Chromatography Forum

I wish to analyze fruit volatiles by SPME.
Q1. While searching for the literature, i came to know that people add pulverized (in liquid nitrogen) fruit powder, to the NaCl solution and then analyse the mixture by SPME. Can anybody comment on why NaCl is used? Is there any literature available for answer of this question?
Q2. IF amount of internal standards (IS) required e.g. per g fruit is around 500 micrograms, how to add such less quantity of liquid IS which is non-polar? If added as a solution in organic solvent (eg. DCM), will the solvent itself not saturate the fiber?
Thanks
Ram

Dear Ram,

As you already know the principle of SPME is partition (absorption or adsorption) of analytes between the matrix and the SPME fiber coating. So regarding your questions:
1. When one does SPME of volatiles (dissolved in water) it is convenient and smart to add NaCl or other salt to the sample matrix in order to enhance the "escaping" tendency of the volatiles from the matrix (salting out effect) and hence to enhance the partition in to the SPME fiber (or to acheve higher enrichment factors). As you mentioned, your fruit analytes are volatile, therefore adding salt to the water solution would "push out" the analytes in to the headspace (if you're doing headspace (HS) SPME - very convenient SPME procedure for volatile analysis) or onto the fiber coating itself (if you're doing direct immersion (DI) SPME - which is less convenient when doing SPME analysis of volatiles, due to the higer risk for fiber fouling, higher equilibration times, etc)

2. Internal standard must be added in the sample matrix prior the SPME. Because your sample matrix is aqueous, you should choose an appropriate (water miscible) solvent in which you going to dissolve the internal standard, initially (eg. methanol). DCM is not miscible with water (I assume that you're adding significant volumes of DCM solution). Choose another solvent that will efficiently dissolve your fruit volatiles and in the same time will be miscible with water.
Organic solvents wouldn't "saturate" the fiber, but may cause fiber fouling if used in higher concentrations. I tend to keep the organic solvents % in the water matrix below 1%. Higer %s of organic solvents in water can cause swelling of the fiber polymer phase. When polymer swelling occurs, the polymer coating may be stripped off the SPME fiber silica core during the retraction of the SPME fiber in to the SPME fiber assembly.
The fiber fouling effect of the organic solvents added to the water samples is more pronounced if you're doing direct immersion SPME, and it's less pronounced with HS-SPME.

What SPME mode (DI or HS) do you use? What type of SPME fiber coating do you use? What thickness?

Hope this helps

Regards

Thanks for the info
I have not yet done any expt. with SPME although have done lot of work with solvent extraction. Soon, I will be doing HS-SPME with PDMS fiber of 100 um thickness. Solvent extraction-GC shows that our samples are rich in monoterepne hydrocarbons (~80%), sesquiterpene hydrocarbons, lactones (about 8 compounds) etc. Is PDMS fiber suitable for such blend?

Dear Ram,

Because you're planning to analyze volatile non-polar analytes with molecular mass smaller than 300 Da, yes, it is recommended to use PDMS fiber with 100um thickness. PDMS coating is very suitable for wide range of non-polar volatile and semivolatile analytes. The distribution process of the analytes into the liquid PDMS coating is predominantly governed by absorption rather than adsorption. Generally, 100um PDMS coating is used when one plans to do analysis of volatile substances with molecular mass smaller than 300 Da and with distribution (partition) constants, K, smaller than 10000.
Also, when extracting small volatile molecules like your terpenes and sesquiterpenes, the 100um PDMS coating will give you higher enrichment factors and hence, lower detection limits. You could extract your analytes with 30um PDMS also, but you'll never get the enrichment factors for 100um PDMS, but you'll definitely cut the equilibration time to smaller values (times), required for establishment of the partition equilibrium between the analyte concentrations in headspace and the PDMS coating.

So, as you see every decision comes with some "trade-off". Picking the right fiber coating thickness is determined by your goals, properties of the sample and the sample matrix, properties of your analytes, the concentration of your analytes in the sample to be analyzed, time, cost of the analysis...

As you already know, the other SPME method parameters must be optimized after the picking of the SPME fiber coating (and thickness) like: vial size, sample and headspace volume size, temperature of extraction, pH of the sample, the addition of salt (salting out effect), equilibration time, etc. All these parameters must be adjusted also, if you're doing liquid-phase microextraction (or single-drop microextraction), as I understand from your previous post, that you're actually familiar with it.

Some references, which I hope will be helpful for you:

http://www.sigmaaldrich.com/Graphics/Su ... 0/4547.pdf

http://www.sigmaaldrich.com/etc/mediali ... /10942.pdf

Good luck with your SPME analysis

Thanks a lot for the information
Ram

I did my first SPME analysis...
1) I had added 4 internal standards as a solution in total 70 ul of methanol to the total ~3ml matrix and saw a big peak of methanol in TIC. Apart from being harmful to SPME fibre (is that true?), would methanol be also deleterious to the GC column (SLB5-MS)?
2) I could not see any of the 7 lactones which I had seen in the TICs of direct solvent extracts. SPME conditions: 1g tissue+2ml 25% NaCl, equilibration time: 15 min, extraction time: 15 min, all at RT, magnetic stirring.
Can anybody suggest any kind modifications to be able to see the lactones? I will be doing another extraction today at little higher temp. 40 C

1)So your solution is ~2,3 v/v% with respect to methanol. This shouldn't be a problem for the SPME fibers. You could "eliminate" the absorbed methanol onto the fiber coating by dipping the fiber into a clean (HPLC) water (for about several seconds) after the extraction of the analytes from the sample. In that way you'll eliminate the possibility of swelling the fiber coating (due to the presence of methanol in the sample) and prolong the SPME fiber lifetime.
Generally methanol isn't harmful for the SPME fibers when it is used in v/v% smaller than 2-3%. Methanol (present in your extracted sample) doesn't have negative effects on your column (you can use it without concern).

2)You could try the higher equilibration temperature and/or higher equilibration time. Because you're using PDMS 100um thick fiber coating (right?) you may need longer time to achieve the extraction equilibrium and to gain decent sensitivity of your analysis. Increasing the temperature of the sample too much, could have reverse effect = although the higer temp. the higher Henry law's constants of your analytes (and higher HS concentrations of the analytes) when doing HS-SPME, further increasing of the sample temperature can lower the partition coefficients from the headspace to the fiber coating for your analytes (because the absorption of the analytes from the HS into the coating is exothermic process). Try to optimize your SPME method by altering your extraction time, temperature, vial volume (sample to HS volume) etc, and report to us your progress.

Good luck!

1)So your solution is ~2,3 v/v% with respect to methanol. This shouldn't be a problem for the SPME fibers. You could "eliminate" the absorbed methanol onto the fiber coating by dipping the fiber into a clean (HPLC) water (for about several seconds) after the extraction of the analytes from the sample. In that way you'll eliminate the possibility of swelling the fiber coating (due to the presence of methanol in the sample) and prolong the SPME fiber lifetime.
Generally methanol isn't harmful for the SPME fibers when it is used in v/v% smaller than 2-3%. Methanol (present in your extracted sample) doesn't have negative effects on your column (you can use it without concern).

2)You could try the higher equilibration temperature and/or higher equilibration time. Because you're using PDMS 100um thick fiber coating (right?) you may need longer time to achieve the extraction equilibrium and to gain decent sensitivity of your analysis. Increasing the temperature of the sample too much, could have reverse effect = although the higer temp. the higher Henry law's constants of your analytes (and higher HS concentrations of the analytes) when doing HS-SPME, further increasing of the sample temperature can lower the partition coefficients from the headspace to the fiber coating for your analytes (because the absorption of the analytes from the HS into the coating is exothermic process). Try to optimize your SPME method by altering your extraction time, temperature, vial volume (sample to HS volume) etc, and report to us your progress.

Good luck!

Thank you for the information
Wouldn't the traces of water adsorbed on the fiber be harmful to the GC column?

You shouldn't worry about water absorption on PDMS fiber. Water is highly polar compound and PDMS is nonpolar polymeric phase, so the affinity of water to PDMS coating is negligible or none. Taking into consideration the fact that only ng amounts from the analytes with similar polarity (to the PDMS fiber coating) are absorbed onto the PDMS fiber, the possibility for significant water absorption and subsequent harmful effects on the capillary GC column is none, though.

Regards[/i]