Hello Thomas,

MT InLab Micro pH electrode works well also in small volumes (1 mL). I haven't experienced any problems so far. Make sure that you are using a small magnetic stirring bar while adjusting pH. Also consider using buffer.

Hello Maksim thanks for your answer. I Guess this would work, however buying a new pH electrode for an (at this point) one time project of a masters student would be a bit excessive. We vortex in between additions, magnetic stirring is thus not necessary (plus I don’t think we have any stir bars which would fit in our tubes).

I’m unsure about a buffered solution we could try, but I have some doubts for this specific application. It would take a large addition to the originally basic hydrolysate to reach the required buffer condition. However we could try this at least, as it would be a fairly simple solution.

So thank you for your answer so far!

I assume your student is doing a liquid-liquid extraction of the hydrolyzed lipids? If so you can also think of increasing the volume of liquid you work with to have better control of measuring the pH and simply dry the extract down and resuspend in smaller volume as the final step.

Hello Sigurður Smárason, Thank you for your answer, yes it is a Chloroform/H2O (we also tried Chloroform/90% MeOH) extraction. However, volume is exactly the Problem. Downscaling the volume is the goal we want to achieve, since we have a lot of samples which are supposed to be extracted simultaneously. They also don’t have a lot of skin material to beginn with, which is why we have to reduce the volume a s much as possible, to minimize the loss through to excess volume (even though, our substances are almost insoluable in water).

Hmmm... I would not be too worried about the starting sample amount. Hydrolysis works in dilute solutions. But if increasng the volume is off the table (either due to no possibility to dry down or not wanting to spend so much organic solvent) then I have two other ideas.

1) How sensitive to acid are the lipids you are interested in? Do you get different results if the pH drops to 1 or 2? If they are not so sensitive you might get away with pre-calculating the amount of acid that would drop the pH to below 4 and always add that amount of acid to your samples without pH measurement. You might have to increase the time for the hydrolysis in that case to ensure reproducible hydrolysis recovery but testing for that could also become part of the masters thesis.

2) Rethink the buffer suggestion of Maksim. First you add near equal amount of 6M HCl to neutralize the base and then you add your prepared buffer solution in sufficiently strong concentration to always come within your tolerance of pH 4 no matter the residual acid or base that is in there. This again can be tested in larger volumes so that you can be reasonably sure to have the right pH even if you don’t measure it.

Hello, Sigurður Smárason. The main Problem would be Volume related, since we would have to completely switch to bigger vessels and scale everything up, wasting a lot of solvent and additional time yes… Maybe we can try to increase the aqueous phase a little bit, to make the pH adjustment easier (Without increasing the amount of organic phase), but since we don’t have much room for this, I doubt it will have much impact (but let’s see).

Unfortunately yes, it seems to have a have impact on hydrolysis, even tough, the lipids are not too terribly acid labile.

Adjusting the Volume with a constant amount of added acid was in fact the first thing we tried, hoping that it wold be pretty much constant. However, since these are biological samples from different patients, the pH was quite different, even despite the small amount of skin material actually processed (and I don’t think this was a handling problem).

For the buffering, I am already thinking up a possible way, to apply this. I think adding a buffer concentrate, to reach the wanted buffer condition in the already present aqueous phase and adjusting the pH using concentrated acid afterwards (if even necessary) would make the most sense, since a constant pH of 4 is basically the only needed condition for this step. This would be the most time saving way and should produce a reliable result.

I think you have already thought of all of the more obvious and easily implemented solutions. What about spectroscopic pH detection? You might at least be able to minimize the amount of indicator you are using if nothing else.

Other two rather remote possibilities that popped into my head this morning:

I have no clue about the cost but oceanoptics.com make two products (a cuvette and a sticker) for optical measurement of pH where you are not adding dyes to the sample but rather the pH sensitive dye is embedded in the container/sticker. They seem to favor selling whole systems (incl the measuring device), but maybe they are interested in collaborating given the type of samples you have?

There are also papers out of using nanoparticles that are pH sensitive along with spectroscopy/fluorescense to measure pH in low volume samples. No idea if this is home made or something you can buy. Maybe that avenue is something to investigate if all else fails.

Best of luck with the project.

Again, thank you very much for your suggestions, very interesting ideas there!

Yes, I thought about using some kind of indicator. However, as suggested in my original question, I would need an indicator which is soluble in water and at best more soluble in water than in Chloroform, otherwise we will extract a lot of indicator in our organic phase later on, which is not too desirable for our densitometric measurements. With all indicators I’m which have a shift at pH=4 (which I’m aware of at this point) are extremely hydrophobic and would enrich together with our lipids. We do separate them later on using HPTLC, but it is quite possible, that the indicator has a comparable retention to our substances of interest with no way of getting rid of it later on.

Exactly for that reason, this optical probe sounds interesting, however again the problem, buying a new probe for a 6 month project would be a bit excessive (especially since I can imagine, that this innovative new type would be quite expensive).

Nanoparticles could be a viable option, I have to do a bit of research on this kind of method if our current approach (simply trying out the buffer suggestion) should not work. Coupling of a dye to an activated starch nanoparticle shouldn’t be too complicated (if they are not for sale or unreasonably overpriced). I have a friend who recently even produced his own DNA-probe magnetic beats.

I’ll post a short response, describing the solution which finally worked out for us, as soon as we get results on that.

Even though I think with these ideas we’ll be able to figure out a way to get this to work, of course I’m still open for suggestions, if anybody should have more ideas

Best

Thomas