Dear Tawatchai,

Please read the following text:

A - Acid-catalyzed esterification

The most common derivatives of fatty acids are the methyl esters obtained by heating free fatty acids with a large excess of anhydrous methanol in the presence of a catalyst, boron trifluoride (Morrison et al J Lipid Res 1964, 5,600). It must be noticed that O-acyl lipids are transesterified very rapidly with the same reagent. 

Reagent

14% Boron trifluoride in methanol (Alltech or Sigma) (keep refrigerated under nitrogen and discard after 3 months or when solids appear at the bottom of the vial).

Pentane, chloroform.

Procedure

As a general procedure, an aliquot of lipid extract (about 10 mg) is dried under nitrogen in a screw-capped glass tube and 1 ml of BF3/methanol is added.

If triacylglycerols or sterol esters are analyzed alone or are abundant in the extract, the dry lipids are dissolved in 0.75 ml of chloroform/methanol (1/1, v/v) and 0.25 ml BF3/methanol are added. If possible, the tube is closed after flushing with nitrogen.

Heat in boiling water (or at 100°C in a sand bath) the time indicated for the respective lipid:

After cooling, add 1 ml water and 2 ml pentane. Vortex for 1 min, centrifuge at low speed and collect the upper phase. Pentane is evaporated and the residue is immediately dissolved in 50-100 µl hexane. The solution is ready for injection in the gas chromatograph.

After TLC, spots containing fatty acid-based lipids may be scraped, collected and treated with the BF3/methanol solution directly in a glass tube. It was reported that selective loss of unsaturated fatty acids was observed oon certain brands of plates (Sowa JM et al., J Chromatogr B 2004, 813, 159). Thus, the authors determined that no loss occurred in both neutral and phospholipids with Alltech or Merck silica gel plates.

Alternative methods :

A sulfuric acid-methanol method was used with success to derivatize very long chain fatty acids (C24:0-C36:0) before gas chromatography analysis (Mendez Antolin E et al., J Pharm Biomed Anal 2008, 46, 194).

Acidic conditions generated by 3M HCl in dry methanol or methanolic sulfuric acid have been also described.

Preparation of fatty acid methyl esters from various sources using commercial aqueous HCl was also described (Ichihara K et al., J Lipid Res 2010, 51, 635). Yields of FAME were the same as those obtained with boron trifluoride method. Furthermore, the reagent is very convenient, safe and cheap.

Shortly, the reagent is made from 9.7 ml commercial concentrated HCI (35%, w/w) diluted with 41.5 ml of methanol and was stored in a refrigerator. A lipid sample was dissolved in 0.20 ml of toluene, then 1.50 ml of methanol and 0.30 ml of the reagent solution were added in this order. The tube was vortexed and then heated at 100°C for 1 h. After cooling, 1 ml of hexane and 1 ml of water were added for extraction of methyl esters in the hexane phase.

An improved method for determining medium- and long-chain in lipid samples using one-step transesterification with acetyl chloride has been reported (Xu Z et al., Lipids 2010, 45, 199). The data suggest that the method can be easily used to accurately determine fatty acids (C6–C24) in functional foods and lipid emulsions.

B - Base-catalyzed transesterification

Fatty esters form with a base (alcoholate) form an anionic intermediate which is transformed in the presence of a large excess of the alcohol into a new ester. Free fatty acids are not subject to nucleophilic attack by alcohols or bases and thus are not esterified in these conditions.

Derivatizations in the presence of basic catalysts have the advantages of speed and mild heating conditions. Thus this type of catalysis is recommended in samples with short-chain fatty acids or labile fatty acids (polyunsaturated, cyclopropane rings, conjugated unsaturations...). 

The most useful basic transesterifying agents are 1 to 2M Na or K methoxide in anhydrous methanol. These solutions are stable for several months at 4°C until a white precipitate of bicarbonate salt is formed. Glycerolipids are rapidly transesterified (2-5 min) at room temperature.

An improved rapid procedure to analyze fatty acid esters from triacylglycerols and phospholipids is described below (Ichihara K et al., Lipids 1996, 31, 535) :  

Reagents:

Hexane, 2 M methanolic KOH, capped plastic tubes.

Procedure:

Up to 10 mg of lipids are dissolved in 2 ml hexane followed by the addition of 0.2 ml of 2 M methanolic KOH. The tube is vortexed for 2 min at room temperature. After a light centrifugation, an aliquot of the hexane layer is collected for GC analysis.

It must be pointed out that sterol esters and waxes do not react under these conditions.

A modification and an adaptation of that procedure has been proposed allowing the direct preparation of fatty acid methyl esters from polar lipids (phospholipids) in lipid mixtures without prior isolation (Ichihara K et al., Lipids 2010, 45, 367). No obvious differences were found between the fatty acid compositions of phospholipids determined by that method and those determined by conventional methods, including lipid extraction with chloroform/methanol followed by isolation of polar lipids by chromatography.

An alternative base-catalyzed methodology in mild conditions was adapted for milk or seed lipids using K tert-butoxide and 2-methoxyethanol (Destaillats F et al., Lipids 2002, 37, 527): 

Reagents:

1 M K tert-butoxide in THF (Aldrich), 2-methoxyethano, hexane, Na sulfate.

Procedure:

100 ml of a solution of K tert-butoxide in THF are added to 200 ml anhydrous 2-methoxyethanol in a closed vial. After homogenization, up to 10 mg of lipid in 1 ml hexane are added. Keep the mixture at 40°C for 15 min. After cooling, 1 ml water and 2 ml hexane are successively added. After 5 s vortexing and a short centrifugation, the organic phase is collected, dried over anhydrous Na sulfate and analyzed by GLC.

We have adopted another approach for some labile samples. A rapid and mild method which avoids the formation of oxidation products was described by Piretti et al. (Chem Phys Lipids 1988, 47, 149). We have most precisely adopted this procedure for the analysis of highly unsaturated lipids since higher amounts of polyunsaturated fatty acids were found when compared to the BF3/methanol procedure.

Furthermore, if hydroperoxy fatty acids are present, they are reduced into the corresponding hydroxy components.

Reagents:

2M NaOH, NaBH4, anhydrous Na2SO4.

Ethyl acetate, methanol, hexane.

Procedure:

2 mg of neutral lipids or up to 100 mg polar lipids are dried in a glass tube.

Add 1 ml of the reagent made in dissolving immediately before use 400 mg NaBH4 in 10 ml of the mixture methanol/2 M NaOH (19/1, v/v).

The mixture is stirred for 20 min at room temperature. After adding 2 ml water, the methanol is eliminated under nitrogen. The methyl esters are recovered from the aqueous phase by extracting 3 times with 1 ml ethyl acetate. The organic phase is then washed 3 times with 1 ml water and dried by adding Na2SO4. After vortexing and centrifugation, the ethyl acetate is evaporated and the residue dissolved in a small amount of hexane for GLC analysis.

Comments : A 30-min, micro-base-catalyzed method for vegetable oil fatty acid determination has been proposed using a novel fatty acid derivatization method (Lall RK et al., JAOCS 009, 86, 309). The sensitivity was improved for relatively small pure oil samples without loss of accuracy.

Silylation

If methyl esterification with BF3/methanol has been the most widely used derivatization method, other approaches were described to correct various defects such as reagent instability, destruction of epoxy, cyclic fatty acids, hydroxy groups, and non-derivatization of unsaponifiable materials. Trimethylsilyl derivatization is known to be an efficient method but it has some faults like thermal instability and partial hydrolysis of the derivatives. To overcome these defects, the ter-butyldimethylsilyl (tBDMSi) derivatization method for GC analysis was developed (Woo KL et al., J Chromatogr A 1999, 862, 199). These derivatives were shown to have a high thermal and hydrolytic stability and they improve the sensitivity and the selectivity of the analyses.

Procedure :

To fatty acids dissolved in 200 ml of hexane, a known amount of internal standard solution, 75 ml of N-methyl-N-(ter-butyldimethylsilyl)trifluoroacetamide and 5 ml of triethylamine are added. After tightly capping, the contents are maintained at 75°C for 30 min before injection.

The separation is done with a HP-1 capillary column (50 m x 0.2 mm ID) with a temperature program as follows : 40°C for 1 min and then after increasing to 70°C with 60°C/min, held for 2 mn. After increasing to 205°C with 5°C/min, held for 25 min and then increased to 285°C with 5°C/min and held for 1 min. Injector and detector are at 300°C.

Comments :

In all fatty acids, the peak responses for these derivatives are higher by 1.5-6.3-times than for methyl esters. In contrast, the stability was shown to be reduced practically to no more than 3 days.

Verification of the desired methyl ester:

The product can be verified using TLC, HPLC, LC-MS or NMR.

TLC is the fastest method; if the product has Rf value different from the reactant it indicates that the methylation process went as expected (planned).

For more details on the topic of your question, please use the following link:

http://www.cyberlipid.org/fattyt/fatt0001.htm

Hoping this will be helpful,

Rafik

Dear Tawatchai,

Please read the following text:

A - Acid-catalyzed esterification

The most common derivatives of fatty acids are the methyl esters obtained by heating free fatty acids with a large excess of anhydrous methanol in the presence of a catalyst, boron trifluoride (Morrison et al J Lipid Res 1964, 5,600). It must be noticed that O-acyl lipids are transesterified very rapidly with the same reagent. 

Reagent

14% Boron trifluoride in methanol (Alltech or Sigma) (keep refrigerated under nitrogen and discard after 3 months or when solids appear at the bottom of the vial).

Pentane, chloroform.

Procedure

As a general procedure, an aliquot of lipid extract (about 10 mg) is dried under nitrogen in a screw-capped glass tube and 1 ml of BF3/methanol is added.

If triacylglycerols or sterol esters are analyzed alone or are abundant in the extract, the dry lipids are dissolved in 0.75 ml of chloroform/methanol (1/1, v/v) and 0.25 ml BF3/methanol are added. If possible, the tube is closed after flushing with nitrogen.

Heat in boiling water (or at 100°C in a sand bath) the time indicated for the respective lipid:

After cooling, add 1 ml water and 2 ml pentane. Vortex for 1 min, centrifuge at low speed and collect the upper phase. Pentane is evaporated and the residue is immediately dissolved in 50-100 µl hexane. The solution is ready for injection in the gas chromatograph.

After TLC, spots containing fatty acid-based lipids may be scraped, collected and treated with the BF3/methanol solution directly in a glass tube. It was reported that selective loss of unsaturated fatty acids was observed oon certain brands of plates (Sowa JM et al., J Chromatogr B 2004, 813, 159). Thus, the authors determined that no loss occurred in both neutral and phospholipids with Alltech or Merck silica gel plates.

Alternative methods :

A sulfuric acid-methanol method was used with success to derivatize very long chain fatty acids (C24:0-C36:0) before gas chromatography analysis (Mendez Antolin E et al., J Pharm Biomed Anal 2008, 46, 194).

Acidic conditions generated by 3M HCl in dry methanol or methanolic sulfuric acid have been also described.

Preparation of fatty acid methyl esters from various sources using commercial aqueous HCl was also described (Ichihara K et al., J Lipid Res 2010, 51, 635). Yields of FAME were the same as those obtained with boron trifluoride method. Furthermore, the reagent is very convenient, safe and cheap.

Shortly, the reagent is made from 9.7 ml commercial concentrated HCI (35%, w/w) diluted with 41.5 ml of methanol and was stored in a refrigerator. A lipid sample was dissolved in 0.20 ml of toluene, then 1.50 ml of methanol and 0.30 ml of the reagent solution were added in this order. The tube was vortexed and then heated at 100°C for 1 h. After cooling, 1 ml of hexane and 1 ml of water were added for extraction of methyl esters in the hexane phase.

An improved method for determining medium- and long-chain in lipid samples using one-step transesterification with acetyl chloride has been reported (Xu Z et al., Lipids 2010, 45, 199). The data suggest that the method can be easily used to accurately determine fatty acids (C6–C24) in functional foods and lipid emulsions.

B - Base-catalyzed transesterification

Fatty esters form with a base (alcoholate) form an anionic intermediate which is transformed in the presence of a large excess of the alcohol into a new ester. Free fatty acids are not subject to nucleophilic attack by alcohols or bases and thus are not esterified in these conditions.

Derivatizations in the presence of basic catalysts have the advantages of speed and mild heating conditions. Thus this type of catalysis is recommended in samples with short-chain fatty acids or labile fatty acids (polyunsaturated, cyclopropane rings, conjugated unsaturations...). 

The most useful basic transesterifying agents are 1 to 2M Na or K methoxide in anhydrous methanol. These solutions are stable for several months at 4°C until a white precipitate of bicarbonate salt is formed. Glycerolipids are rapidly transesterified (2-5 min) at room temperature.

An improved rapid procedure to analyze fatty acid esters from triacylglycerols and phospholipids is described below (Ichihara K et al., Lipids 1996, 31, 535) :  

Reagents:

Hexane, 2 M methanolic KOH, capped plastic tubes.

Procedure:

Up to 10 mg of lipids are dissolved in 2 ml hexane followed by the addition of 0.2 ml of 2 M methanolic KOH. The tube is vortexed for 2 min at room temperature. After a light centrifugation, an aliquot of the hexane layer is collected for GC analysis.

It must be pointed out that sterol esters and waxes do not react under these conditions.

A modification and an adaptation of that procedure has been proposed allowing the direct preparation of fatty acid methyl esters from polar lipids (phospholipids) in lipid mixtures without prior isolation (Ichihara K et al., Lipids 2010, 45, 367). No obvious differences were found between the fatty acid compositions of phospholipids determined by that method and those determined by conventional methods, including lipid extraction with chloroform/methanol followed by isolation of polar lipids by chromatography.

An alternative base-catalyzed methodology in mild conditions was adapted for milk or seed lipids using K tert-butoxide and 2-methoxyethanol (Destaillats F et al., Lipids 2002, 37, 527): 

Reagents:

1 M K tert-butoxide in THF (Aldrich), 2-methoxyethano, hexane, Na sulfate.

Procedure:

100 ml of a solution of K tert-butoxide in THF are added to 200 ml anhydrous 2-methoxyethanol in a closed vial. After homogenization, up to 10 mg of lipid in 1 ml hexane are added. Keep the mixture at 40°C for 15 min. After cooling, 1 ml water and 2 ml hexane are successively added. After 5 s vortexing and a short centrifugation, the organic phase is collected, dried over anhydrous Na sulfate and analyzed by GLC.

We have adopted another approach for some labile samples. A rapid and mild method which avoids the formation of oxidation products was described by Piretti et al. (Chem Phys Lipids 1988, 47, 149). We have most precisely adopted this procedure for the analysis of highly unsaturated lipids since higher amounts of polyunsaturated fatty acids were found when compared to the BF3/methanol procedure.

Furthermore, if hydroperoxy fatty acids are present, they are reduced into the corresponding hydroxy components.

Reagents:

2M NaOH, NaBH4, anhydrous Na2SO4.

Ethyl acetate, methanol, hexane.

Procedure:

2 mg of neutral lipids or up to 100 mg polar lipids are dried in a glass tube.

Add 1 ml of the reagent made in dissolving immediately before use 400 mg NaBH4 in 10 ml of the mixture methanol/2 M NaOH (19/1, v/v).

The mixture is stirred for 20 min at room temperature. After adding 2 ml water, the methanol is eliminated under nitrogen. The methyl esters are recovered from the aqueous phase by extracting 3 times with 1 ml ethyl acetate. The organic phase is then washed 3 times with 1 ml water and dried by adding Na2SO4. After vortexing and centrifugation, the ethyl acetate is evaporated and the residue dissolved in a small amount of hexane for GLC analysis.

Comments : A 30-min, micro-base-catalyzed method for vegetable oil fatty acid determination has been proposed using a novel fatty acid derivatization method (Lall RK et al., JAOCS 009, 86, 309). The sensitivity was improved for relatively small pure oil samples without loss of accuracy.

Silylation

If methyl esterification with BF3/methanol has been the most widely used derivatization method, other approaches were described to correct various defects such as reagent instability, destruction of epoxy, cyclic fatty acids, hydroxy groups, and non-derivatization of unsaponifiable materials. Trimethylsilyl derivatization is known to be an efficient method but it has some faults like thermal instability and partial hydrolysis of the derivatives. To overcome these defects, the ter-butyldimethylsilyl (tBDMSi) derivatization method for GC analysis was developed (Woo KL et al., J Chromatogr A 1999, 862, 199). These derivatives were shown to have a high thermal and hydrolytic stability and they improve the sensitivity and the selectivity of the analyses.

Procedure :

To fatty acids dissolved in 200 ml of hexane, a known amount of internal standard solution, 75 ml of N-methyl-N-(ter-butyldimethylsilyl)trifluoroacetamide and 5 ml of triethylamine are added. After tightly capping, the contents are maintained at 75°C for 30 min before injection.

The separation is done with a HP-1 capillary column (50 m x 0.2 mm ID) with a temperature program as follows : 40°C for 1 min and then after increasing to 70°C with 60°C/min, held for 2 mn. After increasing to 205°C with 5°C/min, held for 25 min and then increased to 285°C with 5°C/min and held for 1 min. Injector and detector are at 300°C.

Comments :

In all fatty acids, the peak responses for these derivatives are higher by 1.5-6.3-times than for methyl esters. In contrast, the stability was shown to be reduced practically to no more than 3 days.

Verification of the desired methyl ester:

The product can be verified using TLC, HPLC, LC-MS or NMR.

TLC is the fastest method; if the product has Rf value different from the reactant it indicates that the methylation process went as expected (planned).

For more details on the topic of your question, please use the following link:

http://www.cyberlipid.org/fattyt/fatt0001.htm

Hoping this will be helpful,

Rafik

The problem with SCFA is that they are volatile and making them into methyl esters makes them even more volatile! This means that the sample contents are easily lost and can be biased toward the longer chain acids. You would have to incorporate a heavy isotope standard for each fatty acid to control or account for this. If you must make methyl ester derivatives I suggest using excess diazomethane in ether solution. The persistence of the yellow color of diazomethane indicates an excess of the reagent is present and that the formation of the methyl esters is complete. Use extreme caution since diazomethane is toxic, volatile and explosive!

Better yet don't use a derivative, use a Wax column which is compatible with GC/MS to analyze the SCFA's directly: http://www.phenomenex.com/Application/Detail/15883?returnURL=/Application/Search

Dear Bruce Levison

Your suggestion is very interesting. The method that uses the color to indicate the successful formation of the methyl ester is easy, but diazomethane is too dangerous. So, are there any chemicals that can replace diazomethane?

Thank you

Tawatchai

An easy simple method to make the methyl esters is to dissolve the compounds in anhydrous methanol with a catalytic amount of HCl. I had a lecture bottle of HCl that I used to bubble the gas into some methanol (to get 1M by weight) then add this solution to the samples. There is no indicator for the end point and water will slow or inhibit the esterification. If you remove the reagent and solvent under a stream of nitrogen (you don't want to inject HCl into a GC or GC/MS as it will strip the bonded phase from the column and corrode the metal parts in the injectors and  detectors) you will loose the C1, C2  and C3 acid derivatives along with some of the C4 chains. You could try extracting the methanol reaction with pentane and analyzing the pentane layer; you won't see formate since methyl formate boils at 32C and pentane boils at 35C, some HCl will still be in the pentane layer.

Or use BCL3 in methanol visit: http://www.sigmaaldrich.com/analytical-chromatography/analytical-products.html?TablePage=105120181 Again there is the problem of injecting a reactive compound BCL3 into the GC or GC/MS or losing the lower esters upon evaporation.

With the diazomethane you can analyze the ether solution directly without worrying about the excess reagent which is easily be removed by adding a much less volatile acid (Salicylate or Phthalate) until the solution turns colorless. With diazomethane in ether you won't see formate since methyl formate boils at 32C and ether boils at 35C. For some notes on diazomethane visit: http://www.masterorganicchemistry.com/2011/11/05/reagent-friday-diazomethane-ch2n2/

It is always preferable not to have to make a derivative since you have to establish the adequate formation and stability of the derivative for your analysis; it is well worth the time to purchase and use a bonded polyethylene glycol (Wax) column and analyze the short chain fatty acids directly.