Standard addition

[CSV]

Hello!
I'm currently trying to analyse a substance in a complex matrix. This matrix possibly contains the analyte it self. Is it possible to use standard addition??? I've tried but the concentration found is approximately 10 times larger.

[Peter Apps]

In principal you can do this by standard addition, but only if you know whether the peak that you think is the analyte really is the analyte.

Peter

[danko]

Hi CSV,

How did you perform the standard addition, in practice?
And if you quantitated an unknown, how would you know it was 10 times larger?

Best Regards

[grzesiek]

"I've tried but the concentration found is approximately 10 times larger" - than what?

[CSV]

Thank's for the replies!

The product nominally contains 5.67 umol/unit. 5 units are extracted with 50 ml of extraction medium. 20 ml is diluted to 25 ml during a hydrolysis reaction. 4 ml of the hydrolysed sample is diluted to 5 ml during standard addition (100ul 18mM std addition solution is added and dilution to volume with solvent).
The results were:
A=area of sample without std addition=43.219
AS= Area of sample with std addition=47.363
Cs=Added concentration of std addition to sample=100ul*18mM/50ml=0.36mM

C=0.36E-3*43.219/(47.363-43.219)=3.75mM
*5/4=4.69 mM
*25/20=5.86mM
*50E-3=0.293 mmole
/5=58.6 umole/unit =(10*label)

[grzesiek]

calculations look ok

what does hydrolysis reaction do to the analyte? should't standard be also hydrolised?

the product is the same as standard? have you tried the procedure for the standard itself (think of standard as a sample)?

[danko]

Hi CSV,

Yes it’s more than a good idea to treat the standard as if it was a sample – at least one portion of it (kind of process control).
Otherwise it is of utmost importance to perform the standard addition procedure in a controlled fashion. Here I’m thinking primarily of the range. In order to assure the linearity/proportionality one needs to generate a “calibration curveâ€

[grzesiek]

as I'm trying to visualize this - your std addition in the place where you begin to lose the linear response, I think that danko's comment can be regarded as the solution, at least for now, untill you provide more data, see the response for the added concentration will be smaller than it should be, so your slope is not so steep, and so the intercept is higher (10 times for instance )

are you in the linear response for the detector? how much is the absorbance for your peaks?

[CSV]

Hi, again!

Alitle bit more info!
The analyte is a fatty acid esther and the same fatty acid is also present in the formulation, both free and as oil. The amount of the fatty acid in the matrix is approximately 10 times larger than the amount derived from the analyte after hydrolyzation.
I've tried to analyse the intact esther but it is very unstable and breaks down to several products (it's already degraded in the sample and the degradation proceeds during analytical handling). The only stable part of the molecule is the fatty acid, but due to the matrix it's hard to analyse.
My main question to this forum is if it's possible to use standard addition to determine the content derived from the esther in a sample already containing 10 times the amount of the targeted analyte? I can't get the theory right!
If it's not possible the only thing left I can think of is to quantify the fatty acid in a placebo formulation and then subtract the result from the results of the actual samples!

[lmh]

I'm getting the impression this thread is going in the wrong direction.

Am I right in summarising the following:

You want to measure a fatty acid ester in a sample that contains 10 units of fatty acid for every one unit of fatty acid ester. You can't analyse the ester directly because it is too unstable, so you are hydrolysing it to the fatty acid, which means you now have 11 units of something, but you only want to measure the one unit that was originall esterified.

If so, I'd think:

(1) You can, in theory, measure fatty acid content with and without hydrolysis, and assign the difference to the esterified version.
(2) You should check this works by spiking samples with fatty acid ester and checking you get realistic recoveries of the spike.
(3) Because the unesterified fatty acid exceeds the esterified by a factor of ten, the approach is likely to lose some precision. The error bars on individual measurements are likely to be large compared to the difference (i.e. measuring small differences between big numbers is generally less precise than measuring a small number itself).
(4) Therefore if you can get rid of the unesterified fatty acid in any way, it would be a jolly good thing. (If the esterified version is so unstable you can't even separate it, then how do you know it isn't hydrolysing in your unhydrolysed sample? This will be tricky...)
(5) The method of standard addition is probably not really relevant to this problem.

Good luck!

[CSV]

.....and alittle more info.

Since the analyte is a fatty acid I'm using an ELSD and not an UV-detector. I know, the response is generally not very linear on this type of detector, but atleast I can detect my analyte. Maybe that's where my problem is!? I would attach calibration plots if I knew how to, but for know I can say that the plot curves somewhat and has a "perfect" quadratic fit (Signal=2.42E6*C(sq)*1.02E4*C+43.3, r(sq)=1.000). (the linear fit is: Signal=1.55E4*C+42.1, r(sq)=0.992).

Does this help?

Regards,

CSV

[CSV]

Hi, Imh!

Yes, you have understod the problem correctly. My latest effort has been to try to separate the oil and free fatty acid from the esther prior to hydrolysis. For this I'm using SAX-SPE (The risk is, as you mentioned that the analyte has already hydrolysed in the sample. Then it to will bind to the SAX and get lost). I get some results tomorrow!

[HW Mueller]

Now that there are some 12 entries we almost know what you are doing, except for what these possibly strange esters are or why they are left in solutions which render them unstable.

[CSV]

Hello!

There is really no need for secrecy. The product contains ascorbyl palmitate (ACP) as an oxidant and it needs to be quantified to asure that the correct amount has been added. However, the ACP degrades very fast, presumably by oxidation, and therefore the product must be analysed very close to the production. My problem is that I've recieved a few old batches on my table for which the client wants to know if the right amount ACP has been added. The content of ACP is between 10 to 50% of label due to degradation and I've to come up with an alternative way to determine the content at time 0, so to say. As previously described, my idea has been to hydrolyse the ACP and degradant products to analyse the more stable fatty acid. However, during this effort I've learned that the product contains atleast two additional sources of plamitic acid which makes quantification hard.
Any idea of how I should do is more than welcome.

Regards

CSV

[HW Mueller]

You mean antioxidant?
There are two main reasons that I am aware of for esterifying ascorbic acid: To stabilize it against oxidation and to make it fat soluble.
So at "time = 0" one just needs to make sure that the medium is neither strongly acidic nor basic. There should be an extensive literature by now on handling and analysing of the ascorbic palmitate, the lit. on ascorbic is overwhelming. Unless both are present in trace amounts, I don´t see much of a problem in analysing both. Forget about the palmitic and hydrolysis, concentrate on stopping any hydrolysis.