How to identify your actual peak?I am confused by looking at so many peaks that which is the my peak which I am looking forward to it.
I used HP-5 column
injection temp-50`C
Split ratio- 20:1
column temp-250`
Hi Shikha, you have two options: 1. Buy FAME standards, inject and see where they elute under your GC conditions, 2. Interpret MS spectra and compare them with commercial spectra libraries (e. g. NIST). - Markus -
P.S.: Some resources of FAME standards:
http://www.restek.com/catalog/view/6098
https://www.sigmaaldrich.com/catalog/product/supelco/47885u?lang=de®ion=DE
https://www.sigmaaldrich.com/catalog/product/supelco/18921amp?lang=de®ion=DE
https://www.merckmillipore.com/DE/de/products/analytics-sample-prep/chromatography-for-analysis/gas-chromatography-gc/gc-reference-substances/xzab.qB.P5cAAAE_b.V3.Lxi,nav
https://www.researchgate.net/post/Which_standard_reference_should_be_used_working_on_GC_FAME_chromatograph
I harvested e.coli cells in the late exponential phase with oleate as a carbon source. I centrifuged the cells, lyophilized them and then followed FAME protocol to convert fatty acid to methyl esters. I extracted FAME in hexane. I run the samples in GC-HRMS with HP-5ms column with injection temp-50`C and column at 250`C. I didn't run standards but through NIST library I got some clues about the compounds which gave peak. I got two different peaks for oleate. I also got peaks for C12, C14, C16 FAMEbut my substrate was oleate(C18:1). Many papers have claimed that when oleate is used as a substrate, C4 and C6 FA gets accumulated in the cell.Ideally, I should have got the peaks for it but I didn't get any peak of them.I am stuck now that what to do. I have one more question, I have got the FAME mix standards concentration of each compound in that mixture is 1000ug/ml in hexane. How much concentration of standards should I prepare?
Thankyou so much christ, davies and carney
Hi Shikha, the concentration of your FAME mix standard depends on the expected concentration.For identification purposes (retention time), the concentration is of secondary importance. There's something else if you want to quantify. I guess that 1mg/ml is more than enough. I would probably dilute the standard by a factor of 10-100. If the concentration is too high, you may overload your column. The result is a bad peak shape.
- Markus -
Shikha,
Perhaps you need to start the task with parameter optimization like injector temp, oven temp, split, injection volume of your sample, ion source and transfer line temperature etc. You may be well aware about total nos of FAMEs compounds present in target sample. First, you note down the total number of baseline separated peaks in your sample, than try to improve separation if number is less. Once, all desired peaks are there, identification will not have a problem.
What I see from you question is that you are working with isothermal conditions. temp programmed is best followed practice rather than iso-thermal column oven condition . Besides, the injector set point temp is also below. Many of FAMEs could not get enough temperature to become vapors, I guess. HP-5 may be good to start with method developement. Otherwise FAME specific FSCAP columns are available with all manufacturers. For the rest, Markus and Noel has already suggested points for improvement.
Good Luck
Dr Chanotiya
Indeed, I agree with earlier answer, you should buy FAME standard in order to determinate your peak, then you identify and set up your standard toward your sample peak. thanks
I have run the FAME mix standard (C4-C24) and run on HP-5 column with injection temp-50`C and column temp-280`C. I got all the peaks of standard so I think i should not tweak the protocol but in sample I should be able to get C4 and C6 peak which I didn't get.
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