Determination of 37 kinds of

Fatty Acid Methyl Esters (FAME)

by Fully Automated ChroZen GC-PAL RTC
 GC Application

Abstract

There are various ways to intake fatty acids such as edible oils,
processed food, health functional food, etc. Due to the
presence of double bonds, it is required to optimize the sample
preparation to separate various types of fatty acids including
isomers, and gas chromatography is commonly used to
determine fatty acids.

If the fatty acids are injected in an underivatized form, it could

lead to adsorption issues in the column, result in tailing peaks,
and also affect the durability of a gas chromatograph. So it has
to go through the process of the esterification of fatty acids to
fatty acid methyl esters which is very complicated to conduct
manually.

In this study, the fully automated ChroZen GC/PAL RTC enables

to perform the full process of fatty acid methyl esters (FAMEs)
analysis including sample preparation (esterification of fatty
acids), injection, separation, identification, and quantitation
with an unmanned operation. This can reduce human error
causing poor repeatability and data reliability while enhancing
productivity.
 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

Automated ChroZen GC-PAL RTC [Instrument and Software]

Item Description Part No.

Oven ChroZen GC Mainframe Assembly with UPC Detector Board Unit 6701012502

Inlet Capillary Inlet Assembly for ChroZen GC 6701012550

Detector FID Assembly for ChroZen GC 6901012110

Install. kit Start-up kit 6701012590

Column DB-FastFAME (30 m x 0.25 ㎜ x 0.25 ㎛) -

ChroZen PAL RTC system 6501011610

Mounting Kit for ChroZen GC PAL3-Kit-YI6700

PAL Dilutor Option PAL3-DilutorMulti

Autosampler Universal Liquid Syringe Tool x 2 ea PAL3-TH-D757

Vortex Mixer PAL3-VortexMixer

Large Wash Module for 2x 100mL solvent vials and one waste position PAL3-WashLarge

Fast Wash Module for two solvents PAL3-WashFast

YL-Clarity software for single instrument of YL GC 5301011020

CDS
Autosampler control of YL-Clarity 5301011040

ChroZen PAL System Vial 2CV, 1.5mL Clear Glass with Label Vial-1.5-ND9-CG-100

ACC Cap-ND9-St-SP10-
ChroZen PAL System Screw Cap 2CV
100

Fig 1. ChroZen GC-PAL RTC

 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

Solvents/Reagents

1. 1, 4-Dioxane, p.A. (CAS 123-91-1)

2. Methyl tert-butyl ether (MTBE), p.A. (CAS 1634-04-4)

3. Heptane, p.A. (CAS 142-82-5)

4. Dimethylformamide (DMF), p.A. (CAS 68-12-2)

5. Water, p.A. (CAS 7732-18-5)

6. Methanol, p.A. (CAS 67-56-1)

7. Sodium Methoxide p.A., CH3ONa (CAS 124-41-4)

5 g /100 mL methanol (5%), also called sodium methylate, NaOMe

8. Disodium hydrogen citrate, p.A. (CAS 144-33-2) 15 g /100 mL water

9. Methyl tridecanoate (CAS 1731-88-0) _ Internal Standard

10. 37-Component FAME Standard MIX _ Standard material

Fig 2. Dilutor module for 5 different solvents

 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

Methods of Analysis

GC/FID Condition

Column DB-FastFAME (30 m x 0.25 ㎜ x 0.25 ㎛)

Temperature : 250 ℃
Split Ratio : 10 : 1
Inlet
Injection Volume : 1 ㎕
Carrier Gas : He (99.999%)

Oven temperature program:

Oven 50 ℃, 1 min 25 ℃/min to 190 ℃, 1 min
3 ℃/min to 240 ℃, 3 min

Detector (FID) Temperature: 280 ℃

Table 1. ChroZen GC/FID Condition

[Table 1] indicates the analysis condition of GC with FID. ChroZen GC-PAL RTC provides a fully
automated process including the esterification of fatty acids. We also have the video to show the
operational procedure for your better understanding of how the derivatization process works.
[https://youtu.be/pnBIZgMxTA4]

As the sample preparation (esterification of fatty acids) is the critical step for the analysis of
FAMEs, it is required to conduct it while ensuring repeatability as well as reliability.

The following workflow describes all the process step by step to show how the automation
really works in it in detail.
 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

START
Automated workflow for FAME determination

Derivatization
Hardware Check Aspirate Citrate Solution
Check if all required hardware is configured correct Sodium citrate solution is aspirated in the dilutor tubing

Change Tool Dispense Citrate Solution

Get the dilutor tool Dispense sodium citrate solution in the derivatization vial

Prime Dilutor Tubing Heptane Addition

Prime the dilutor tubing with Dioxane Deliver Heptane to the derivatization vial

Dioxane Addition Vortex Vial

Vortex derivatization vial
Deliver Dioxane to the raw sample vial

Change Tool Needle Cleaning

1. Clean outside of dilutor tool needle with wash 2
Get the liquid tool for IS addition 2. Clean outside of dilutor tool needle with wash 1

IS Addition Prime dilutor tubing

Transfer IS solution to the raw sample vial 1. Purge the dilutor tubing with MeOH
2. Purge the dilutor tubing with water

Vortex Vial
Vortex raw sample vial Injection
Change tool
Change Tool Get the tool for the GC injection
Get the dilutor tool

Transfer Wait for timer

Wait for end of phase separation time
Transfer an aliquote of the raw sample with IS to the
derivatization vial

Get sample
Prime Dilutor Tubing Get sample for the injection from the derivatization vial
Purge the dilutor tubing with MeOH

Inject sample
Sodium Methylate Addition Inject sample in the GC and set the injected signal
Transfer NaOMe solution to the derivatization vial

Syringe cleaning (optional)

Vortex Vial Post clean the syringe
Vortex derivatization vial

Prime Dilutor Tubing

1. Purge the dilutor tubing with MeOH
Analysis Run / End of Method
2. Prime the dilutor tubing with Heptane

Table 2. Workflow Diagram of the Automated FAME Transesterification Procedure

 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

Result

Fig 3. 37-Component FAME Standard Mixture Chromatogram

The DB-FastFAME column is dedicated to the analysis of FAMEs and it delivers excellent
separation of 37-component FAMEs within the fast analysis time.

Depending on the type of samples and the matrix, the column can be varied. You can
refer to other application notes for FAME analysis through our website.
[https://eng.youngincm.com/goods/list.php?M2_IDX=18659&SC_ALL=&SC_SC1_IDX=&S
C_SC2_IDX=&SC_SF_IDX=Array&SC_SM_IDX=&SC_TYPE=ALL&SC_WORD=fame&SC_TI_I
DXS=&SC_PA_LEN=&SC_LIST_TYPE=&SC_BOOKMARK=N&SC_ALL=&SC_SC1_IDX=&SC_
SC2_IDX=&SC_LIST_TYPE=SPD]
 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

1. Olive Oil

Fig 4. Chromatogram of FAMEs in Olive Oil

Reten.Time [min] Fatty Acids Response [pA*s] Area [%]

7.643 C13:0 (Internal Standard) 3276.203

9.415 C16:0 978.072 13.1

9.642 C16:1 77.936 1.0
11.192 C18:0 150.574 2.0
11.552 C18:1 cis 5487.796 73.5
11.993 C18:2 cis 672.457 9.0
12.655 C18:3n3 40.958 0.5
13.387 C20:0 29.113 0.4
13.747 C20:1 23.46 0.3
16.133 C22:0 8.915 0.1
 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

2. Butter

Fig 5. Chromatogram of FAMEs in Butter

Reten.Time [min] Fatty Acids Response [pA*s] Area [%]

7.615 C13:0 (Internal Standard) 788.534

3.212 C4:0 266.09 1.6

4.287 C6:0 270.435 1.6
5.332 C8:0 254.562 1.5
6.272 C10:0 526.303 3.2
7.148 C12:0 1027.374 6.2
8.155 C14:0 2019.843 12.1
8.367 C14:1 240.41 1.4
8.71 C15:0 190.424 1.1
9.498 C16:0 5051.856 30.3
9.682 C16:1 347.844 2.1
10.198 C17:0 118.636 0.7
10.467 C17:1 57.427 0.3
11.263 C18:1 trans 1739.833 10.4
11.543 C18:1 cis 4138.976 24.8
11.993 C18:2 cis 331.845 2.0
12.685 C18:3n3 81.11 0.5
 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

3. Coconut oil

Fig 6. Chromatogram of FAMEs in Coconut Oil

Reten.Time [min] Fatty Acids Response [pA*s] Area [%]

7.608 C13:0 (Internal Standard) 342.602

4.29 C6:0 92.445 0.5

5.347 C8:0 1295.817 7.1
6.28 C10:0 1109.535 6.0
7.203 C12:0 8684.863 47.3
8.172 C14:0 3506.203 19.1
9.438 C16:0 1711.9 9.3
11.17 C18:0 559.584 3.0
11.462 C18:1 cis 1127.404 6.1
11.96 C18:2 cis 276.498 1.5
 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

4. Omega-3

Fig 7. Chromatogram of FAMEs in Omega-3

Reten.Time [min] Fatty Acids Response [pA*s] Area [%]

7.602 C13:0 (Internal Standard) 245.81

5.333 C8:0 6.816 0.1

6.268 C10:0 6.696 0.0
7.133 C12:0 51.583 0.4
8.11 C14:0 29.396 0.2
9.38 C16:0 56.897 0.4
9.633 C16:1 37.399 0.3
10.128 C17:0 14.988 0.1
10.415 C17:1 73.242 0.5
11.112 C18:0 38.857 0.3
11.47 C18:1 cis 19.977 0.1
11.93 C18:2 cis 25.623 0.2
12.65 C18:3n3 21.443 0.2
13.483 C20:0 24.641 0.2
13.778 C20:1 102.304 0.8
14.493 C20:2 36.967 0.3
15.13 C20:04n6 254.451 1.9
15.388 C20:03n3 12.572 0.1
16.28 C20:5n3(EPA) 7518.743 55.4
16.603 C22:1 534.694 3.9
17.743 C23:0 317.411 2.3
19.383 C24:0 684.375 5.0
19.73 C22:6(DHA) 3700.289 27.3
 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

5. Krill

Fig 8. Chromatogram of FAMEs in Krill

Reten.Time [min] Fatty Acids Response [pA*s] Area [%]

7.602 C13:0 (Internal Standard) 247.743

7.132 C12:0 26.835 0.6

8.127 C14:0 562.273 12.3
8.415 C14:1 11.865 0.3
8.697 C15:0 22.074 0.5
9.42 C16:0 1095.426 24.0
9.653 C16:1 337.827 7.4
10.172 C17:0 130.907 2.9
10.417 C17:1 19.789 0.4
11.125 C18:0 68.526 1.5
11.43 C18:1 cis 575.103 12.6
11.94 C18:2 cis 100.432 2.2
12.267 C18:3n6 9.289 0.2
12.652 C18:3n3 56.058 1.2
13.745 C20:1 43.264 0.9
15.1 C20:04n6 26.652 0.6
16.088 C22:0 927.398 20.3
17.697 C23:0 31.389 0.7
19.272 C24:0 30.265 0.7
19.558 C24:1 491.753 10.8
 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

6. Sesame Oil

Fig 9. Chromatogram of FAMEs in Sesame Oil

Reten.Time [min] Fatty Acids Response [pA*s] Area [%]

7.6 C13:0 (Internal Standard) 240.418

7.132 C12:0 7.222 0.1

8.108 C14:0 17.522 0.2
9.412 C16:0 876.756 8.6
9.637 C16:1 15.87 0.2
10.177 C17:0 6.407 0.1
11.21 C18:0 488.127 4.8
11.527 C18:1 cis 4031.928 39.8
12.092 C18:2 cis 4444.905 43.8
12.672 C18:3n3 125.763 1.2
13.397 C20:0 51.62 0.5
13.75 C20:1 26.095 0.3
15.29 C20:03n3 9.232 0.1
16.017 C22:0 22.434 0.2
9.503 C24:1 15.228 0.2
 Determination of 37 kinds of Fatty Acid Methyl Esters (FAME) by Fully Automated ChroZen GC-PAL RTC

Conclusion Reference

The analysis of FAMEs (Fatty Acid Methyl Esters) 1. 2.1.5.4 Fatty Acids_Standard Test
requires the optimized sample preparation
Methods_Korean Ministry of Food and
procedure because it could lead to adsorption
Drug Safety
issues in the column, result in tailing peaks,
and also affect the durability of a gas
2. Analysis of Fatty Acid Methyl Esters
chromatograph when it’s injected in
(37mix) by YL6500 GC _Young In
underivatized form.
Chromass

In this study, the determination of FAMEs was 3. Automated Determination of Fatty Acid

conducted by ChroZen GC-PAL RTC. The entire Methyl Esters_ CTC Analytics
process was performed fully automatically, so
it reduces human error causing poor
repeatability and data reliability while
enhancing productivity regardless of the
sample matrix.

60, Anyangcheondong-ro, Dongan-gu,

This also ensures that the ChroZen GC-PAL RTC
Anyang-si, Gyeonggi-do, 14042, Korea
system is suitable to analyze FAMEs in various
TEL: +82-31-428-8700
samples with high resolution and superior
FAX: +82-31-428-8787
sensitivity.
E-mail: [email protected]
Homepage: www.youngincm.com

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