{"id":43541,"date":"2020-10-21T00:00:00","date_gmt":"2020-10-21T00:00:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/highly-reproducible-detailed-cistrans-fames-analysis-ensured-by-new-optimized-rt-2560-column-manufacturing-and-application-specific-qc-test\/"},"modified":"2026-01-28T15:53:58","modified_gmt":"2026-01-28T15:53:58","slug":"highly-reproducible-detailed-cistrans-fames-analysis-ensured-by-new-optimized-rt-2560-column-manufacturing-and-application-specific-qc-test","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/it\/articles-ja\/ffar2589\/highly-reproducible-detailed-cistrans-fames-analysis-ensured-by-new-optimized-rt-2560-column-manufacturing-and-application-specific-qc-test","title":{"rendered":"Highly Reproducible Detailed cis\/trans<\/em> FAMEs Analysis Ensured by New Optimized Rt-2560 Column Manufacturing and Application-Specific QC Test"},"content":{"rendered":"\n

Restek\u2019s Rt-2560 GC column is commonly used for detailed analysis of fatty acid methyl esters (FAMEs) in foods; for the determination of trans<\/em> 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 \u00b5m 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\u2019s 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<\/em> FAMEs analysis.<\/p>\n\n\n\n

Redesigned Process Produces Consistent Column Performance<\/h2>\n\n\n\n

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 \u00b0C.<\/p>\n\n\n

Figure 1:<\/strong>\u00a0Rt-2560 columns manufactured under Restek\u2019s optimized procedure exhibit highly reproducible column-to-column performance for detailed\u00a0cis\/trans<\/em>\u00a0FAMEs analysis.<\/div>
\n
\"Lot-to-Lot<\/div>

GC_FF1263<\/p>

Peaks<\/h4>\n
<\/th>Peaks<\/th>tR<\/sub> (min)<\/th>Conc.
(mg\/mL)<\/th>		Structural Nomenclature<\/th><\/tr><\/thead>\n
1.<\/td>Methyl butyrate<\/a><\/td>11.92<\/td>40<\/td>C4:0<\/td><\/tr>\n
2.<\/td>Methyl caproate<\/a><\/td>14.13<\/td>40<\/td>C6:0<\/td><\/tr>\n
3.<\/td>Methyl octanoate<\/a><\/td>17.85<\/td>40<\/td>C8:0<\/td><\/tr>\n
4.<\/td>Methyl decanoate<\/a><\/td>22.80<\/td>40<\/td>C10:0<\/td><\/tr>\n
5.<\/td>Methyl undecanoate<\/a><\/td>25.46<\/td>20<\/td>C11:0<\/td><\/tr>\n
6.<\/td>Methyl dodecanoate<\/a><\/td>28.12<\/td>40<\/td>C12:0<\/td><\/tr>\n
7.<\/td>Methyl tridecanoate<\/a><\/td>30.71<\/td>20<\/td>C13:0<\/td><\/tr>\n
8.<\/td>Methyl myristate<\/a><\/td>33.21<\/td>40<\/td>C14:0<\/td><\/tr>\n
9.<\/td>Methyl myristoleate<\/a><\/td>35.22<\/td>20<\/td>C14:1 (c9)<\/td><\/tr>\n
10.<\/td>Methyl pentadecanoate<\/a><\/td>35.60<\/td>20<\/td>C15:0<\/td><\/tr>\n
11.<\/td>Methyl pentadecenoate<\/a><\/td>37.57<\/td>20<\/td>C15:1 (c10)<\/td><\/tr>\n
12.<\/td>Methyl palmitate<\/a><\/td>37.90<\/td>60<\/td>C16:0<\/td><\/tr>\n<\/tbody><\/table>\n\n
<\/th>Peaks<\/th>tR<\/sub> (min)<\/th>Conc.
(mg\/mL)<\/th>		Structural Nomenclature<\/th><\/tr><\/thead>
13.<\/td>Methyl palmitoleate<\/a><\/td>39.51<\/td>20<\/td>C16:1 (c9)<\/td><\/tr>\n
14.<\/td>Methyl heptadecanoate<\/a><\/td>40.09<\/td>20<\/td>C17:0<\/td><\/tr>\n
15.<\/td>Methyl heptadecenoate<\/a><\/td>41.66<\/td>20<\/td>C17:1 (c10)<\/td><\/tr>\n
16.<\/td>Methyl stearate<\/a><\/td>42.19<\/td>40<\/td>C18:0<\/td><\/tr>\n
17.<\/td>Methyl octadecenoate<\/a><\/td>43.13<\/td>20<\/td>C18:1 (t9)<\/td><\/tr>\n
18.<\/td>Methyl oleate<\/a><\/td>43.54<\/td>40<\/td>C18:1 (c9)<\/td><\/tr>\n
19.<\/td>Methyl linoleaidate<\/a><\/td>44.60<\/td>20<\/td>C18:2 (t9,t12)<\/td><\/tr>\n
20.<\/td>Methyl linoleate<\/a><\/td>45.50<\/td>20<\/td>C18:2 (c9,c12)<\/td><\/tr>\n
21.<\/td>Methyl arachidate<\/a><\/td>46.12<\/td>40<\/td>C20:0<\/td><\/tr>\n
22.<\/td>Methyl linolenate<\/a><\/td>46.94<\/td>20<\/td>C18:3 (c6,c9,c12)<\/td><\/tr>\n
23.<\/td>Methyl eicosenoate<\/a><\/td>47.39<\/td>20<\/td>C20:1 (c11)<\/td><\/tr>\n
24.<\/td>Methyl linolenate <\/a><\/td>47.71<\/td>20<\/td>C18:3 (c9,c12,c15)<\/td><\/tr>\n<\/tbody><\/table>\n\n
<\/th>Peaks<\/th>tR<\/sub> (min)<\/th>Conc.
(mg\/mL)<\/th>		Structural Nomenclature<\/th><\/tr><\/thead>
25.<\/td>Methyl heneicosanoate<\/a><\/td>47.97<\/td>20<\/td>C21:0<\/td><\/tr>\n
26.<\/td>Methyl eicosadienoate<\/a><\/td>49.26<\/td>20<\/td>C20:2 (c11,c14)<\/td><\/tr>\n
27.<\/td>Methyl behenate<\/a><\/td>49.77<\/td>40<\/td>C22:0<\/td><\/tr>\n
28.<\/td>Methyl eicosatrienoate <\/td>50.62<\/td>20<\/td>C20:3 (c8,c11,c14)<\/td><\/tr>\n
29.<\/td>Methyl erucate<\/a><\/td>50.98<\/td>20<\/td>C22:1 (c13)<\/td><\/tr>\n
30.<\/td>Methyl eicosatrienoate <\/td>51.34<\/td>20<\/td>C20:3 (c11,c14,c17)<\/td><\/tr>\n
31.<\/td>Methyl tricosanoate<\/a><\/td>51.48<\/td>20<\/td>C23:0<\/td><\/tr>\n
32.<\/td>Methyl arachidonate<\/a><\/td>51.66<\/td>20<\/td>C20:4 (c5,c8,c11,c14)<\/td><\/tr>\n
33.<\/td>Methyl docosadienoate<\/td>52.75<\/td>20<\/td>C22:2 (c13,c16)<\/td><\/tr>\n
34.<\/td>Methyl lignocerate<\/a><\/td>53.16<\/td>40<\/td>C24:0<\/td><\/tr>\n
35.<\/td>Methyl eicosapentaenoate<\/a><\/td>53.77<\/td>20<\/td>C20:5 (c5,c8,c11,c14,c17)<\/td><\/tr>\n
36.<\/td>Methyl nervonate<\/a><\/td>54.33<\/td>20<\/td>C24:1 (C15)<\/td><\/tr>\n
37.<\/td>Methyl docosahexaenoate<\/a><\/td>58.48<\/td>20<\/td>C22:6 (c4,c7,c10,c13,c16,c19)<\/td><\/tr>\n<\/tbody><\/table><\/div>

Conditions<\/h4>
Column<\/th>Rt-2560, 100 m, 0.25 mm ID, 0.20 \u00b5m (cat.# 13198<\/a>)<\/td><\/tr>
Standard\/Sample<\/th>Food industry FAME mix (cat.# 35077<\/a>)<\/td><\/tr>\n
Diluent:<\/th>Hexane\/dichloromethane<\/td><\/tr>
Conc.:<\/th>1,000 \u00b5g\/mL<\/td><\/tr>
Injection<\/th><\/td><\/tr>
Inj. Vol.:<\/th>1 \u00b5L split (split ratio 20:1)<\/td><\/tr>
Liner:<\/th>Premium 4 mm Precision liner w\/wool (cat.# 23305<\/a>)<\/td><\/tr>
Inj. Temp.:<\/th>225 \u00b0C<\/td><\/tr>
Oven<\/td><\/tr>
Oven Temp.:<\/th>100 °C (hold 4 min) to 250 °C at 3 °C\/min (hold 30 min)<\/td><\/tr>
Carrier Gas<\/th>He, constant flow<\/td><\/tr>
Flow Rate:<\/th>1.3 mL\/min<\/td><\/tr><\/table>
Detector<\/th>FID @ 285 \u00b0C<\/td><\/tr>
Make-up Gas Flow Rate:<\/th>30 mL\/min<\/td><\/tr>
Make-up Gas Type:<\/th>N2<\/sub> <\/td><\/tr>
Hydrogen flow:<\/th>30 mL\/min<\/td><\/tr>
Air flow:<\/th>300 mL\/min<\/td><\/tr>
Data Rate:<\/th>50 Hz<\/td><\/tr>
Instrument<\/th>Agilent 7890B GC<\/td><\/tr><\/table><\/div><\/div><\/div>
<\/path><\/g><\/path><\/g><\/svg>Download PDF<\/a><\/div><\/div><\/div>\n<\/div><\/div><\/div>\n\n\n
<\/div>\n\n\n\n

New QC Test Ensures High-Quality, Detailed cis\/trans<\/em> FAMEs Analysis<\/h2>\n\n\n\n

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<\/em> 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 \u00b0C following elution of the FAMEs.<\/p>\n\n\n\n

Table I:<\/strong> 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.<\/p>\n\n\n\n

Test Mix Component<\/strong><\/th>Structural Nomenclature<\/strong><\/th>Performance Metric<\/strong><\/th><\/tr><\/thead>
Methyl octadecanoate<\/td>C18:0<\/td> <\/td><\/tr>
Methyl elaidate<\/td>C18:1-c9<\/td>Retention index<\/td><\/tr>
Methyl oleate<\/td>C18:1-c9<\/td> <\/td><\/tr>
Methyl nonadecanoate<\/td>C19:0<\/td> <\/td><\/tr>
Methyl eicosanoate<\/td>C20:0<\/td>Retention factor<\/td><\/tr>
Methyl eicosenoate<\/td>C20:1-c11<\/td>Resolution with methyl linolenate<\/td><\/tr>
Methyl linolenate<\/td>C18:3-c9,c12,c15<\/td>Retention index<\/td><\/tr>
Methyl heneicosanoate<\/td>C21:0<\/td> <\/td><\/tr>
Methyl docosanoate<\/td>C22:0<\/td>Retention factor and efficiency<\/td><\/tr>
Methyl eicosatrienoate<\/td>C20:3-c11,c14,c17<\/td>Retention index<\/td><\/tr>
Methyl tricosanoate<\/td>C23:0<\/td> <\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n
<\/div>\n\n\n\n

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<\/em>-alkanes, in order to avoid potential interfacial adsorption effects on the highly polar stationary phase.<\/p>\n\n\n\n

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<\/em> fat, particularly in the separation of the complex C18:1 region, and in the detailed profiling of unsaturated fatty acids.<\/p>\n\n\n\n

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<\/em> 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.<\/p>\n\n\n

Figure 2:<\/strong>\u00a0Analysis 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\u00a0cis<\/em>\u00a0and\u00a0trans<\/em>\u00a0C18:1-9 isomers is obtained on both columns.<\/div>
\n
\"Unsaturated<\/div>

GC_EX1134<\/p>

Peaks<\/h4>\n
<\/th>Peaks<\/th>tR<\/sub> (min)<\/th>Conc.
(mg\/mL)<\/th>		Structural Nomenclature<\/th><\/tr><\/thead>\n
1.<\/td>Methyl octadecanoate<\/a><\/td>11.31<\/td>1.1<\/td>C18:0 <\/td><\/tr>\n
2.<\/td>Methyl elaidate<\/a><\/td>12.24<\/td>0.8<\/td>C18:1 (t9)<\/td><\/tr>\n
3.<\/td>Methyl oleate<\/a><\/td>12.67<\/td>0.6<\/td>C18:1 (c9) <\/td><\/tr>\n
4.<\/td>Methyl nonadecanoate<\/a><\/td>13.40<\/td>1.1<\/td>C19:0 <\/td><\/tr>\n
5.<\/td>Methyl eicosanoate<\/a><\/td>16.13<\/td>1.1<\/td>C20:0 <\/td><\/tr>\n<\/tbody><\/table>\n\n
<\/th>Peaks<\/th>tR<\/sub> (min)<\/th>Conc.
(mg\/mL)<\/th>		Structural Nomenclature<\/th><\/tr><\/thead>
6.<\/td>Methyl eicosenoate<\/a><\/td>18.25<\/td>0.6<\/td>C20:1 (c11) <\/td><\/tr>\n
7.<\/td>Methyl linolenate<\/a><\/td>18.64<\/td>0.8<\/td>C18:3 (c9,c12,c15)<\/td><\/tr>\n
8.<\/td>Methyl heneicosanoate<\/a><\/td>19.70<\/td>1.1<\/td>C21:0 <\/td><\/tr>\n
9.<\/td>Methyl docosanoate<\/a><\/td>24.39<\/td>1.1<\/td>C22:0 <\/td><\/tr>\n
10.<\/td>Methyl eicosatrienoate<\/td>27.93<\/td>0.6<\/td>C20:3 (c11,c14,c17) <\/td><\/tr>\n
11.<\/td>Methyl tricosanoate<\/a><\/td>30.54<\/td>1.1<\/td>C23:0<\/td><\/tr>\n<\/tbody><\/table><\/div>

Conditions<\/h4>
Column<\/th>Rt-2560, 100 m, 0.25 mm ID, 0.20 \u00b5m<\/td><\/tr>
Standard\/Sample<\/th>Unsaturated FAMES QC test mix <\/td><\/tr>\n
Diluent:<\/th>Hexane<\/td><\/tr>
Injection<\/th><\/td><\/tr>
Inj. Vol.:<\/th>1 \u00b5L split (split ratio 200:1)<\/td><\/tr>
Liner:<\/th>Premium 4 mm Precision liner w\/wool (cat.# 23305<\/a>)<\/td><\/tr>
Inj. Temp.:<\/th>250 \u00b0C<\/td><\/tr>
Oven<\/td><\/tr>
Oven Temp.:<\/th>180 °C (hold 40 min)<\/td><\/tr>
Carrier Gas<\/th>H2<\/sub>, constant flow<\/td><\/tr>
Flow Rate:<\/th>2.5 mL\/min<\/td><\/tr><\/table>
Detector<\/th>FID @ 275 \u00b0C<\/td><\/tr>
Make-up Gas Flow Rate:<\/th>50 mL\/min<\/td><\/tr>
Make-up Gas Type:<\/th>N2<\/sub> <\/td><\/tr>
Hydrogen flow:<\/th>40 mL\/min<\/td><\/tr>
Air flow:<\/th>400 mL\/min<\/td><\/tr>
Data Rate:<\/th>50 Hz<\/td><\/tr>
Instrument<\/th>Agilent 7890B GC<\/td><\/tr>
Notes<\/th>GC column cat.# 13199 was used to produce this chromatogram, but it has since been discontinued. For assistance choosing a column for this application, contact Restek Technical Service or your local Restek representative.<\/td><\/tr><\/table><\/div><\/div><\/div>
<\/path><\/g><\/path><\/g><\/svg>Download PDF<\/a><\/div><\/div><\/div>\n\n\n

After Redesign<\/strong><\/p>\n\n\n

\"Unsaturated<\/div>

GC_QA0099<\/p>

Peaks<\/h4>\n
<\/th>Peaks<\/th>tR<\/sub> (min)<\/th>Conc.
(mg\/mL)<\/th>		Structural Nomenclature<\/th><\/tr><\/thead>\n
1.<\/td>Methyl octadecanoate<\/a><\/td>11.68<\/td>1.1<\/td>C18:0 <\/td><\/tr>\n
2.<\/td>Methyl elaidate<\/a><\/td>12.64<\/td>0.8<\/td>C18:1 (t9)<\/td><\/tr>\n
3.<\/td>Methyl oleate<\/a><\/td>13.09<\/td>0.6<\/td>C18:1 (c9) <\/td><\/tr>\n
4.<\/td>Methyl nonadecanoate<\/a><\/td>13.84<\/td>1.1<\/td>C19:0 <\/td><\/tr>\n
5.<\/td>Methyl eicosanoate<\/a><\/td>16.66<\/td>1.1<\/td>C20:0 <\/td><\/tr>\n<\/tbody><\/table>\n\n
<\/th>Peaks<\/th>tR<\/sub> (min)<\/th>Conc.
(mg\/mL)<\/th>		Structural Nomenclature<\/th><\/tr><\/thead>
6.<\/td>Methyl eicosenoate<\/a><\/td>18.85<\/td>0.6<\/td>C20:1 (c11) <\/td><\/tr>\n
7.<\/td>Methyl linolenate<\/a><\/td>19.20<\/td>0.8<\/td>C18:3 (c9,c12,c15) <\/td><\/tr>\n
8.<\/td>Methyl heneicosanoate<\/a><\/td>20.35<\/td>1.1<\/td>C21:0 <\/td><\/tr>\n
9.<\/td>Methyl docosanoate<\/a><\/td>25.17<\/td>1.1<\/td>C22:0 <\/td><\/tr>\n
10.<\/td>Methyl eicosatrienoate<\/td>28.79<\/td>0.6<\/td>C20:3 (c11,c14,c17)<\/td><\/tr>\n
11.<\/td>Methyl tricosanoate<\/a><\/td>31.49<\/td>1.1<\/td>C23:0<\/td><\/tr>\n<\/tbody><\/table><\/div>

Conditions<\/h4>
Column<\/th>Rt-2560, 100 m, 0.25 mm ID, 0.20 \u00b5m (cat.# 13198<\/a>)<\/td><\/tr>
Standard\/Sample<\/th>Unsaturated FAMES QC test mix <\/td><\/tr>\n
Diluent:<\/th>Hexane<\/td><\/tr>
Injection<\/th><\/td><\/tr>
Inj. Vol.:<\/th>1 \u00b5L split (split ratio 200:1)<\/td><\/tr>
Liner:<\/th>Premium 4 mm Precision liner w\/wool (cat.# 23305<\/a>)<\/td><\/tr>
Inj. Temp.:<\/th>250 \u00b0C<\/td><\/tr>
Oven<\/td><\/tr>
Oven Temp.:<\/th>180 °C (hold 40 min)<\/td><\/tr>
Carrier Gas<\/th>H2<\/sub>, constant flow<\/td><\/tr>
Flow Rate:<\/th>2.5 mL\/min<\/td><\/tr><\/table>
Detector<\/th>FID @ 275 \u00b0C<\/td><\/tr>
Make-up Gas Flow Rate:<\/th>50 mL\/min<\/td><\/tr>
Make-up Gas Type:<\/th>N2<\/sub> <\/td><\/tr>
Hydrogen flow:<\/th>40 mL\/min<\/td><\/tr>
Air flow:<\/th>400 mL\/min<\/td><\/tr>
Data Rate:<\/th>50 Hz<\/td><\/tr>
Instrument<\/th>Agilent 7890B GC<\/td><\/tr><\/table><\/div><\/div><\/div>
<\/path><\/g><\/path><\/g><\/svg>Download PDF<\/a><\/div><\/div><\/div>\n<\/div><\/div><\/div>\n\n\n
<\/div>\n\n\n\n

Conclusion<\/h2>\n\n\n\n

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<\/em> 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.<\/p>\n\n\n\n

<\/div>\n","protected":false},"excerpt":{"rendered":"

Even minor variations in manufacturing processes can potentially have detrimental effects on column-to-column reproducibility. 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