Sound like there's either (1) no chemicals from spider mites (unlikely), (2) the concentration of extracts in solvent is too low to be detected (likely), or (3) there's a problem with the GC.  Hexane should work well and at least extract some cuticular hydrocarbons, so I bet it's (2).  I would try to extract more mites using less solvent and skip the evaporation step (you should probably run solvent through a filter before GC injection) since some compounds would be lost during this step.

Good luck!

Hi Justin,

Thanks for the repsonse. I can confidently say that the GC-MS is completely fine and operational as Agilent have recently come to service the machine (less than two weeks ago) and they reported no issues. So that leaves me with your first two hypotheses. Hypothesis (1) cannot be true as there has been a paper publish (unfortunately in Spanish) whereby they managed to obtain cuticular hydrocarbons from spider mites. Hoewever, the authors don't state sample size, solvent volume, or extraction time in their methods so i'm a little dubious to be honest. I have attached their paper to this message. The issue I have with hypothesis (2) is that my solvent extract is already a frightening shade of green, which cannot be good for the GC and I worry that increasing the sample size from 25.0 mg will only exacerbate this issue (picture attached to this message). 

I have considered trying a different range of solvents: diethyl ether, ethyl acetate, dichloromethane and hexane to see if there are different results.

Joe

Dear dr. Joseph Roberts , are you going to extract xenobiotic chemicals bound with the animal cuticle or you are extracting chemicals from the cuticle composition? Any how in the both cases colored material could be a mater of interest if comes from the same sort. As you are encountering with problem , if you have plenty  of sample go to double extraction with a bigger amount then  first extraction you make with 2:3 acetone: hexane then ether + dichloromethane mix both and bring to dryness and re-dissolve in a suitable diluents tor run on gc

Iso pentane (2 methyl butane) is a good option. I like it. It extracts similar compounds than hexane (even more) and easy to evaporate (27.7°C) without loosing volatile molecules. It also evaporates quickly in bioassays, and you can get rid off the solvent very quickly. Also interesting for the GC using the retention gap (see Jennings publications) or cold trapping on a precolumn.

Dr Khan:

Thank you very much for your response. You're right it is quite possible that the coloured material may be of interest to me. I am looking to surface hydrocarbons and fatty acids to trial in bioassays in order to determine whether they are feeding cues for predatory mites. I can increase my sample size from 25.0 mg to 50.0 mg without any issues and I will try the solvent mixtures you suggested. What are the benefits of using multiple solvents to extract one sample and what solvent would I reconsitute my extract in once I have brought it to dryness?

Dr Thiery:

Thank you very much for your response, I will investigate retention gaps and cold trapping for the GC as well as investigate the use of iso pentane as a solvent.

Best wishes,

Joe

I'm agree with the idea of Denis Thiery. I have used n-pentane before to extract cuticular components from beetles with good results. I hypothesize that the amount of solvent that you used is quite large and probably you need to reduce it and/or increase the number of individuals. After that I suggest to adjust the time of extraction. The longer the extraction time the deeper in the cuticle the solvent may act. So if your idea is to identify some signals in the cuticle you should reduce the time.

All the best,

Ruben

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Thanks for your reply Dr Palma, it is much appreciated. What are the benefits of using n-pentane over hexane? The volume of solvent is quite high, but it only just covers the sample so I think it is the minimum I can use to extract 25.0 mg of mites. I am thinking that combining extracts before evaportating to dryness may be a good idea, as is reducing the extraction time. Thank you for this suggestion.

Best wishes,

Joe

Concerning how to use less solvent. You may extract in micro vials (like a sealed tip of a pastor pipette) or place the tip of the pipette filled with solvent on the body of your mite and extract by capillary effect

I have some 350 ul glass micro vials for conducting my extractions in, maybe I'll investigate the use of smaller vials.

Dear colleague,

Pentane or hexane were the solvents usually used for cuticular hydrocarbon extraction from insects. You can find in the attached  publications some informations on the extraction procedures of CHs using these two solvents.

Best regards N. Soltani 

Hello,

See also the attached publication.

Good luck !

N. Soltani

Dear dr. Joseph Roberts  you ask:

·         What are the benefits of using multiple solvents to extract one sample?

Obviously, different solvents have different solubility for the different compounds; moreover, for the cuticle one solvent may remove one compound to make accessible another bound compound to the second solvent.

·         and what solvent be used to reconstitute the  extract once brought it to dryness?

My experience is with acetone for the plant extracts. I find it fairly fine.

You should use isopropanol-n-hexane (60:40) it is best solvent to extract oily substances.

Hello, any article on the chemical composition of mites cuticule ?