THE INTERNATIONAL UNIVERSITY (IU) – VIETNAM NATIONAL UNIVERSITY – HCMC

FINAL EXAMINATION – CLASS BTFT18

Date: .....18/06/2021.................................
Duration: 90 minutes
Student ID: BTFTIU18165 Name: HUYNH LE TUYET MY

SUBJECT: FOOD ANALYSIS

For Dean of School of Biotechnology Lecturer
Signature: Signature:

Full name: Dr. Đặng Quốc Tuấn Full name: Dr. Phạm Văn Hùng
GENERAL
INSTRUCTION(S)

1 This is opened book examination

2. No talking during the exam
3. No mobile phone used during the exam
4. No dictionary
GOOD LUCK!

QUESTIONS (please read the question carefully and show your understanding about that)
1. In order to analyze amino acid composition, a researcher should select an appropriate
equipment and know how to analyze the sample. In your case, what do you do? Please write
clearly your opinion. (25 points)
2. In order to analyze fatty acid composition, a researcher should select an appropriate
equipment and know how to analyze the sample. In your case, what do you do? Please write
clearly your opinion. (25 points)
3. A HPLC running condition information as follows. Explain the following information in
detail: Phenolic compounds in whole wheat were quantified using a reverse phase HPLC
procedure employing a Supelcosil LC-18-DB, 150 mm × 3.2 mm column. Isocratic elution
was conducted with 0% to 20% acetonitrile in water in 45 min and then increased to 100%
acetonitrile in 5 min, at a flow rate of 0.6 mL/min. This was delivered using a Waters 515
HPLC pump (Waters Corp., Milford, MA). A Waters 2487 dual wavelength absorbance
detector (Waters Corp.) was used for UV detection of analytes at 280 nm. Data signals were
acquired and processed on a PC running the Waters Millennium software, version 3.2 (1999)
(Waters Corp.) (25 points).
4. Explain what you understand about the following information: The extraction of
volatile aroma compound was carried out using the GC/MS (electron ionisation, EI) on a
GCMS-QP2010 (Shimadzu, Milan, Italy). The GC equipped with a fused silica capillary
column SLB-5MS (5% diphenyl:95% methylsiloxane) 30m × 0.25 i.d. × 0.25mm film
thickness (Supelco, Milan, Italy); carrier gas He at a constant linear rate 30cms-1 (30.6 kPa);
 split/splitless injector port; injector temperature 250oC; injection mode split (split ratio
100:1). The oven temperature programme: 50oC, hold 3min; 3oCmin-1 to 240oC; 15oCmin-1 to
280oC, hold 1min. Data were handled through the use of GCMS-Solution software and the
peak identification was carried out with NIST21,107,147 Library according to a similarity >
90% and other published mass spectra. Identification of componentswas confirmed by
comparison of experimental linear retention indexes with those available in the literature.
GC/MS analysis was carried out in duplicate.
- THE END –

1/ In order to analyze amino acid composition, a researcher should select an appropriate

equipment and know how to analyze the sample. In your case, what do you do? Please
write clearly your opinion.
Principle of analysis
 After hydrolyzing a protein/peptide into its individual amino acid components, the amino
acid compositions are evaluated using a chromatographic technique.
 Separation techniques include ion exchange chromatography, reversed phase liquid
chromatography, and gas–liquid chromatography.
Procedure for Amino Acid Composition analysis
A. Hydrolysis
1. Overnight in 6 M HCl at 100 oC.
2. Enzymes.
B. Separation by HPLC.
Protein Hydrolysis
 The most frequent technique for hydrolyzing a protein sample before amino acid analysis
is acid hydrolysis; 
 Nevertheless, certain amino acids may be eliminated.
 To determine the beginning concentration of partially damaged or slow to cleave amino
acids, a time-course research (i.e., amino acid analysis at acid hydrolysis periods of 24,
48, and 72 hours) is commonly used.
• Free amino acids can be obtained from proteins by strong acid hydrolysis:
Protein  (6N HCl, 100◦C, 24h) amino acids
• 3 of the standard aas are lost during acid hydrolysis treatment:
Asparagine  Aspartic acid
Glutamine  Glutamic acid
 Amides go to acids
Tryptophan  Decomposed
 Hydrolysis Solution: 6 N hydrochloric acid containing 0.1% to 1.0% of phenol.
(phenol is to prevent halogenation of tyrosine)
 Liquid Phase Hydrolysis
a. Place the protein sample in a hydrolysis tube, and dry.
b. Add 200 μL of Hydrolysis Solution per 500 μg of protein.
c. Freeze the sample tube in a dry ice‐acetone bath, and flame seal in vacuum.
d. To avoid oxidation, samples are generally hydrolyzed at 110°C for 24 hours in a vacuum
or inert environment. If there is a worry that the protein is not fully hydrolyzed, longer
hydrolysis durations (e.g., 48 and 72 hours) are examined.
Separation by HPLC
 One of the most frequent procedures for quantitative amino acid analysis is ion
exchange chromatography with postcolumn ninhydrin detection.
 For the more complicated py g h siological samp, les, a Libased cationexchange
system is utilized, and for the more simplistic amino acid mixes, a quicker Nabased
cationexchange system is used.
 On an ion exchange column, amino acid separation is achieved by a combination of
pH and cation strength variations.
 To improve separation, a temperature gradient is frequently used.
 When an amino acid interacts with ninhydrin, the result is a purple or yellow reactant.
 Amino acids, with the exception of imino acids, have a purple hue and have a
maximum absorption wavelength of 570 nm.
 Imino acids like proline have a yellow hue and have a maximum absorption
wavelength of 440 nm.
 At 440 and 570 nm, the reaction between ninhydrin and the amino acid eluted from
the column is measured.
 and the amino acid composition is determined using the chromatogram obtained.
2/ In order to analyze fatty acid composition, a researcher should select an appropriate
equipment and know how to analyze the sample. In your case, what do you do? Please
write clearly your opinion.
Principle of fatty acid composition analysis
 1. The hydrolytic technique is used to remove fat and fatty acids from food.
2. Fatty acids are formed once fat is degraded.
3. To increase volatility, improve peak symmetry, and lower sample activity, fatty acids are
methylated to fatty acid methyl esters (FAMEs).
4. GC measures FAMEs quantitatively.
Procedure for Fatty Acids Analysis
1. Remove the fat.
2. Saponification (hydrolysis under basic condition).
3. Make a methyl ester (CH3ONa). The esterification is done in methanol with a reagent like
boron trifluoride (BF3 in methanol).
4. Methyl ester chromatography
5. Determine the fatty acid peak regions. The retention period of fatty acids is used to identify
them.
6. Compare to the typical response curve.
Procedure:
Step 1: samples infection and vaponration
Step 2: separation in the column
Step 3: detecting and recording result
3/

Pump:
Waters 515 HPLC Pump ( Water Corp, Milford, MA)
Flow rate: 0.6 mL/min Stationary phase (Reversed phase) Employing Supelcosil LC-18-DB
Length of column: 150mm Inner diameter of column: 3.2mm
Mobile phase: isocratic elution plus 20% acetonitrile in water for 45 mins, then plus 100%
acetonitrile in 5 mins
UV Detector: A Waters 2487 applied at wavelength 280nm
Data system: Waters Millennium software, version 3.2 (1999) ( Water Corps)

4/
Method : gas chromatography mass spectrometry, GC-MS
Machine : GCMS QP 2010
Data system: GCMS – solution software
Peak identification: mass spectra Libraies ( NIST 21, 107, 147) and other published mass spectra
Column: SLB- 5MS capillary GC column
Stationary phase: silica
Length: 30m
Inner diameter: 0.25m
Film thickness: 0.25m
Mobile phase: helium gas
Flow rate: 30 cm/s
Pressure: 30.6kPa
Technique: spilt/ split less injection port
Injector temperature : 250 ◦C
Split ratio: 100:1