Hi everyone,
I have been extracting PAHs from different food matrix but I am constantly facing the problem of low recovery. Please suggest me what may be the probable reason for low recoveries and how I can resolve it for the method below:
I have taken 1gm of sample (spiked at 50ppb, 100ppb and 500ppb using 5000ppb standard and non-spiked) and kept it for 30min at dark in a glass bottle. After 30 min treatment for the spiking, I am adding 10ml of 1M KOH:methanol solution (1:1v/v) and heating the sample at 60 degree celsius for 5h in a waterbath. After 5h, the solution is kept overnight for cooling. Next day, I add up 10ml cyclohexane and 10mL of water and thoroughly mix the solution. Afterwards, the sample is again kept at 60 degree celsius in a waterbath for 1h. After cooling the sample is centrifuged at 4600g for 15min and supernatant layer is collected. The layer is then concentrated to 500-1000uL and clean up is performed.
For clean up, I am using Silica gel 70-230 mesh size (Sigma) with 60 Amstrong specification from Sigma. The method is:
Five grams of silica gel was activated at 180°C for 24h and kept in dessicator. Before use, the powder was deactivated with 1% water and 40mL of n-hexane was added to make the slurry. A packed column was made and concentrated sample extract was poured on the top of the column. The column was first eluted with 10mL of n-hexane. The eluate was discarded and the PAH fraction was further eluted from packed column with 20mL hexane: toluene (50:50 v/v) solution. The fraction possessing PAH was then concentrated to 0.5mL after solvent exchange to acetonitrile. The final volume for each sample concentrate was then made 1mL and passed through nylon filters before chromatographic analysis.
Please find the attachment of the sample chromatograph and matrix blank with and without spiking and with variable sample weight used for extraction. I think, The reason affecting the recovery possibly may be occurrence of a large peak at 5-7min. I am not getting what this peak is and how it can be removed. This peak is even visible when i tried a different method of extraction using hexane followed by treatment with MgSo4 and NaCl combination without any further treatment of sample.
I request to kindly check the chromatograph (Label or nomenclature used and their meanings: S2: sample weight 2gm; S5:sample weight 5gm; Matrix blank: method blank; Blank: pure ACN; Cal_25ppb is standard; spiked ppb: Sample pre-spiked with 25ppb followed by extraction) and suggest me how i should modify my method to enhance recovery and eliminate the interference observed at 5-7.5min retention time.
Thank you in advance for the valuable suggestions.
First of all some questions derived from your method description: it is a fatty food matrix based on your saponification step? Which kind of chromatographic analysis is used, HPLC-UV? Which PAHs are analyzed, e.g. EPA-PAH, PAH based on the SFC guideline or REACH-PAH? This is important to understand your problem.
As a general idea I would recommend to perform a number of spiking experiments. Spiking should be done separately before extraction, after the saponification step and after cleanup with the silica column as well as for the final solution. Then you should be able to elucidate possible losses due to sample prep. This could also help to see the influence of the large peak at 5-7min. If you use a mass spec technique you have further possibilities using labeled standards, but this is another topic.
Sir, thank you for your valuable suggestions. It has given me a very different perspective to work for solving my problem. I am working on 16 priority EPA-PAHs. I am using HPLC_FLR for chromatographic analysis. My food matrix is chicken and coffee. I would try the suggestions provided by you and check thee results. Thank you.
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