CBMS 825 Chemical Analysis II: GC-MS Analysis of Fat in Snack Foods

This experiment focuses on analyzing the types and amounts of fat present in snack foods using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The experiment involves the preparation of fatty acid methyl esters (FAMEs) from snack foods like Twisties and Pretzels through a derivatization process. The FAMEs are then analyzed using both GC and GC-MS to identify and quantify different fatty acids. The report includes a comparison of the two methods, calculations of relative response factors (RRF), and statistical analysis to determine significant differences in saturated FAMEs. The limit of detection (LOD) for C12:0 is also calculated for both GC and GC-MS. The results provide insights into the fat composition of the selected snack foods, highlighting the levels of saturated, monounsaturated, and polyunsaturated fats.

Lab Demonstrator- Ben Ford
Presented by:
Abrar Taif (44182562)
CBMS 825 Chemical Analysis II
Analysis of Fat in Snack Foods by Gas Chromatography and
Gas
Chromatography / Mass Spectrometry
Experiment 3

Experimental
Outline• Introduction
• Experimental Principals
• Procedure
• Results and Discussion
• Conclusion
• Learning Outcomes
• References

Introduction
 Snack foods that contain large amount of fat is of great significant health concern
 Saturated fats increases blood cholesterol in humans1
 High LDL level associated with increased risk of heart disease and stroke in humans1
 Perception of Saturated and Unsaturated fats2
 Saturated Fats have single C-C bond2
 Unsaturated Fats have one more more double C=C bond2
 The principles involving the application of gas chromatography (GC) and gas
chromatography/mass spectrometry (GC-MS)
 The investigation of operational differences between flame ionisation detector that
implements gas chromatography and mass spectrometer detector that implements gas
chromatography/mass spectrometer.
 The significance of derivation technique of fatty acid in snacks while applying gas
chromatography (GC)

AIM
 The aim of the experiment is to analyze
types of fat in snack foods by using gas
chromatography and gas chromatography/
mass spectrometry.

Experimental Principals
Flame Ionisation Detector through application of Gas Chromatography FID
 Mechanism:
- Compounds burned in H-air flam.
- C-containing compounds produce
ions →attracted to the collector
- No. of ions hitting the collector is
measured→ signals generated
 Selectivity:
- Compounds with C-H bonds √
- Poor response for other organic
compounds
 Sensitivity: 0.1-10 ng
 Linear range: 105 – 107
 Gases:
- combustion: H & air
- makeup: He & N
 Temperature: 250-300 ̊ C
(400-450 ̊ C for ↑ temp. analysis)

Principle of GC-MS using mass spectrometer detector

Comparison between GC using flame ionisation
detector and GC-MS using mass spectrometer
detector
Gas Chromatogrpahy (GC) Gas Chromatography-Mass
Spectrometer (GC-MS)
Carrier gas. –Hydrogen
(H2)
Ion Source
Injector Injector
Column Electromagnet
Detector (FID Detector (MS at 74 m/z)
Gas Chromatography Mass Spectrometer

Derivatisation technique of fatty
acid when using GC
 For analysis by GC & GC-MS, analyte must be volatile.
 Fat and oil triglycerides: Not very volatile. Large molecules (800-1000 mw).
 Methods for the conversion of fats and oils into fatty acid methyl esters:
 Acid or base-catalysed trans-esterification where reaction with ethanol in the presence of acid or base catalyst directly (boron
trifluoride/methanol) directly convert triglyceride to fatty acid methyl esters. The acid catalyst is the lewis acid BF3.
 Saponification or acid hydrolysis or enzymatic hydrolysis of the triglacyride to releas fatty acid then by acid catalyzed esterification to give
FAMEs.
• TG
hydrolysis
• fatty acid
Derivatisation
• Fatty acid
methyl esters
(FAME)
Analysis by GC or GC-MS

Procedure
Preparation of FAMEs from fats in snack food
 Two snack foods were selected to be analyzed using GC and GC/MS. Twisties (contains 23.3 g/ 100g total fat) and
Pretzels (contains 3.6 g / 100 g total fat).
 Approximately 5 g were crushed using clean mortar and pestle.
 The nutritional information on the packing was examined for both snack foods and the aliquot mass was calculated
using the formula
 Aliquotmass = (15000 / fat in products g/100 g) mg
 Amount of 643.78 mg of Twisties and 4167.7mg of Pretzels were taken after crushing the sample and aliquoting them
 Internal standard (tridodecanoin - C12:0 triglyceride) was added.
 Aa volume of 20 mL of 1:1 (v/v) chloroform / methanol was added and the mixture was ultrasonicate for 5 minutes.
 The snack foods were Filtered from the1:1 (v/v) chloroform / methanol solution using a Buchner funnel and the beaker
was washed with 5 mL of 1:1 (v/v) chloroform / methanol.
 Remove 1 mL of the filtered and place in clean scintillation vial.
 The sample was evaporated to dryness (oil form) by adding 2 mL of BF3/ methanol derivatisation reagent to the vial ,
the vial was caped and the mixture was shook vigorously.
 The mixture was heated to 60 °C in the oven for 10 minutes and shaking of the mixture was maitained every 2 minutes.
 The vial was removed from the oven and left to cool down. Then, 5 mL of hexanes and 5 mL of saturated NaCl were
added to the vial.
 The vial was caped and the mixture was shook vigorously for a minute.
 After separation of the two phases, 1 mL of the top layer was removed using pasture pipette and transfer into clean
vial. This is the sample to be analyzed contains methyl ester derivatives.
 The computer was prepared to collect data and 0.5 mL of the sample was injected into GC and GC/MS.

Measurement of Fatty Acid Response Factor
 The chromatographic conditions of GC-17A (SHIMADZU) and GCMS-QP5000
(SHIMADZU) were confirmed and the computer was prepared to collect data by
follow-up the steps mentioned in experiment 4 manual.
 After the READY light on GC turns green and click sound on GC-MS was noticed,
0.5 μL of FAME standard was injected using 1μL syringe followed by pressing START
button on front of GC. FAME standard 20 ug/mL contains C12:0, C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, C20:0 and C20:1
 Check to see the run starts, wait for approximately 20 minutes to finished.
 When ready light turn green again, inject the sample again (duplicate).

Results and
DiscussionRaw Data from Gas Chromatography
Fig-1 GC of Fame Std 2
Fig 2 -GC of Twisties
Fig 3- GC of Pretzels

Raw Data from Gas Chromatography-Mass Spectrometer
Fig-4- GC-MS of Fame Std 2
Fig-5- GC-MS of Pretzels
Fig-6- GC-MS of Twisties