New EZGC library: Fatty Acid Methyl Esters on the Rtx-225

While building the EZGC library of residual solvents on the Rtx-225, we learned that the Rtx-225 has been used for separating fatty acid methyl esters (FAMEs) in recently published literature. Despite the relatively low bleed temperature (220^oC), the Rtx-225 has been used to identify FAMEs in both food^1–3 and medicine^4,5 applications. So, to compliment the work that our customers have accomplished, we have added an EZGC library of FAMEs for the Rtx-225.

Table 1: FAMEs included in the Rtx-225 EZGC library.

Name (Carbon:Double Bonds notation)	CAS#		Name (Carbon:Double Bonds notation)	CAS#
C4:0	623-42-7		C18:1 (trans-11)	6198-58-9
C6:0	106-70-7		C18:1 (cis-11)	1937-63-9
C8:0	111-11-5		C18:2  (trans-9,12 )	2566-97-4
C10:0	110-42-9		C18:2  (cis-9,12 )	112-63-0
C11:0	1731-86-8		C18:3  (cis-6,9,12 )	16326-32-2
C12:0	111-82-0		C18:3  (cis-9,12,15 )	301-00-8
C13:0	1731-88-0		C20:0	1120-28-1
C14:0	124-10-7		C20:1  (cis-11 )	2390-09-2
C14:1  (cis-9 )	56219-06-8		C20:2  (cis-11,14 )	2463-02-7
C15:0	7132-64-1		C20:3  (cis-8,11,14 )	21061-10-9
C15:1  (cis-10 )	90176-52-6		C20:4 (cis-5,8,11,14)	2566-89-4
C16:1  (cis-9 )	1120-25-8		C21:0	6064-90-0
C17:0	1731-92-6		C20:3 (cis-11, 14, 17)	55682-88-7
C17:1  (cis-10 )	75190-82-8		C20:5 (cis-5,8,11,14,17)	2734-47-6
C18:0	112-61-8		C22:1  (cis-13 )	1120-34-9
C18:1 (trans-6)	14620-36-1		C22:2  (cis-13,16 )	61012-47-3
C18:1 (trans-9)	1937-62-8		C22:6 (cis-4,7,10,13,16,19)	2566-90-7
C18:1 (cis-6)	2777-58-4		C24:0	2442-49-1
C18:1 (cis-9)	112-62-9		C24:1  (cis-15 )	2733-88-2
C18:1 (trans-11)	6198-58-9

Using the EZGC library, we were able to create a <15min run that separated 35 of the 41 FAMEs included in the library. The six FAMEs that we could not separate were the C18:1 isomers: trans-6, trans-9, trans-11, cis-6, cis-9, and cis-11. Our column was measured to be 29.09m (see tips on calculating column length here), we used an inlet temperature of 250^oC, a Topaz straight inlet liner with wool (cat#23300), and a 20:1 split. The standards used were cat# 35079, 35066, 35078, 35034, 35014, and 35077 diluted to approximately 100µg g^-1 in hexanes. The EZGC speed-optimized oven program was: 60^oC (hold 0.5min) to 200^oC at 18.5 ^oC/min (hold 7 min).

With the help of EZGC, we continued to fine-tuned parameters to achieve better separation of the C18:1 isomers. Compared to the ‘speedy’ (<15min) run, we could improve separation by lowering the final temperature to 205^oC, and/or extending the column length to 60m (Table 2). While we did not have a 60m column on hand, we still wanted to see if there would be a noticeable difference in the separation of these isomers by just lowering the final temperature.

Table 2: EZGC predictions for separation of C18:1 isomers.

Name (Carbon:Double Bonds notation)	‘speedy’ 29.09m (tR)	EZGC 30m (tR)	EZGC 60m (tR)
C18:1 (trans-6)	0.7	0.7	1.1
C18:1 (trans-9)	0.3	0.3	0.5
C18:1 (cis-6)	0.3	0.3	0.4
C18:1 (cis-9)	0	0.1	0.3
C18:1 (trans-11)	0	0.1	0.3
C18:1 (cis-11)	1.4	1.4	1.8
Runtime →	15 min	18.5 min	43.5 min

As it turns out, the answer is ‘somewhat’ (Figure 2). In practice, C18:1 trans-9, cis-6, and cis-9 were the problematic co-elution, with trans-6 and trans-11 separating better than predicted. By decreasing the final temperature, we extended the run time for all 41 compounds by 3.5 min, but there is some noticeable improvement in the separation of C18:1 trans-9, cis-6, and cis-9. Individual peak ‘shoulders’ became more distinguishable when the final temperature was reduced to 205^oC, and the co-elution peak subsequently widened by about 2 seconds, as would be expected if co-eluting peaks were starting to separate. This is certainly a sign that the method can continue to be fine-tuned depending on your application.

So, why didn’t EZGC suggest the method with slightly greater resolution? I had the chromatogram modeler set to optimize the method for speed. Since changing the ramp rate did not indicate sufficiently improved resolution (t_R>1.5), I assume the modeler gave me the quickest method it could while ignoring those co-elutions.

Figure 2: Separation of C18:1 isomers (cat# 35079) using the ‘speedy’ method that ramps to 220^oC (top), compared to a method that ramps to 205^oC (bottom). While the C18:1 isomers elute earlier in the bottom chromatograph, the complete method run-time is 3.5 min longer. Co-eluting C18:1 isomers (peaks 2-4 increased in width as shown in the bracket.

Overall, if you are looking to improve the separation of the C18:1 isomers, try a 60m column instead of 30m. Otherwise, the 30m Rtx-225 can provide the separation you need for FAMEs. EZGC can help you fine-tune separations with either column length, while saving time and resources. If you want to see a 60m Rtx-225 in action, or have some specific applications in mind, let us know in the comments!

 

For further reading:

1. Ahmad, M. N.; Mehyar, G. F.; Othman, G. A. Nutritional, Functional and Microbiological Characteristics of Jordanian Fermented Green Nabali Baladi Olives. Grasas Aceites 2021, 72 (1), e396. https://doi.org/10.3989/gya.1258192.
2. Al-Ismail, K.; Al-Awamleh, S. A.; Saleh, M.; Al-Titi, H. Impacts of Oil Types and Storage Conditions on Milk Fat Quality of Strained Yogurt Immersed in Oil. J. Am. Oil Chem. Soc. 2019, 96 (2), 171–178. https://doi.org/10.1002/aocs.12176.
3. Palma, J.; Mercado, A.; Paredes, A.; Lizama, C.; Pohl, G.; Larrazabal, M. Assessing Properties of Acantholippia Deserticola (Phil.) Moldenke Essential Oil: Comparison between Hydrodistillation and Microwave-Assisted Hydrodistillation Extraction Methods. Qual. Assur. Saf. Crops Foods 2020, 12 (4), 36–49. https://doi.org/10.15586/qas.v12i4.792.
4. Roychowdhury, S.; Glueck, B.; Han, Y.; Mohammad, M. A.; Cresci, G. A. M. A Designer Synbiotic Attenuates Chronic-Binge Ethanol-Induced Gut-Liver Injury in Mice. Nutrients 2019, 11 (1), 97. https://doi.org/10.3390/nu11010097.
5. Ichihara, K.; Kohsaka, C.; Tomari, N.; Yamamoto, Y.; Masumura, T. Determination of Free Fatty Acids in Plasma by Gas Chromatography. Anal. Biochem. 2020, 603, 113810. https://doi.org/10.1016/j.ab.2020.113810.