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Post by biokemist89 on Mon Jun 30, 2008 5:08 pm

1. Im having a problem with my analysis of FAMEs, often I got a recovery of <90 or >100 using internal standard method. The requirement is >90 but <100. What are the possible cause of having a total FAME of <90 and >100? Sad

2. For analysis of total FAME in biodiesel (coconut based). Also I cannot get the min specification of 96.6% FAME, the method is based on EN 14103 (IS method) but we modify part of it to compensate the coconut methyl esters. The required column is used, since then we got low FAME content more then the usual (97 or 98). What are the probable cause of getting low FAME? Sad

Thanks Very Happy

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Re: Tanong po?

Post by Admin on Sun Jul 06, 2008 7:58 am

Hi Biokemist89,

Thank you for the posts. Our group was quite busy with LabTec-Cebu in the past few days, and I was hoping you would understand my late post.

With regards to FAME analysis:

1. In order to improve sensitivity of the GC analyses it is important to test the effect of carrier gas flow rate, as well as gas (the gas used to burn the flame, usually a mixture of Air and Hydrogen) flows in FID, in order to reduce the noise from the Hydrogen flame (Laakso et al, 2002 ). In general, most serious inaccuracies result not from detection, but from losses during esterification or injection.

2. The amounts of FAMEs are determined via the peak area. The peak area of a given amount of FAME is not influenced by the peak shape provided that extremely strong loading or tailing does not occur (Christie, 1989 ). The linearity should be checked with various dilutions of standard mixtures which should be selected according to FAME concentrations of the samples to be analyzed for quantitation (Arbertyn et al., 1982 ).

3. Quantitation based on peak height has the disadvantage that even minor changes in the peak shape distort the result. Problems of quantitation occur if two peaks are not completely separated, especially if one component is visible only as a minor shoulder or broadening of a major peak. In these cases, either the operating conditions must be altered or a column with higher resolution capability must be used (Christie, 1989 ).

4. Quantitation of FAMEs is commonly carried out after calibration of the system with standards containing known amounts of FAMEs. FAME mixture standards should be similar in composition to the samples to be analyzed (Shantha et al., 1992 ). A popular method for absolute quantitation is the addition of a FAME not present in the sample as an internal standard (IS). The internal standard should be selected according to the sample analyzed. Because since C16:0 in most biological samples are predominant, C17:0 is a popular internal standard in the analysis of FAMEs . If the sample contains C17:0, other FAMEs such as C19:0 or C21:0 can be used as the internal standard (Eder et al., 1992). If boiling-point-dependent sample discrimantion can not be eliminated by optimization of the equipment and injection technique (section 2.3.5.A), the use of more than one internal standard can improve the accuracy and precision of results (K Eder, 1995 ).

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Post by biokemist89 on Mon Jul 07, 2008 2:54 pm

1. Actually the application is for trans fatty acids in fats and oils but we are also considering the FAME content to for fat and oil blends. Eversince we are using C13 as internal standard because according to some study C17 might be present in some animal fat. Does it affect the result if we use low-boiling IS in our analysis?

Thank you very much! Laughing

You're a great help Laughing

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Re: Tanong po?

Post by LFerrer on Thu Jul 17, 2008 9:04 am

Let me try to address your issues:
1. Assuming the method that you are using is validated there must be no issue of low recovery. First, how low is low recovery for you? If you are expecting between 90 to 100% recovery and you are getting say 85% recovery - you must check the robustness of your method? Is the method your using exhibit 90% robustness? If so - then you must expect that your result will be off by at least 10%. Please be guided that GC is a relative technique meaning results are tested agaisnt known standard. If for instance - after method calibration - you injected the standard and consider it as sample and the result is with-in acceptable recovery, then you're problem is on the matrix effect of the actual sample. To be able to resolve this - please try to do standard addition experiment and then check if recovery become acceptable. If this happen, then your problem can be easily solve by simple baseline subtraction technique.
2. On issue of IS. Please check first why are you using IS on your method? IS in low or high boiling part does not matter - for as long as there is no overlapping peak. For as long that it does not overlap on anyway with any adjacent peak, it will have no effect on quantification whatsover.
3. Also- please check how off is your recovery? How much are you getting? Compare to what you expect. Also - a sample and standard chromatogram will help us alot to troubleshoot your problem. Maybe you can email me some chromatogram so i can more or less give some specific solution to this issue.

Thanks

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Re: Tanong po!

Post by cromwell on Thu Jul 17, 2008 2:14 pm

biokemist89 wrote:1. Im having a problem with my analysis of FAMEs, often I got a recovery of <90 or >100 using internal standard method. The requirement is >90 but <100. What are the possible cause of having a total FAME of <90 and >100? Sad

2. For analysis of total FAME in biodiesel (coconut based). Also I cannot get the min specification of 96.6% FAME, the method is based on EN 14103 (IS method) but we modify part of it to compensate the coconut methyl esters. The required column is used, since then we got low FAME content more then the usual (97 or 98). What are the probable cause of getting low FAME? Sad

Thanks Very Happy

Can you email me your procedure of EN 14103, for Fames. Actually, my company is producing fatty acid, we are using AOCS method.

Can u also email me chromatograms of your C13 alone used as your calibration and of your sample w/ C13 IS.

I hope that the amount of C13 you spike for your sample is the same amount you used for your C13 for the calibration.

My email address: cromwell_flores97@yahoo.com

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Re: Tanong po?

Post by cromwell on Wed Jul 23, 2008 2:58 pm

we encountered that before too biokemist, if you are using manual injection, be careful in injecting your sample, the tip of the syringe must not touch the glass liner, there is a possibility that your sample will stick on it, that's only our observation if our two trials do not match.

sample preparation does really matter, especially in diluting your sample, few are using the term, "end volume dilution".

thank you.

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Re: Tanong po?

Post by biokemist89 on Thu Jul 24, 2008 12:06 pm

so its the matter of mode of injection? but before we had autoinjector in our system and still the same problem we experienced.

actually the method is for trans- fatty acids, and we use %FAME recovery to assure that our analysis is accurate and precise becuase our customer demands for % FAME for every oil blend we deliver to them. The standard we used is a mixed standard in an ampoule (36 glc from nu check), and we correlate it to the sample based on the response factor of each FAME. We are getting an RSD of >2.5% as required by the customer but when we are to calculate the result sometimes we are gettig <90 or >100?


regarding on en method we only have a hard copy oi it, is it ok with u if i type it in details?

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Re: Tanong po?

Post by cromwell on Thu Jul 24, 2008 1:56 pm

ok. pls type the method.

If you still encounter the same problem, pls inject the sample right after you extract or prepare the sample, because some fatty acids will precipitate immediately (higher chain. if you want, use nitrogen to preserve the sample in the succeeding injections and put it in the chiller. If you can't get a repeatable results, then you must prepare a new extract/sample to inject from the same sample.


Last edited by cromwell on Thu Jul 31, 2008 5:44 pm; edited 1 time in total

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Re: Tanong po?

Post by cromwell on Thu Jul 24, 2008 2:21 pm

The mixed standards is only to determine the peaks of fatty acids present in your sample. The internal standardsyou use is C13, right? It must be pure, C13. For example, if you take 50mg of pure C13 and put it together into your sample and dilute it to volume, end volume dilution. There's a corresponding area generated on the chromatogram report, let us say, the area is "1000 ". In your succeeding preparation, take 50mg exactly of pure C13 and dilute it in the same manner with your sample. It must generate an area of 900 to 1000, so that your recovery will fall on 90 to 100%. If you cant weigh 50mg exactly, you can have a ratio and proportion to correct for your calculation. I hope this will help you.

thank you


Last edited by cromwell on Sat Jul 26, 2008 12:31 pm; edited 1 time in total

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Re: Tanong po?

Post by cromwell on Fri Jul 25, 2008 4:27 pm

Just an addition. The mixed analytical reference standard for correlation is used to get a correction factor based on the result of your chromatogram with the known composition of your mixed analytical reference standard, and you use this result in calibrating your sample. Does your mixed standrad contain C13? tnx. please include in your mixed standard, C13 as part of the mixture so that you can also establish a correction factor for C13.

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