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Posted: Wed Jun 02, 2010 9:51 am
by Bruce Hamilton
I don't recall an obvious problem with oxygen on any FAMEs capillary columns, but I was using H2 carrier with two Oxy-traps.
I do recall that SP-2330 gave co-elution issues ( C18:3 and C20:1 ? - it's decades ago ), and I moved from it quickly. It was also the first glass capillary column we purchased, and I moved to a different polarity fused silica column, so the co-elution issue may have been subsequently addressed.
There are several polar silicone-based columns available, each with differing selectivity. CW20M is popular for C8+ FAMES because it usually resolves groups of common unsaturates within even carbon numbers and by unsaturation, but it's not always the most suitable for shorter chain lengths.
Posted: Wed Jun 02, 2010 1:24 pm
by AICMM
leo1352,
As Don notes, three minutes is way, way too long. Half a minute or 0.75 minutes will work even for a 0.25 column.
Has anyone used a LowOx for this application? Seems like it might be a good match.
Best regards.
Posted: Wed Jun 09, 2010 11:34 pm
by leo1352
Thanks for the strong response. I have tried this method and column DB-225 (I used a 60m instead) coupled with MS just to make sure what I am getting is Butyric. .
http://www.chem.agilent.com/cag/cabu/pdf/fabfames.pdf . it eluted as you said early on 3.4 min and the peak was broad. meanwhile the BP21 column from SGE company looks to have good separation of Butyric acid without methylating it.
http://www.sge.com/products/columns/gc- ... -bp21-ffap . does any one of you friends have any idea how can I simply separate Butyric acid out of Milk. it should mostly be in aqueous phase, but I need to separate it from glycerides in milk into free form as well.
Posted: Wed Jun 09, 2010 11:46 pm
by leo1352
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
The carrier gas is He, you mean that the pressure is way high for such a column length?
Posted: Thu Jun 10, 2010 3:02 am
by Don_Hilton
Separation of butyric acid from milk. I wo uld suggest looking around on line. DO any of these help?
http://www.cyberlipid.org/acylglyt/acyl0006.htm
Molkentin, J., Precht, D. (1998) Precision of milk fat quantitation in mixed fats by analysis of butyric acid. Chromatographia 48: 758-762
http://www.doaj.org/doaj?func=abstract& ... o=15&toc=1
http://lipidlibrary.aocs.org/GC_lipid/0 ... index2.htm
While they may not direclty answer your question, they may cite other works which do or they may give you some ideas.
Posted: Thu Jun 10, 2010 7:18 am
by Peter Apps
Hi Leo
Yes, 3 bar is way too high for the column dimensions that you give. If you really do have this pressure in the inlet, then either your flow rate through the column will be excessive or you have some major plumbing problem that needs to be sorted out before you worry about separating particular pairs of peaks.
Injecting a plug of gas from a cigartte lighter and measuring its retention time is a quick and easy way of checking carier gas flow - on a 30 m column with helium you need a retention time of 60 - 90 s for ordinary conditions.
Peter
Butylation how do you think?!
Posted: Fri Jun 11, 2010 2:27 am
by leo1352
I had some literature review, some advises are to use BUTANOL instead of METHANOL with sulfuric acid for making esters of fatty acids especially the short chain ones. this way they will have heavy molecular weight and thus longer retention times that make it's peak clear off the solvent.
Posted: Fri Jun 11, 2010 11:52 pm
by Bruce Hamilton
I need to ask:- What are you trying to measure?
Is it:-
1. The free butyric acid content?.
2. The butyric acid present in neutral lipids?
3. The total amount of butyric-containing molecules in milk.
Each requires a different protocol, and a sample-preparation technique and it's important that that the preparation is specific.
Forming butyl esters can result in better separation of the short chain fatty acids from solvents, but it also can delay the longer chain compounds, making methods much longer.
As I noted above, the dairy science literature has several publications covering the analysis of milk and milk-lipids. A good literature seach should locate them.
Posted: Sat Jun 12, 2010 10:13 am
by leo1352
I need to ask:- What are you trying to measure?
Is it:-
1. The free butyric acid content?.
2. The butyric acid present in neutral lipids?
3. The total amount of butyric-containing molecules in milk.
Each requires a different protocol, and a sample-preparation technique and it's important that that the preparation is specific.
Forming butyl esters can result in better separation of the short chain fatty acids from solvents, but it also can delay the longer chain compounds, making methods much longer.
As I noted above, the dairy science literature has several publications covering the analysis of milk and milk-lipids. A good literature seach should locate them.
yes, I am measuring both free and glycerol form of fatty acids which butyric is the most volatile of them in milk. so sulphuric acid helps hydrolysing the tri,di or mono glycerides and at the same time butylating the free fatty acids. I guess longer GC times is the price I should pay for as you said to have a Butyric acid separated. I believe it should elute around 7 min with DB-WAX with my settings. I will let you know about that. One another advantage of longer elution time of Butylated Butyric acid is that if it elutes at later than 5min our GC-IRMS detector will be safe from solvents as it is very sensitive to solvents containing halides. thats why our friend operating the instrument use a back flash of He until 4 minutes to make sure no solvent is coming through.
Posted: Tue Jun 22, 2010 9:20 am
by leo1352
I have done the butylation of fatt acids, the good news is the butyric acid is eluting at 7.2 min with a 60m DB-225 colum. The bad news is that the butylated sultion with sulfuric acid canno't readly separate from added Hexane to the tubes . That means the sulfuric acid, buyl esters and Hexane make a unique phase from water phase and it makes it too dilute to take this phase for GC-MS. I did now propylation with Isopropanol and I get good separation with addede Hexane. I will let you know about the retention time and quality of the peaks soon.
Posted: Wed Jun 23, 2010 6:51 am
by leo1352
Well, butylation has some drawbacks. it(sulfuric acid-Butanol) can not be separated out when you add Hexane to collect your fatty acid esters. I did the esterification with Sulfuric acid _isopropanol instead, which was mentioned in some old articles, I got very good separation of Hexane phase plus the final concentration of fatty acid propyl esters are almost 3 times more than fatty acid methyl esters by FID, especialy on C 4:0 till C 8:0. Butyric Isopropyl ester eluted around 7.7 min on a DB-5 column which is safe from solvents now, especially for GC-IRMS analysis.
how to program DB-WAX for Butyric Acid in Milk FAPE
Posted: Fri Jun 25, 2010 7:05 am
by leo1352
Hi again,
As I said I had very good separation from my sample with a 45m DB-5 which belonged to another lab and I do not have everyday acess to it. Now I have problem with Butyric acid separation on DB-WAX 25m , with the same sample I can only have caproic acid C 6:0 and later fatty acids of C 8:0 ,C 9:0 . C 6:0 is eluting at 6 min but I get no peaks for butyric acids. my program is 40min hold for 3min , then ramping to 220 in 15C/min holding 2min at 230 and finaly 8C/min to230. I do really appreciate if anyone can help me out of this nightmare.
Posted: Fri Jun 25, 2010 8:07 am
by Peter Apps
Hi Leo
It is a recurrent theme in recent threads that trying to do a separation on the wrong column usually wastes more resources than would be spent on just buying the right column. In this case you are in the happy position of knowing exactly which column you need to do the separation.
Peter