ISO 12966-1-2014 pdf free download.Animal and vegetable fats and oils – Gas chromatography of fatty acid methyl esters – Part 1: Guidelines on modern gas chromatography of fatty acid methyl esters.
5 Columns
At the time of publication of this part of ISO 12966, for the separation of FAM Es, wall coated open tubular (WCOT) capillaries are used as they offer a number of advantages over a packed column. This includes vastly improved separations with higher resolution, reduced time of analysis, smaller sample size and higher sensitivities. All this is enabled by the possibility of using very long columns with a large number of theoretical plates.
Sample capacity increases with the column diameter, smaller diameters giving greater efficiency and better resolutions. Therefore for complex samples columns with small diameters and small sample capacity are used. Common available internal diameters (ID) are 0,1 mm (fast GC columns) to 0,53 mm (wide bore column), film thickness is between 0,1 pm and 0,3 tim. The column diameter shall suit the type of sample inlet system: 0,20 mm, 0,25 mm or 0,32 mm ID column are for split and splitless injection systems. 0,32 mm ID for splitless and on-column injections, and 0,53 mm ID for direct injection systems. Retention and sample capacity increase with increasing film thickness, at the same time the column efficiency decreases. Film thickness is inversely related to plate number, but directly proportional to the time of analysis. This also means that a greater film thickness gives greater retention, which requires a higher oven temperature in isothermal conditions.
The length of the columns is 10 m to 100 m depending on the required resolution and separation problem. With short columns limited, but fast, information can be obtained, in process control, etc. Different types of capillary columns with non-polar, polar, and highly polar stationary phases are used for the separation of FAMEs. The elution order for columns with different polarity are shown in ISO 12966-4 (see Annex B and Annex C).
Fused silica columns coated with highly polar stationary phases of cyanoalkyl polysiloxane are used for the analysis of samples with complex mixtures of geometrical and positional isomers of polyunsaturated fatty acids (PUFA). The main advantage of these high-polar phases compared to non-polar phases is their high-resolution capability of unsaturated FAME, especially for the separation of cis and trans FA isomers. However, the polarity of this column shows less thermal stability compared to other stationary phases.
Column length is a significant factor for the separation of FAMEs, especially ifas mentioned in this clause, the separation of isomers is required, A longer column will provide a better resolution compared with a shorter column, nevertheless there are some practical limits for increasing the column length. Doubling column length (e.g. 30 m to 60 m) will increase the resolution by a factor of i12 = 1,41, which is 40 % only. At the same time, the time of analysis will increase as well as the head pressure. Shorter columns are a compromise between speed and resolution. However, the separation of geometrical and positional isomers of FAMEs requires in some cases a 100 m column. The use of 100 m, 0,20 mm or 0,25 mm ID, 0,20 pm film thickness columns with a highly polar biscyanopropyl (SP-2560) or a highly substituted cyanopropyl stationary phase (CP-Sil 88) is recommended, as the separation capacity of these columns is sufficient to separate most C18:1 trans- and cis-isomers. Some 50 m or 60 m long columns may also achieve this separation mostly for vegetable oils. Other types of columns with different cyanopropyl polysilpehylene-siloxanes (BPX70, DB-23, HP-23, Rtx-2330, SP-2330, SP-2380, SLB-IL111, etc.) may also be used, but a change in the elution order is possible. The AOCS method Ce 1h-05 (2009) recommends a 100 m SP-2560 or CP-Sil 88 capillary columns for the determination of cis-, trans-, and unsaturated FAMEs in vegetable or non-ruminant animal oils. For the determination of FAMEs in marine and other oils containing long-chain PUFAs, the AOCS method Ce 1i-07 (2009) recommends a highly polar Supelcowax 10 capillary column with a polyethylene glycol phase; and AOCS method Ce 1b-89 (2009) uses a Omegawax for the determination of long-chain omega-3 FAMEs.2)ISO 12966-1 pdf download.