HPLC sample density

[fuzzytiger]

Hi,
We use HPLC for QC on the soft drinks we make at work. A question has come up, and we keep going round in circles when we try to answer it!

We make a standard solution of sweeteners and preservatives, in water using mg/L as ppm.

When we test non-fruit based drinks all is well and results are (usually) as expected. However when we test a fruit based drink we can struggle to produce product at mimimum specification.

Would the density of the sample have anything to do with it? Fruit based drinks are much higher density.


The argument goes something like this...

We use mg/L as ppm in the standard and inject a fixed volume into the HPLC so the HPLC won't take density into account....

But...
The standard can also be said to be mg/Kg and because the density of the fruity sample is higher this is a more accurate measure of ppm.

Or is it just because we use a filter on fruit products?

Many Thanks

Fuzzytiger

[Noser222]

Are these sweeteners that you are adding to a drink made from a fruit concentrate?

If you make standards based on a volumetric concentration then the result from HPLC is also a volumetric concentration (mg/L), regardless of sample filtration. You then have to adjust that result with the density of your solution if you want the result in mg/kg, resulting in a lower number if your density is greater than water.

If your test results have beenn lower than expected with fruit products, are you sure that your method has good recovery in that matrix?

[fuzzytiger]

Hi,
The sweeteners are all artificial, aspartame and saccharine mainly.

Although we make the standard up to 1L of water to give a volumetric concentration. Could it not be said that this is also the same as making the standard up to 1Kg (as density of water is virtually 1g/mL.
Thus giving the standard results and beverage sample results in mg/Kg.

Or would this only hold up if we injected a fixed mass intot he HPLC rather than a fixed volume?

As for the method, I doubt it's been tested properly for each range of products! - We've told the powers that be but they don't appear to be too willing to actually put a long term fix in place. Or even test the method properly.

[zokitano]

Hi fuzzytiger

I agree with Noser222. When you're using solution of sample that has density value which is differssignificantly from 1mg/cm3 (as your sample), you must take the density value of the sample in the calculations
of the analyte content (whoever it is, but present in your sample).
If you're weighing grams or miligrams of your sample when you're preparing the sample solution,you must take in to consideration the density of your sample (soft drink) if you want to express the content of the analyte of interest (sweeteners or preservatives) in to mass/volumen units. Otherwise, you'll constantly make a systematic error when calculating the content of sweeteners or preservatives in your sample. Thus your calculated results will differ from the actual (true) results (values).
I suppose that your soft drinks contain sugar. Did you use solid phase extraction to eliminate the sugars from your sample solution prior to injection in to HPLC? Removing the sugars from your sample significantly will affect the sample solution density.

Best regards

[fuzzytiger]

Thanks for your help.
I might change the HPLCs to display in mg/L to avoid any more confusion.

The next conundrum is why does the fruity matrix give such bad results......
And how do we get round it!


Fuzzytiger

[Noser222]

Preparing spiked samples and measuring recovery would be a good start.

[Bruce Hamilton]

The next conundrum is why does the fruity matrix give such bad results......
And how do we get round it!
Fuzzytiger

The answers above address your calculation issues.

To get a high density on a clear fruit juice solution, you will have more dissolved solids, and those compounds may not only affect density, but also viscosity, and may also bind to some of your sweeteners.
Undissolved solids ( trapped on the filter ), could also bind to some of your sweeteners.

If you dilute the samples more ( eg 2x the normal amount ) before filtration, do your calculated results improve?. It may be that you just need to dilute the fruit drink samples more - depending on your method sensitivity.

You could rinse ( or preferably ultrasonicate ) a used filter with some warm water or suitable solvent, and analyse the rinse to check that no sweetener is present.

Note that spiking ( as suggested by Noser222 ), should be performed on fruit soft drink samples without the sweeteners, as the fruit material may only bind a certain amount of the sweeteners.

If you use soft drink already containing the normal amount of added sweeteners, then you may not discover the problem.

Please keep having fun,

Bruce Hamilton

[fuzzytiger]

Thanks Bruce,
I've just tried a few of your suggestions:
Diluting the sample made no difference to the results I diluted 50/50 v/v and results were half of the 'normal' sample.

I then put 10mL of a sample through a filter (just) and removed the membrane from the plastic housing. I placed the membrane in 10mL of water and put it in an ultrasonic bath for a few minutes.
Analysed it there was a few ppm of both sweetener and presevative!

I'll try spiking some fruit juice later, it's now nearly 6am and shift end is nearly here!! And I'm on holiday for the next few days
I'll try it when I get back.

Many Thanks
Fuzzytiger

[DR]

If you want to know the difference between your gravimetric ppm and your volumetric mg/L, simply prepare your standard in a tared flask. Your final weight's deviation from 1kg will be pretty close to your error...

[pharmason]

This is Calculation issue

suppose you making 50 ppm of standard
50 mg of standard to 100 mL of Diluent , further dilueting it 5 mL to 50 mL will give you 50 ppm.
now suppose your sample having known density of 1.2030 gm/mL.
suppose your sample contains 1 mg/mL of preservative.

Sample Preperation logic
if you take 5 mL of sample and dilute it to 100 ml that will give you 50 ppm concentration of preservative in to the sample.(5mg to 100 mL).

now instand of takin 5 ml sample you take sample by weight that will be
5 * 1.2030 = 6.015 gm.
so about 6 gm of sample to 100 ml of diluent.

Calculation
% Assay = (Area of Sam x 50 mg x 5 mL x 100 mL x 1.2030 x 100)/(Area of Std x 100 mL x 50 mL x 6.0 gm x 1 mg/mL)

i have not considered purity of standard you using asuming it is 100%.

if i understood your problem correctly then this is the answer.

[HW Mueller]

Having enormous trouble, lately, in understanding many contributions (because of slang used) I wonder what the problem is here.
Fuzzytiger, your company puts x amounts of sweetener and preservative per liter of softdrink, some containing "fruit", some not? Now you analyse these preparations and find less than x/L only in the drinks wich contain "fruit"? If so you have a workup problem. I don´t see how density plays any role here.

This could be a splendid demonstration for why molarity was invented.