Butyric acid separation

[leo1352]

Hi everyone I am new to here and just star asking questions. I am planning to analyse milk fatty acids to check it’s fatty acid isotopic signature with GC-IRMS. Before that I need to get a good extraction of milk fatty acids and analyse its profile with GC-FID. I am using DB-WAX 30m for the time being. My problem is butyric acid is co-eluting with my solvents which are Hexane(for the extraction) and chloroform-methanol(2:1) which I spiked the latter to each vial in 100 µL with my internal standard of Methyl Tricosanoic C23:0 dissolved in it.
My solvent mixtures are present almost till 4.5 minutes. I wonder if someone has any suggestion about how to get a separate peak of Butyric acid. Do I have to change my solvents, column or GC timing program.

[Don_Hilton]

Tell us a bit more abuout your chromatographic conditions, particularly are you making a splitless injection, how large, what inlet temperature, whick liner, what carrier gas, and what is the linear velocity in the column?

[leo1352]

Tell us a bit more abuout your chromatographic conditions, particularly are you making a splitless injection, how large, what inlet temperature, whick liner, what carrier gas, and what is the linear velocity in the column?

thanks for the reply, I made a splitless injection of 1 micro litre. I forgot to tell that the assay Tat I talked about, has been made with four standard fatty acids of C4:0 , C 14:0 , C16:0 and C18:1(100micro gram/100 micro litre each) which got methylated prior to injection with the internal standard of C23:0. the inlet temperature is 210C ,the carrier gas is He , I do not remember the liner velocity but I will check the lab tomorow to see it.

[mahdiebrahimi]

The solvent (hexane) can be overlapped by butyric acid. You can change your solvent from hexane to petroleum ether.

[leo1352]

The solvent (hexane) can be overlapped by butyric acid. You can change your solvent from hexane to petroleum ether.

You mean to extract my fatty acid metyl esters with petroleum ether instead?

[Don_Hilton]

A splitless will suffer from the effects of putting the entire contens of the inlet onto the column and can result in a tail on the solvent peak. A shorter puge off time can shorten the solvent tail - but if too short, with the cost of discrimination in the inlet. So, what is your liner volume, what is your current purge off time, what kind of liner are you using, what is the oven temperature at injection - and ramps, and what is the carrier flow rate in ml/in? (The linear velocity is important because you need to be in a reasonable range. And carrier flow rate is an important consideration in selecting purge off time.)

Petroleum ether can be a good solvent. Given that this is a petroleum fraction, keep an eye on the background. If there are any higher boilers in the petrolelum ether try pentane.

The matter of changing solvent brings up another question: Are you trying to follow a previously developed procedure or are you creating a new procedure?

[mahdiebrahimi]

You mean to extract my fatty acid metyl esters with petroleum ether instead?

The extraction by chloroform: Methanol (2:1) is good enough. Just at the end of the methylation process you can use petroleum ether as a solvent.

[Consumer Products Guy]

Have you tried n-butanol esterification (catalyzed with either sulfuric acid or BF3) ?

How about DMF and trimethylsilyl derivatization? We used the TMS derivatives for hexanoic acid in fatty acid samples.

[leo1352]

A splitless will suffer from the effects of putting the entire contens of the inlet onto the column and can result in a tail on the solvent peak. A shorter puge off time can shorten the solvent tail - but if too short, with the cost of discrimination in the inlet. So, what is your liner volume, what is your current purge off time, what kind of liner are you using, what is the oven temperature at injection - and ramps, and what is the carrier flow rate in ml/in? (The linear velocity is important because you need to be in a reasonable range. And carrier flow rate is an important consideration in selecting purge off time.)

Petroleum ether can be a good solvent. Given that this is a petroleum fraction, keep an eye on the background. If there are any higher boilers in the petrolelum ether try pentane.

The matter of changing solvent brings up another question: Are you trying to follow a previously developed procedure or are you creating a new procedure?

Ok, Inlet temperature is 290C ,purge time 3 min ,purge flow 18.5ml/min , flow is pressure controled. colum pressure 3 bar, tempertature program is: 4.5 min 30C then from 30C -220C in 16.3 min , from 220 to 230 in 4 min. I am thinking maybe as you said splitless could help me. but I have to separate butyric acid from my solvent and it's been a reall problem for me.

[leo1352]

Have you tried n-butanol esterification (catalyzed with either sulfuric acid or BF3) ?

How about DMF and trimethylsilyl derivatization? We used the TMS derivatives for hexanoic acid in fatty acid samples.

Do you mean use your suggested solevnts instead of Hexan? I haven't tested them so far. the final solvent has always been Hexane in the GC vials. what's your idea about using them? is it because they might elute faster than Butyric acid?

[Don_Hilton]

Two things I don't know yet from you: flow through the column in cm/sec and flow in ml/min. Without your column dimensions, I can not calculate these.

But, assuming a 0.25 mm ID column or a 0.32 mm ID column, three minutes is a fairly long purge off time. You may be able to shorten this down. I would take the volume of the GC inlet liner you are using and determine the time it takes for three to four volumes to pass through the inlet at the flow rate you are using and try that time for the purge off time. If the tail of the hexane peak and the analyte peaks are well separated, then I would see if you can use a slightly longer purge off time. The reason to use as long a purge off time as you can tollerate is to avoid discrimination.

But, before making this adjustment, the reason I ask about linear velocity is that you get the best separation with an optimum flow (linear velocity) through the column. In a capillary GC column, I like to see a linear velocity in the 22 to 70 cm/sec range.

And there is a reason why I asked if you are trying to follow a previously developed procedure or if you are creating one. If you are trying to set up a method that has alredy been proven and is used by others - we need to be sure that it is not a minor setup problem that is causing you a problem. If you are developing a method on your own, there are many more options - but the path may be a bit longer than implementing a method that has already been developed.

[BZK]

Methyl ester of butyric acid could not be analysed by splitless without be overlapped to the solvent, even if very low boiling solvents such as pentane or petrol ether are used. Due to low boiling point of methyl ester of butyric acid no focusing is possible into the top of GC column and peak is then very broad and mixed with solvent front. You have only two way to solve the problem. First (better) : use on-column injection and pentane as solvent. Second : use split mode injection with at least 1:20 of split ratio, with 20 time loss of sensitivity.

[Bruce Hamilton]

There are plenty of papers that discuss the issues of analysing the fatty acid profile of dairy lipids. The technique that I use is derived from the procedure published by Bannon et al in 1985, as part of their series on FA analysis.

Analysis of Fatty Acid Methyl Esters with High Accuracy and Reliability. IV. Fats with Fatty acids containing Four or More Carbon Atoms.
Cecil D. Bannon, John D. Craske, Audrey E. Hilliker.
JAOCS Vol 62, No 10 ( Oct 1985 ), p1501-1506.

The major issues are partitioning of short chain acids, but I found that vigorous mixing during derivatisation was critical to ensure quantitative conversion. That's contrary to their subsequently reported misgivings about the ambient temperature derivatisation ( JAOCS Vol 65 No 2 ( Feb 1988 ) p262-266 ), which used a 22m x 0.25mm x 0.2um DEGS capillary column.

However, their initial method is so quick and simple, and easy to downsize to smaller samples that it remains my preferred method for neutral lipids, with freshly-generated diazomethane for derivatisation of free fatty acids.

They used iso-octane and a DEGS packed column. I also prefer using iso-octane, but I've also used n-hexane, on polar and non-polar capillary columns. I tend to use split, and for short chain would consider both more or less polar capillary stationary phases than Carbowax, depending on the presence and profile of unsaturates. Using longer, less polar, columns may retain the FAMEs, often moving shorter chains away from the solvent, as does using more polar, such as DEGS, OV275, Silar 10CP, etc..

The dairy science literature has plenty of papers discussing techniques to analyse the fully fatty acid profile, starting with C4.

Bruce Hamilton

[Consumer Products Guy]

They used iso-octane and a DEGS packed column. I also prefer using iso-octane, but I've also used n-hexane, on polar and non-polar capillary columns. I tend to use split, and for short chain would consider both more or less polar capillary stationary phases than Carbowax, depending on the presence and profile of unsaturates. Using longer, less polar, columns may retain the FAMEs, often moving shorter chains away from the solvent, as does using more polar, such as DEGS, OV275, Silar 10CP, etc..

DEGS !!! Better keep oxygen away. We used DEGS packed columns in the 1970s, switched then to SP-2330 cyanopropyl silicone columns, had same order of elution but tons more stable and rugged for fatty acid methyl esters.

[Peter Apps]

[quote= colum pressure 3 bar, [/quote]

3 bar on a 30m column, something is wrong here . You need to check for leaks (using a leak seeker) and then measure the time that it takes an injection of lighter gas to pass through the column.

What carrier gas are you using ?

Peter