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Topic: Calibration problem  (Read 3028 times)

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Offline shafaifer

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Calibration problem
« on: May 09, 2015, 07:27:51 AM »
I want to know the concentration of D3-vitamin in food when adding 1 gram of vitamin D3 premix:

I have a calibration range spanding 0.1026666667-10.62833333 mAU min or 5-0.05 mcg / mL. 1 gram of D3 premix gives an area of 732,40 mAU min, so 1 mcg should give 732.4 mAU min / 10^6 = 0.0007324000000 mAU min, but this value is not included in the calibration range, so couldn't I multiply it by 300 mcg for example? This gives me an area of 0.2197200000 mAU min. Inserting this value in the calibration equation gives:

A = 2.1269c - 0.0108 <=> c = (A + 0.0108) / 2.1269 = (0.2197200000 mAU min + 0.0108)/2.1269 = 0.1083830928 mcg / mL - now, multiplying it by 10^6/300 should give me the concentration in 1 gram = 361.2769760 mcg / mL. If I repeated this procedure with 150 mcg instead I would obtain a concentration of 378.2030185 mcg / mL. My thinking is the concentrations should be the same after scaling up as long as you choose a mass-value of premix that has an area within the calibration range. I do not know which mistakes I have made not to obtain same results :(
« Last Edit: May 09, 2015, 11:06:19 AM by shafaifer »

Offline Arkcon

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Re: Calibration problem
« Reply #1 on: May 09, 2015, 10:52:40 AM »
Your question is a little hard to follow, and I'd like you to work on these problems first, before we get to your real question.

You have to use significant figures intelligently.  No way in hell are six figure optical uinits in any way correlateable to masses of actives humans can measure on an analytical balance, and they are in no way statistically significant to any animal model.  So stop doing that, right now, because it just looks silly.

I don't recognize your units -- "mAU min", maybe you mean "mAU/min" or some other missing punctuation.  You should mention your instrument -- is this some sort of kinetic assay?  Let us know, so an expert can find this thread, and help you out.  You mention calibration area -- is this an HPLC or GC method?  In that case, we tend to identify such a method, and just call it peak area, without mentioning the chromatogram's units.

But briefly, no, if you want to evaluate an unknown, you have to be with a recently calibrated range, or be using a previously validated method known to cover that range.  But I can't read your numbers, so I have to take you at face value that you're outside the range.
« Last Edit: May 30, 2015, 07:15:47 PM by Arkcon »
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

Offline shafaifer

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Re: Calibration problem
« Reply #2 on: May 09, 2015, 11:17:47 AM »
Calibration range* (not area, sorry). Thanks, what I mean by mAU min is mAU * min. HPLC is used to analyze the vitamin. This is a case / theoretical proeject my group has been given, so the problem is we cannot change the calibration range because we are not supposed (maybe not allowed also) to go to the laboratory and do that. But using less mass of premix is indeed a dilution - but again, this is only a theoretical project so I cannot practically do it. This is why I want to calculate what amount of premix needs to be added to get an acceptable area and concentration and then scaling up - but I cannot do this? Are there ways to correctly calculate it theoretically if this is not okay?

Offline mjc123

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Re: Calibration problem
« Reply #3 on: May 09, 2015, 01:26:52 PM »
I don't like your peak area of 732.4 mAU min per 1g. Is this an experimental value, or a figure extrapolated from a measurement on a smaller quantity? Assuming your peak is not more than 1 min wide (and if it is, you should consider using a different chromatographic method) it must be well over 0.7 AU high, and so probably outside the reliably linear response range. (The calibration range you are given is what it is for a reason.)
Apart from that, your discrepancy seems to be the result of assuming your peak area will be exactly proportional to the amount of D3, while using a calibration curve with a non-zero intercept (i.e. the area is not exactly proportional to the concentration). Without knowing more about your procedure, I don't know if that is justified, but it will certainly not give the same answer every time.

Offline shafaifer

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Re: Calibration problem
« Reply #4 on: May 09, 2015, 02:13:34 PM »
Thanks. It is an experimental value.

Offline Arkcon

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Re: Calibration problem
« Reply #5 on: May 09, 2015, 04:46:22 PM »
So you have a standard curve with observed values of 0.1 to 10.6 AU (I like to call those arbitrary units -- heh) for 0.5 to 5 μg.  Your unknown has area of 0.0007 AU.  Extrapolate if you wish to for a high school project, but this is not the way analytical chemistry is done.  There are many things wrong here, and mjc123: pointed out an important one.  You can't even expect the instrument to respond the same over 3 orders of magnitude -- it would be worrisome if you had 0.7 AU, and bad for 0.07 AU, but you're too far away.
« Last Edit: June 02, 2015, 01:30:31 PM by Arkcon »
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

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