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Highly Reproducible Detailed cis/trans FAMEs Analysis Ensured by New Optimized Rt-2560 Column Manufacturing and Application-Specific QC Test

By Kristi Sellers and Rebecca Stevens

Restek’s Rt-2560 GC column is commonly used for detailed analysis of fatty acid methyl esters (FAMEs) in foods; for the determination of trans fat content in foods and edible oils; and for profiling of fatty acids in edible oils. The column is well suited to this purpose as it contains a highly polar, nonbonded, biscyanopropyl polysiloxane stationary phase housed in a 100 m x 0.25 mm x 0.20 µm column format. The length and polarity of the column allow it to separate FAMES based on chain length and degree of unsaturation, as well as the positional and geometrical isomerism of double bonds. However, as a consequence of the column’s length and high polarity, even minor variations in the manufacturing process can potentially have detrimental effects on column-to-column reproducibility. Restek has optimized the manufacturing process and implemented a new, application-specific, QC-testing procedure for all new Rt-2560 columns (cat.# 13198) in order to ensure consistent performance for detailed cis/trans FAMEs analysis.

Redesigned Process Produces Consistent Column Performance
The optimized manufacturing process for Rt-2560 columns results in highly consistent performance from one column to the next without affecting important features of the original column, such as selectivity, sample loading capacity, stationary phase bleed, and thermal stability. Figure 1 demonstrates the excellent column-to-column reproducibility that is typically observed across different lots of Rt-2560 polymer. In addition, low bleed levels are consistently obtained, even at the upper temperature limit of 250 °C.

Figure 1: Rt-2560 columns manufactured under Restek’s optimized procedure exhibit highly reproducible column-to-column performance for detailed cis/trans FAMEs analysis.

PeakstR (min)Conc.
(µg/mL)
Structural Nomenclature
1.Methyl butyrate11.9240C4:0
2.Methyl caproate14.1340C6:0
3.Methyl octanoate17.8540C8:0
4.Methyl decanoate22.8040C10:0
5.Methyl undecanoate25.4620C11:0
6.Methyl dodecanoate28.1240C12:0
7.Methyl tridecanoate30.7120C13:0
8.Methyl myristate33.2140C14:0
9.Methyl myristoleate35.2220C14:1 (c9)
10.Methyl pentadecanoate35.6020C15:0
11.Methyl pentadecenoate37.5720C15:1 (c10)
12.Methyl palmitate37.9060C16:0
13.Methyl palmitoleate39.5120C16:1 (c9)
14.Methyl heptadecanoate40.0920C17:0
15.Methyl heptadecenoate41.6620C17:1 (c10)
16.Methyl stearate42.1940C18:0
17.Methyl octadecenoate43.1320C18:1 (t9)
18.Methyl oleate43.5440C18:1 (c9)
19.Methyl linoleaidate44.6020C18:2 (t9,t12)
20.Methyl linoleate45.5020C18:2 (c9,c12)
21.Methyl arachidate46.1240C20:0
22.Methyl linolenate46.9420C18:3 (c6,c9,c12)
23.Methyl eicosenoate47.3920C20:1 (c11)
24.Methyl linolenate 47.7120C18:3 (c9,c12,c15)
25.Methyl heneicosanoate47.9720C21:0
26.Methyl eicosadienoate49.2620C20:2 (c11,c14)
27.Methyl behenate49.7740C22:0
28.Methyl eicosatrienoate 50.6220C20:3 (c8,c11,c14)
29.Methyl erucate50.9820C22:1 (c13)
30.Methyl eicosatrienoate 51.3420C20:3 (c11,c14,c17)
31.Methyl tricosanoate51.4820C23:0
32.Methyl arachidonate51.6620C20:4 (c5,c8,c11,c14)
33.Methyl docosadienoate52.7520C22:2 (c13,c16)
34.Methyl lignocerate53.1640C24:0
35.Methyl eicosapentaenoate53.7720C20:5 (c5,c8,c11,c14,c17)
36.Methyl nervonate54.3320C24:1 (C15)
37.Methyl docosahexaenoate58.4820C22:6 (c4,c7,c10,c13,c16,c19)
Lot-to-Lot Reproducibility of the Rt-2560 Stationary Phase Polymer
GC_FF1263
ColumnRt-2560, 100 m, 0.25 mm ID, 0.20 µm (cat.# 13198)
SampleFood industry FAME mix (cat.# 35077)
Diluent:Hexane/dichloromethane
Conc.:1,000 µg/mL
Injection
Inj. Vol.:1 µL split (split ratio 20:1)
Liner:Premium 4 mm Precision liner w/wool (cat.# 23305.1)
Inj. Temp.:225 °C
Oven
Oven Temp.:100 °C (hold 4 min) to 250 °C at 3 °C/min (hold 30 min)
Carrier GasHe, constant flow
Flow Rate:1.3 mL/min
DetectorFID @ 285 °C
Make-up Gas Flow Rate:30 mL/min
Make-up Gas Type:N2
Hydrogen flow:30 mL/min
Air flow:300 mL/min
Data Rate:50 Hz
InstrumentAgilent 7890B GC

New QC Test Ensures High-Quality, Detailed cis/trans FAMEs Analysis
In order to verify that the optimized process and original process both produce Rt-2560 columns of equivalent performance, a new application-specific QC test was designed and stringent specifications were set. The test mix used in the new QC test contains both saturated and unsaturated FAMEs that are commonly encountered in fats and oils analysis. Because every column manufactured under the improved process is tested, only high-performance columns suitable for detailed cis/trans FAMEs analysis are certified for sale. Table I lists the components of the test mix and some of the chromatographic performance metrics that are calculated for each. Stationary phase bleed is measured at 250 °C following elution of the FAMEs.

Table I: Application-specific components in the new QC test are used to verify the performance of Rt-2560 columns (cat.# 13198) by measuring critical chromatographic performance metrics. Components are listed in elution order.

Test Mix Component

Structural Nomenclature

Performance Metric

Methyl octadecanoate

C18:0

 

Methyl elaidate

C18:1-t9

Retention index

Methyl oleate

C18:1-c9

 

Methyl nonadecanoate

C19:0

 

Methyl eicosanoate

C20:0

Retention factor

Methyl eicosenoate

C20:1-c11

Resolution with methyl linolenate

Methyl linolenate

C18:3-c9,c12,c15

Retention index

Methyl heneicosanoate

C21:0

 

Methyl docosanoate

C22:0

Retention factor and efficiency

Methyl eicosatrienoate

C20:3-c11,c14,c17

Retention index

Methyl tricosanoate

C23:0

 


The retention index values measured for the series of unsaturated FAMES are used to characterize stationary phase selectivity and confirm that each column exhibits the same elution pattern and separation capabilities. These indices are measured using the bracketing provided by homologous saturated FAMEs, rather than by n-alkanes, in order to avoid potential interfacial adsorption effects on the highly polar stationary phase.

Efficiency and retention factor measurements are used to verify proper stationary phase deposition and consistent separation performance. Columns with low efficiency will show significantly reduced performance in the speciation of trans fat, particularly in the separation of the complex C18:1 region, and in the detailed profiling of unsaturated fatty acids.

The baseline resolution of methyl eicosenoate (C20:1-c11) and methyl linolenate (C18:3-c9,c12,c15) is a critical separation required by standard methods, such as AOAC 996.06 and AOCS Ce 1j-07, in order to demonstrate system suitability. Using an optimal column and standard conditions, C18:3-c9, c12, c15 elutes after C20:1-c11 with baseline resolution. If these conditions are met, peaks eluting between C20:0 and C20:1-c11 can be tentatively identified as C18:3 isomers containing at least one trans double bond. In Figure 2, the new QC test is analyzed on columns manufactured using the original and the optimized procedures. This comparison demonstrates that equivalent separation characteristics are obtained both before and after process improvement. In addition, Figure 2 shows baseline resolution of C20:1-c11 and C18:3-c9, c12, c15.

Figure 2: Analysis of the FAMEs QC test mix confirms that Rt-2560 columns from before and after the manufacturing process redesign show equivalent separation characteristics. Baseline resolution of C20:1-c11 and C18:3-c9, c12, c15 as well as cis and trans C18:1-9 isomers is obtained on both columns.

Before Redesign


PeakstR (min)Conc.
(mg/mL)
Structural Nomenclature
1.Methyl octadecanoate11.311.1C18:0
2.Methyl elaidate12.240.8C18:1 (t9)
3.Methyl oleate12.670.6C18:1 (c9)
4.Methyl nonadecanoate13.401.1C19:0
5.Methyl eicosanoate16.131.1C20:0
6.Methyl eicosenoate18.250.6C20:1 (c11)
7.Methyl linolenate18.640.8C18:3 (c9,c12,c15)
8.Methyl heneicosanoate19.701.1C21:0
9.Methyl docosanoate24.391.1C22:0
10.Methyl eicosatrienoate27.930.6C20:3 (c11,c14,c17)
11.Methyl tricosanoate30.541.1C23:0
Unsaturated FAMES QC Test Mix on Rt-2560 Before Manufacturing Process Redesign
GC_EX1134
ColumnRt-2560, 100 m, 0.25 mm ID, 0.20 µm (cat.# 13199)
SampleUnsaturated FAMES QC test mix
Diluent:Hexane
Injection
Inj. Vol.:1 µL split (split ratio 200:1)
Liner:Premium 4 mm Precision liner w/wool (cat.# 23305.1)
Inj. Temp.:250 °C
Oven
Oven Temp.:180 °C (hold 40 min)
Carrier GasH2, constant flow
Flow Rate:2.5 mL/min
DetectorFID @ 275 °C
Make-up Gas Flow Rate:50 mL/min
Make-up Gas Type:N2
Hydrogen flow:40 mL/min
Air flow:400 mL/min
Data Rate:50 Hz
InstrumentAgilent 7890B GC




After Redesign


PeakstR (min)Conc.
(mg/mL)
Structural Nomenclature
1.Methyl octadecanoate11.681.1C18:0
2.Methyl elaidate12.640.8C18:1 (t9)
3.Methyl oleate13.090.6C18:1 (c9)
4.Methyl nonadecanoate13.841.1C19:0
5.Methyl eicosanoate16.661.1C20:0
6.Methyl eicosenoate18.850.6C20:1 (c11)
7.Methyl linolenate19.200.8C18:3 (c9,c12,c15)
8.Methyl heneicosanoate20.351.1C21:0
9.Methyl docosanoate25.171.1C22:0
10.Methyl eicosatrienoate28.790.6C20:3 (c11,c14,c17)
11.Methyl tricosanoate31.491.1C23:0
Unsaturated FAMES QC Test Mix on Rt-2560
GC_QA0099
ColumnRt-2560, 100 m, 0.25 mm ID, 0.20 µm (cat.# 13198)
SampleUnsaturated FAMES QC test mix
Diluent:Hexane
Injection
Inj. Vol.:1 µL split (split ratio 200:1)
Liner:Premium 4 mm Precision liner w/wool (cat.# 23305.1)
Inj. Temp.:250 °C
Oven
Oven Temp.:180 °C (hold 40 min)
Carrier GasH2, constant flow
Flow Rate:2.5 mL/min
DetectorFID @ 275 °C
Make-up Gas Flow Rate:50 mL/min
Make-up Gas Type:N2
Hydrogen flow:40 mL/min
Air flow:400 mL/min
Data Rate:50 Hz
InstrumentAgilent 7890B GC

Conclusion
The improved manufacturing process and application-specific QC test both ensure that Rt-2560 columns provide a consistently high level of performance for detailed cis/trans FAMEs analysis and are suitable for standard methods of fat speciation in food, such as AOAC 996.06 and AOCS Ce 1j-07. Rt-2560 columns produced using the optimized process exhibit the same selectivity expected from a high cyanopropyl column with little column-to-column variability, while the new QC test ensures a high-quality product that meets method performance requirements.