROCEDURE (PROTEIN ANALYSIS)
On the water pipe and ensure the water
is in continuous flowing
Check 4 containers (1 at instrument, 3
under instrument). If empty, DO NOT
start any analysis and inform the lab
assistant.
On the instrument by pressing the main
switch and wait until the instrument is
in ready status and “SELECT FUNCTION”
is shown.
CLEANING PROCESS
Place the Digestion Tube and lower the
lid.
Press Manual switch (‘Hand’ switch),
choose ‘ADD WATER’ and press Enter
( ) and ‘ON’ will be appeared on
screen. Wait until ‘ON’ disappeared. At
this moment, water will flow into
digestion tube.
Choose ‘STEAM ON’ and press Enter.
Run the process for 3-5 min. To end this
process, press again Enter and ‘ON’ will
disappear.
Run this cleaning process for 3 times
before starting any analysis.
After cleaning, choose ‘ADD RECEIVER’
and press Enter to ensure flow the
receiver until all solution is purple
colour. Container receiver should be
shaken prior to this.
Run the cleaning process after all
analysis has completed. Then, close the
instrument.
After running Blank Test, sample is
ready to be analysed. Change result to
protein and key in the weight of the
sample. Place the digestion tube
containing digested sample, and close
the lid to start the process.
Run the BLANK TEST. For this process,
digested blank will be used. Digested
blank is sulphuric acid + catalyst that
has been digested.
SAMPLE ANALYSIS PROCESS
After blank test, for Recovery, change
‘Result’ to recovery and enter the
weight of Ammonium Iron (II) Sulphate
(0.5XXX). Digestion Tube containing
Ammonium Iron (II) Sulphate is placed
and lid is lowered. Process will begin an
ensure the accepted result is between
99.5% - 100.5%. If fail, the process is
repeated until obtaining the % set.
Close the lid and process will begin and
stop automatically.
Blank Test is performed by placing clean
Digestion Tube and press analysis
button. On the result screen, change to
‘BLANK’ by pressing left and right
button. Enter the sample weight
0.0000g.
7
PROSES RECOVERY
When the instrument is
closed, insert the digestion
tube that contains distilled
water and close the lid. At the
titration part, (add receiver
part), fill distilled water until
the second level of electrode.
CAUTION!!
DO NOT run analysis if your
sample is not digested
completed, that the sample
still contain dark solids and the
liquid is not cleared. The dark
solids will clog and damage the
instrument. To avoid this
condition, ensure the all
sample either liquid or solid is
placed at the bottom of the
digestion tube without sticking
at the wall of the digestion
tube.
Catalyst to be used:
Selenium Tablet – to produce
clear liquid in yellow.
Kjeltabs Cu-3.5 – to produce
clear liquid in blue or blue
greenish.
 Lab 3: Fat Content Determination

Determination of Crude Fat Content – Soxhlet Method

Apparatus:
Soxhtec 2050 and its equipment:
- Extraction cup
- Thimble
- Cotton wool
- Thimble ring
Analytical balance
Oven
Desiccators

Food Sample: 2g

Chemical reagents:
Petroleum Ether, 90mL**handle inside fumehood

Procedures:
1. Weigh blank extraction cup and record as a.
2. Weigh sample, and record near to four decimal points – record as c.
3. Put the sample into the thimble and cover with cotton wool. Attach thimble ring to the thimble.
4. Attach thimble into the Soxhtec 2050.
5. Pour petroleum ether into extraction cup and put into Soxhtec 2050.
6. Start Soxhtec 2050 by pushing the button start and it will start extracting for approximately 1
and a half hour.
7. After extraction, place extraction cup into the oven at 105°C for 30 minutes.
8. Cool extraction cup in desiccators prior to weighing and record weight as b.
9. Calculate fat content using the formula:

𝑏𝑏 − 𝑎𝑎
𝐹𝐹𝐹𝐹𝐹𝐹 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐, % = × 100
𝑐𝑐

8
 Procedure on Soxtec 2050 – Fat Determination

Sample Preparation:
• Solid – Grind
• Semi Solid – Dried within 24hrs.
• Liquid – Must add 1g of Celite 566.
Sample Weight:
• 0 – 20% Fat: 1.5g
• >20% Fat: 1g

Weigh extraction cup and record in g.

Weight sample and put into thimble (label it). Insert defatted cotton and also thimble adapter (thimble rings).

Pour 90mL of Petroleum Ether into the extraction cup.

RUNNING ON SOXTEC 2030

Turn on the tap water and make sure the water is flowing. If not, do not start any process and inform lab asst.

Switch ‘on’ the machine. On button is on the controlling system. (2 switch to be on).

Wait until it’s ready and switch the button hot plate. It will heat up until 135C

Press the up and down button until you can adapt the thimble onto the magnet rings.

Again press the up and down button so that the thimble is bring up and you can put the extraction cup on to the
hot plate.

Slide down the cover and press the start button ( ).

Extraction process will take about 1:30hrs. The process will end without any alarm to inform.

Press the up and down button so that you can take out the extraction cup. The extraction cup should be dried out.

Weigh extraction cup again and use the formula to get fat content.

Warning:
Petroleum Ether is highly volatile. While pouring it into extraction cup, make sure it is done in the fume hood.
Inhaling PE is not good for your health.
Wash the extraction cup after using it. Unclean extraction9 cup may interfere with the extraction process and also
the result. Best, put the extraction cup prior extraction.
 Lab 4: Crude Fiber Content Determination

Determination of Crude Fiber Content

Apparatus:
Fiber bag
Hot plate
Beaker
Glass watch
Glass Rod
Thongs
Crucible
Oven
Furnace
Desiccators

Food Samples: 2g

Reagents: 12.5g/L NaOH, 7mL/L H2SO4, Litmus paper (Red and Blue), Hot water, ice cubes.

Procedures:
1. Weigh blank fiber bag, record as m1.
2. Weigh sample and record as m2.
3. Put the sample into the fiber bag and tied it tightly.
4. While weighing, boil NaOH solution on the hot plate. When it is boiling, put fiber bag into the
NaOH solution. Cover the beaker with the glass watch and put ice cubes upon the glass watch
to create a condensation system. Watch for the ice as it may melt, continues to replace the
ice. This step required 30 minutes upon completion.
5. After 30 minutes, take out fiber bag and wash it with hot water and test with red litmus paper.
Wash until the red litmus paper does not turn to blue.
6. Repeat step 4 and 5 using H2SO4 solution and test with blue litmus paper.
7. After completing, place fiber bag in a crucible and dry with the oven at 105°C overnight.
8. After drying, cool the fiber bag, weigh and record as m3 (with crucible).
9. After that, place fiber bag together with crucible in the furnace (550°C) overnight.
10. The ash of the fiber bag will be cool down and weigh as m4.
11. The crude fiber content calculates as below formula.

(𝑚𝑚3 − 𝑚𝑚1) − (𝑚𝑚4 − 𝑚𝑚5)

𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹, % = × 100%
𝑚𝑚2

Note:
m5 is the blank value for fiber bag. For this, ash a blank fiber bag overnight and substract the
weight of crucible. (m5 = Ashed fiber bag – weight of crucible)

10
 Lab 5: Dietary Fiber Determination - Demonstration

Determination of Dietary Fiber in Food Samples

Apparatus:
Flask (FOSS)
Shaking water bath
Micropipette
Aluminium foil
Measuring cylinder
pH meter
Glass rod/ spatula
Thermometer
Crucible (FOSS glass crucible for dietary fiber)
Foss Fibertec 1023 for filtering aid

Chemical reagents:
Phosphate buffer solution, 0.08M with pH 6.0 (±0.2)
- Prepared by dissolving 1.4g of sodium phosphate anhydrate (Na2HPO4) (or 1.753g dehydrate)
and 9.68g sodium phosphate monobasic monohydrate (NaH2PO4) (or 10.94g dihydrate) in
approximately 700mL distilled water. Dilute to 1L with water. Check pH.

Sodium hydroxide solution 0.275N

- Prepared by dissolving 11.00g NaOH in approx. 700mL of distilled water and dilute to 1L water.

Hydrochloric acid solution 0.325N

- Dilute 325mL of 1.0N HCl to 1L distilled water.

Megazyme Enzymatic Kit

- α-amylase (thermostable), 50uL
- protease, 100uL
- amyloglucosidase, 200uL

Chemicals:
Each flask will need 280mL of 95% ethanol.
10mL of 78% ethanol, 95% ethanol, and acetone for washing purpose.
Celite 545 (Diatomaceous Earth)

Sample preparation:
Samples with high fat needed to be defatted with petroleum ether.
Samples with >50% of carbohydrate needed to sugar extracted with ethanol 78%
Weight of samples needed: 1.000g

11
Procedures:
1. Weigh duplicate 1g samples, accurate to 0.1mg into the flask. Sample weights should be
different by less than 20mg from each other. Add 50mL phosphate buffer to each beaker and
check pH (pH must be 6.0±0.1). Adjust pH if required.
2. Add 50uL α-amylase solution to the flask.
3. Cover beaker with aluminium foil and place in boiling water bath for 30 minutes.
4. Cool solution to room temperature.
5. Adjust to pH 7.5±0.1 by adding 10mL 0.275N NaOH solution. Check pH with a pH meter.
6. Add 100uL of protease.
7. Cover the beaker with aluminium foil and incubate at 60°C with continuous agitation for 30
min.
8. Cool and add 10mL of 0.325N HCl solution to adjust to pH to 4.5 ±0.2. Check pH with a pH
meter.
9. Add 200uL amyloglucosidase, cover and incubate 60°C for 30 min.
10. Add 280mL of 95% ethanol preheated to 60°C and let precipitate form at room temperature.
11. Weigh crucible (blank) and add Celite 545 1g then wet with 78% ethanol using Fibertec
filtering aid machine. Suction is applied when using the Fibertec machine, this is to achieve
even mat of celite.
12. Filter precipitation of fiber by using Fibertec filtering aid. (Procedure are based on the Fibertec
equipment).
13. Residue from the filtering will be wash with three 20mL portions of 78% ethanol, two 10mL
portions of 95% ethanol and two 10mL portions of acetone.
14. Dry crucible overnight in 105°C air oven.
15. Cool in dessicator and weigh. Substract crucible and celite weights to determine the weight of
residue.
16. Analyze residue from one sample of duplicate for protein (using Kjeldahl, N x 6.25 conversion
factor), and one for ashing (5 hours at 525°C furnace).
17. Calculate dietary fiber content using the formula:

Calculations:
The uncorrected average blank residue
(UABR)
Blank protein residue (BPR)
Blank ash residue (BAR)
Corrected blank (CB)
Uncorrected average sample residue (USAR)
Samples protein residue (SPR)
Samples ash residue (SAR)
Corrected samples residue (CSR)
% TDF
= Average blank residue of duplicate blanks
in mg
= UABR x % protein in blank/100
= UABR x % ash in blank/100
= UABR-BPR-BAR
= Average sample residue of duplicate in mg
= USAR x % protein in samples/100
= USAR x % ash in samples / 100
= USAR-SPR-SAR-CB
= 100 x CSR/mg sample

12
 Lab 6: Spectrophotometer

Standard Operating Procedures (SOP)

1. Switch on the spectrophotometer for about 30 minutes before used to allow warm-up Turn on the
associated PC and control software UV WinLab.

2. Choose and select the required method from the Application menu.
Scan: for scanning spectra
Time Drive: for absorbance measurement over time
Wave Prog: for absorbance measurement at a different wavelength
Conc: for concentration from the calibration curve

3. Set the various parameters. Select each tab by clicking on it. Go to Configuration to select the
directory to save method and results. Save the method. Click Set UP. The start button becomes
green indicating that the instrument is ready to use.

4. Depending on the method selected, a blank sample may be required to auto-zero. Insert cuvettes
with blank solutions in the reference and sample cell holders. Click OK to auto-zero.

5. Remove the blank and insert a cuvette containing sample solution in the sample holder. Click OK.
The absorbance reading and wavelength are shown on the display.

6. Remove both cuvettes from the sample compartment. Shut down the software and switch off the
instrument.

13
 Lab 7: Inductively Coupled Plasma Mass Spectrometry (ICPMS) – Demonstration

Standard Operating Procedures (SOP)

1. The sample is digested with acid (e.g nitric acid).

2. Make sure the ICP vacuum is switch on 24 hours before the run.

3. ICP-MS is tuned using tune solution following the guidelines.

4. A standard curve is build using standard solution.

5. The liquid sample is pumped into a nebulizer within spray chamber using the peristaltic pump.

6. The ICP torch is a magnetic field whereby it ionizes the sample.

7. The ions were detected.

14
 Lab 8a: High-Performance Liquid Chromatography – Demonstration

Standard Operating Procedures (SOP)

1. Before running the sample, the purge valve located on pump module is opened by loosening it. This
step is important to remove any residues or contaminants inside the HPLC tubing. This HPLC is purge
for 30 minutes.

2. After that, the purge valve is tightening up.

3. Setting up the methods in the METHOD parameter in the CHEMSTATION.

4. From the METHOD menu, select <Edit Entire Method>.

5. Parameters namely the injection volume, mobile phase, flow rate, temperature, and detectors are
set up.

6. After finish setting up the method, save, and name the method.

7. Next, go to <run sequences> to set up the samples information. The sample vial is placed in the
sample tray.

8. After setting up the HPLC method and sample information, click <Switch Pump On> to initiate the
pump.

9. Injection of the sample can be conducted as all parts are in ready condition.

10. For data analysis, chromatogram showing the retention time and area is obtained.

11. After finish analyzing the sample, flush the HPLC before off the module.

15
 Lab 8b: Ultra Performance Liquid Chromatography-Mass Spectroscopy (UPLC-MS) –
Demonstration

Standard Operating Procedures (SOP)

1. The computer is switch on.

2. The UV detector and HPLC pump are switched on.

3. The LC solution icon is doubled click on the desktop, followed by the HPLC icon.

4. Check if there is sufficient elution solvent. Top up if insufficient. Only HPLC grade solvent is used to
prevent clogging of the system.

5. If HPLC had not been used for a while, air bubbles in the system are needed to purge out. The
knob on the HPLC is turned a quarter, and the purging process will take 10 minutes.

6. Once the purging is finished, the knob is turned back.

7. The column is attached in the correct flow detection.

8. The required method file is opened and builds a new method file. Ensure that the wavelength, flow
rate, elution time, and elution proportion are correct.

9. The baseline is monitored by clicking the Baseline Icon in the program. The tubing connection is
checked to ensure that there is no leakage.

10. Once the baseline is flat, the system is ready for sample injection.

11. The sample is prepared in the appropriate solvent and filtered using an appropriate syringe filter.

12. The sample is filtered into the sample vial and place in the sample tray. Ensure no bubble in the
solution.

13. The Single Run icon is clicked once ready.

14. After the run is finished, the system is flushed with an appropriate solvent for at least 30 minutes.

16
 Lab 9: Gas Chromatography (GC) & Gas Chromatography Mass Spectroscopy (GC-MS) –
Demonstration

Standard Operating Procedures (SOP)

1. During the sample preparation, the sample must be filtered out. The sample must be free from any
particles and moisture.

2. Switch on the GC and place the sample vial at the sample tray.

3. Depending on the method used, the parameters are set up.

4. Click on the start button after all the parameters have been key-in.

5. There are two detectors used in the laboratory which are Flame Ionization Detector (FID) and
Thermal Conductivity Detector (TCD). In the laboratory, TCD is used because it is a universal detector
and has a wide variety of detection of the sample. For FID, it is specific and preferred to hydrocarbon
sample.

6. In the laboratory, the standard heated port (split or splitless) is used starting from autosampler to
the inlet, column, and detector. Split is used for a concentrated sample whereas splitless is used for
diluted sample.

7. The gas used in the laboratory is helium gas, but it might replace with other gas which must be an
inert gas.

8. Finally, chromatogram will be obtained.

9. After the experiment end, “wash” has to be carried out, where the whole system is flushed with
absolute propanol or ethanol. The “wash” procedure also carried out before the experimental start.

17