Release of Volatiles at different pH levels

[bethlynn]

Hi all.

Would someone please verify my logic below?

I use a sodium salt to raise the pH of a citral solution from 2.8 to 4.2. I analyzed both samples on GCMS - static headspace. Looking at response of terpenes, the lower pH has a higher signal response than higher pH. Salt was used to raise the pH not for purposes of salting out. So I am looking to understand the effects individually.

1. The addition of salt should release more volatiles but this did not occur - response was greater win lower pH which had no salt.
2. The lower pH is less neutral therefore the volatiles can be carried easier into headspace - this did occur.

So the effects of pH on the release of terpene compounds into headspace are greater than the salting out effect. Does this make sense or have I missed some interactions?

Thanks.

[MichaelVW]

I don't know nothin' about nothin' - but could you use look at the effect of pH independently of the salt by using HCl/NaOH or something?

How much was the differnece between the two runs?

[rb6banjo]

From your post, it's not totally clear what your analytes are but if it's only citral then I think this makes sense.

Citral is an alpha-beta unsaturated aldehyde. It can tautomerize to make an ene-ol. Ene-ol's are more acidic than your standard alcohol. It is likely that raising the pH of your solution could have an adverse affect on your ability to detect citral because you've titrated some of the citral inadvertently. Perhaps other analytes in your mixture can exhibit this chemistry as well?

Did you try a neutral salt (sodium chloride perhaps) to see if you can achieve the "salting out" that you appear to be seeking?

[bethlynn]

Michael - I could have used something different but experiment was already set.....

rb6banjo - hmm a wee bit over my head. I could use a beginner chemist's view. Analytes are terpenes specifically limonene, myrcene, cymene etc. The salt I used to increase the pH was Na citrate. At point of analysis, i trialed different levels of NaCl with marginal increases in signal counts at a 10%w/v dosage. When you say "titrated out" exactly what does that mean - there is no visible precipitate on the bottom nor has colour altered. There is a clear aroma distinction between the samples. The highest pH (most amount of Sod.citrate) has barley any citral/lemon aroma which co-incides with the analytical results.

i am just looking to understand better (chemistry wise) how the citrate affected the sample.

Thanks.

[rb6banjo]

Adding salt of any type isn't going to change the solubility of those hydrocarbons a great deal. They are already quite insoluble in water. That's why you didn't see a tremendous increase in signal when you added the sodium chloride.

Ene-ol's are more acidic than standard alcohols (like methanol and ethanol). When you increase the pH you are increasing the concentration of hydroxide in the water. The more abundant hydroxide would then be available to react with the more acidic ene-ol, thus decreasing the availability for the citral to partition into the headspace. Hence, the amount detected would be expected to be lower. This argument becomes moot when you are only looking for hydrocarbons. They can't form ene-ol's.

Is the citral concentration - as determined using your chromatographic method - lower at higher pH?

I expect that the hydrocarbons would be substantially unaffected by salt and pH.

[bethlynn]

rb6banjo - thank you for reply. Yes all of my compounds of interest are lower at higher pH in the control samples. I also subjected these samples to warmer temperatures and photo conditions - which interesting enough has created very different results....as you would expect.

so if it is a moot point regarding the hydrocarbons - i guess i still do not understand why the volatiles are do not partition into headspace the more neutral the solution becomes. At one point I thought I understood but I seem to be a wee bit confused again.

Thanks.

[Peter Apps]

Salting out works best for solutes that are more water soluble than your terpenes, and pH changes affect compounds that are themselves acidic or basic, which your compounds are not, so there is no reason to expect any difference due to your changes in conditions. Are you sure that the differences you see are not just due to analytical variation - the repeatability of equilibrium headspace can be poor if conditions are not optimised.

Peter

[bethlynn]

Peter,
I am sure the results are not due to analytical variation as I have analyzed in replicates of 4 and 3 separate time intervals spanning every two weeks. I ran PCA with 85% variance in 3 components, second component being effect of pH (3 separate pH levels). Searching thru internet left me with as pH increases the nonpolar terpenes want to stay in solution and not partition to headspace. I just cant fully grasp the reasons especially when i have used salt to increase the pH - although I think rb6banjo has tried to explain.

Thanks.

[rb6banjo]

My explanation would only apply to compounds like citral.

https://flic.kr/p/o19Bmt

The behavior of the hydrocarbons does not apply. I do not readily see how adding sodium citrate to this seemingly simple system should affect the partitioning of the hydrocarbons at all.

You mention that the increased pH caused a suppression in the "lemon aroma" of your solution. If that's the essence of the problem here, could it be that those hydrocarbons are not the active flavor/aroma ingredients?

[rb6banjo]

As part of a project I'm working on, limonene was of interest. So, I calibrated for limonene in water using just water and water containing 5% sodium citrate (pH measured to be 8-9 with 0-14 pH strips). Here is the result:

https://flic.kr/p/ohHX3X

No great enhancement of the limonene response with added salt - as predicted by the fact that hydrocarbons are not terribly soluble in water in the first place. The response factors are within 4% of each other ("enhancement factor" at the bottom of the table):

https://flic.kr/p/ohx8DN

Raising the pH to 8-9 ish in water only, didn't show any strange chemistry (i.e., loss) for limonene.

I believe that salting is a sample prep. step that is not needed for this analysis. You can certainly get very low detection limits without the added complexity.

[bethlynn]

rb6banjo - i like the flic.kr application - i will learn.

I want to back track a little as I may have confused things a bit. I wasnt using the the sodium citrate as a salting out agent but a pH modifier in attempts to make the limonene more stable - i commonly work at low pH. The three pH levels I am working at are 2.8,3.5 & 4.2, all three contain increasing amount of sodium citrate. My bad - i didnt think to mention that the limonene is not pure limonene - it is a cloud emulsion dosed in a 5% ethanol solution that also contains citric acid. I am guessing that was propbably important - sorry.

When I tried the sodium chloride as a salting out agent, the 10% level showed a marginal increase. This was first project that I used NaCl for the salting out effect - only after did I make the connection of the two salts. I am learning as i go.

I tried to insert a bitmap of chromatogram of the three levels compared....but wasnt able to.

When I evaluate the aroma of the three samples-there is a clear difference in intensity level, with the highest pH not very volatile. This was an immediate response - so the sodium citrate is somehow holding the volatiles in solution. Also when I adjust the pH down on this 4.2 sample with citric acid - the volatiles re-emerge.

Also, I am not so concerned at this point with low levels of detection - i want to understand the reactions at the different pH levels as the limonene breaks down over time and other compounds are created.

Thanks.

[rb6banjo]

Your observation regarding the aroma of your higher-pH samples is why I'm thinking that the active ingredients are not these hydrocarbons.

No matter what ionic material you add to a solution, you will get the "salting out" effect. All added salts increase the ionic strength of the medium. Organic molecules generally don't like water as well as water does. The hydrocarbons are already pretty insoluble in water and marginally soluble in solutions that are substantially water (5% ethanol), so adding salt will have a marginal effect on those compounds. Where the salt can pay off is for materials that show a good degree of water solubility (other alcohols). In your case, sodium citrate addition is a pH adjuster AND a salting out agent. I don't know it for sure but I'd bet that sodium chloride is a better salting out agent than sodium citrate because I have a hunch that for solutions of equal concentration of the salts, the conductivity of the NaCl solution will be larger.

The raising of the pH is creating reactions in your solution that are messing with the aroma-active species.

My data shows that limonene (and likely the others as well) is substantially unaffected by salt and pH as far as headspace analysis goes (I used SPME to generate the data I've displayed). If you're after the aroma changes, then looking at the hydrocarbons is probably not the route you want travel.

[bethlynn]

I am at a loss to explain further what I see in this experiment....comes with further research and continued learning of flavour chemistry I guess.

At this point I am unsure of alternate compounds other than the limonene and other minor terpenes in this solution. The flavour used is almost entirely limonene. I spend a fair amount of time evaluating the aroma profile of single flavours or multi flavour systems and the aroma is definately of limonene nature. I just lack the understanding of joining the chemistry, gc profile and sensory attributes together.

I appreciate your insight rb6banjo and if I discovery anything new, I will share.

Thanks.

[Peter Apps]

Is it possible that changing the pH (and/or ionic strength) changes the properties of the emulsion, and liberates more limonene to the aqeuous phase and the headspace ?

And, yes, the extra detail at the outset would have been very useful.

Peter