1. Establish a standard curve:

First, it is necessary to measure the gas chromatogram of the standard solution of different concentrations, and record the retention time (tR) and peak area of ​​each component. Then draw a standard curve based on the concentration of the standard solution and the corresponding peak area. Generally speaking, there is a linear relationship between peak area and concentration. With the peak area as the ordinate and the concentration as the abscissa, a linear equation can be obtained, such as y = kx + b, where y represents the peak area, x represents the concentration, and k and b are the slope and intercept of the linear equation.

2. Analyze the sample to be tested:

Analyze the sample to be tested by gas chromatography to obtain its gas chromatogram. Record the retention time and peak area of ​​each component in the sample to be tested.

3. Confirm the component:

By comparing the retention time of each component in the sample to be tested with the retention time of the component in the standard curve, the component in the sample to be tested can be confirmed.

4. Calculate the mass concentration:

Using the peak area of ​​each component in the sample to be tested and the standard curve equation, the mass concentration of each component in the sample to be tested can be calculated. The specific calculation method is to substitute the peak area of ​​the sample to be tested into the standard curve equation and solve it to obtain the concentration x. The calculation formula is: x = (y - b) / k

Where x is the mass concentration of the component in the sample to be tested, y is the peak area of ​​the component in the sample to be tested, and k and b are the slope and intercept of the standard curve equation.

Through the above steps, the mass concentration of each component in the sample to be tested can be obtained. It should be noted that in the actual operation process, it may be affected by various factors such as instruments and operators, so it is necessary to ensure the accuracy of the operating specifications and instruments.

Hi, Chen. Thanks for your reply. I briefly explained the protocol and the issues we faced. Kindly go through it and give your suggestions.

We are estimating lipid content in shoot tissues of rice using GC-MS. For this, we have extracted individual lipid classes (galactolipid and phospholipid) and esterified the lipid samples by adapting the protocol of Wang et al., 2011 (doi:10.3791/2518). During the esterification process, we added pentadecanoic acid (C15:0) as an internal standard. After this, we ran our samples in Shimadzu's GC-MS instrument. Initially, we ran Supelco 37-Component FAME Mix (SP™-2560) to determine the retention time of different carbon compounds. Next, we ran pentadecanoic acid (C15:0) and other lipid samples separately.

Now, we are facing issues in incorporating the results for estimating lipid content in individual samples.

1) How to calculate % total FAME and % mol FAME using our data?

2) Because, the internal standard pentadecanoic acid (PD) (C15:0) gives multiple peaks at different retention times, instead of a single peak. So, while normalizing lipid content, do we need to consider the cumulative peak area of PD (or) only the area of the major peak of PD that matches with the retention time of C15:0 in Supelco 37-Component FAME Mix?

3) We have added 50 ug/ml of PD as an internal standard in all the lipid samples. So, while estimating the lipid content of these samples, do we need to subtract the peak area generated by PD?

Chen Jingwen