ESTD ESTD% NORM% Agilent HPLC

[Paul_1002]

Hello,

I am new here so how are You, Chromatography Community? Hope, everything is well.

I have a question according to a ESTD, ESTD% and NORM% value. I ran the sample (Preparation: 300 mg in 30 ml of solution what is 10 mg/ml concentration or 1% if consider percent). I put Dilution as 1 and Multiplier as 1 (I created calibration table in mg/ml).
However, when I create result ESTD based on Area I have result 10 mg/ml but ESTD% I was expecting 1% but I got 3% [I know Formula = 100*(Amount of Compound/Sample Amount) for ChemStation]. My concern is to whatever sample amount I weight like for example 5 gr, 1 gr and 0.1 gr to will get the same ratio of my compound 1% but I do not know how to achieve this. Should I use NORM%?

I would appreciate if someone could help me.

[vmu]

What Sample Amount did you enter in the software? Does "got 3%" mean "got 3.00%" or does it mean "got 3.33%"? Obviously, to obtain 1% from 10 (mg/ml), it is necessary to use Sample Amount = 1000. This is just a change of a dimension unit. The only question is the purpose of this calculation. Describe your procedure in detail (sample preparation, standard preparation, the values you enter in the software) and define clearly what relative to what you are trying to obtain as a result expressed in percent.

[Paul_1002]

Hi

Sample amount I put 300 mg. Yes, 3% mean 3.33333%.
Way of preparation calibration table:
- 5 mg to 50 ml [0.1 mg/ml = 0.01%]
- 150 mg to 50 ml [3 mg/ml = 0.3%
- 300 mg to 50 ml [6 mg/ml = 0.6%]
- 500 mg to 50 ml [10 mg/ml = 1%]
Solution that I used was Acetonitrile:Water in ratio 60:40 (Recommended for sugar sample). Sample was just fructose, I used to check that my calibration table works fine and I was expecting 10 mg/ml for ESTD and 1% for ESTD%.
Sample preparation - I put 300 mg to 30 ml of this solution, dissolved everything so concentration is 10 mg/ml and 1% so why result is only right for ESTD.
Value in software:
Response: 1343607.25
RF = (software shows 0.00001 but to calculation it use 10/1343607.25 is 7.442 x 10-6 so I do not understand that)
Multiplier - 1
Dilution - 1
Sample amount - 300 (because I dissolve 300 mg and my calibration table is in the same unit).
What I need is that whatever sample I take to testing like 1 gram, 2 gram, 5 gram I do not need real, absolute value but If sample get 5% fructose I want to obtain 5% in 1 gram, 2 gram and 5 gram

[Consumer Products Guy]

We almost always wanted our results to be in "% of sought-for component in the sample". And Agilent GC and HPLC used the same software and set up.

We got our results in an automated Sequence Summary Report at the end of the sequence. We used ESTD%. In the Calibration Table, the concentration of standard was entered in grams/ml. In the Sample Table, the grams sample weighed went into the Amount column, and the number of ml that was diluted to was entered in Dilution. Any additional dilutions of sample like 5ml to 100ml went into the Multiplier column as "20".

First line of sequence table was standard and set to replace the calibration in the computer memory, two injections, first was a throw-away.

Second line was standard and set to Average. No entries for amount or dilution for the standard injections.

Then would come sample injections.

Agilent had a PDF or two with instructions how to set up, we learned by trial and error, and checked to make sure that the results were the same.

[Paul_1002]

So it possibile to get same percent of our looking compound whatever amount of sample I weighted? Because 300 mg in 30 ml it is 1% concentration and this result I am expecting. What should I change? I have tried doing calculation on that in Excel however any combination I put I never get proper results.

[vmu]

You calibrate using the concentration of fructose in the calibration solution expressed in mg/ml as the Amount of Compound. Therefore, the Amount of Compound you obtain for your test solution in the ESTD calculation is the concentration of fructose in this test solution in mg/ml. Then the ESTD% calculates exactly what it can calculate using the value you enter: 100 * 10 (mg/ml) / 300 (mg) = 3.3333 (% 1/ml). This is a meaningless calculation. So, if you know the calculation formula in the soft, why do you enter that value 300 mg and perform this meaningless calculation?

Write down the formulas you would use to find the result without the software, and you will understand what you should enter in the soft to obtain the correct result.

If you need the fraction of fructose in your solid sample expressed in % w/w, you have to enter the value m/V as the Sample Amount, where V is the volume of the test solution in ml, and m is the mass of the sample in mg. In your example, V = 30 ml, m = 300 mg, m/V = 10 mg/ml. If you obtain the Amount of Compound 10 mg/ml for the test solution, you will further obtain the ESTD% = 100%, meaning that the tested fructose sample is 100% pure (within the accuracy of the method). Another option is to set Sample Amount = 300 (mg), Multiplier = 30 (ml).

If you just want the result as % w/v in the solution, you have at least two options:
1. Use the concentrations in % w/v for the calibration instead of mg/ml. You will obtain the Amount of Compound in % w/v. For your 300 mg/30 ml sample you will obtain the value 1% (in ESTD calculation).
2. Calibrate as you are doing it now (using mg/ml) and use the value 1000 as the Sample Amount. This will simply recalculate mg/ml to % w/o (in ESTD% calculation). For your 300 mg/30 ml sample you will obtain the value 1%.

Value in software:
Response: 1343607.25
RF = (software shows 0.00001 but to calculation it use 10/1343607.25 is 7.442 x 10-6 so I do not understand that)

If the software can display only 5 digits after the decimal point, it displays 5 digits. The value 0.00001 is nothing else but the rounded 7.442 x 10-6. Of course, the accurate value of RF (without rounding) is used in the calculations.

[Paul_1002]

Thank You for answers.

It is not that I do not put anything except 300 mg. I prepared table on Excel and according to Agilent formula R x RF x M x D I put diffrent numbers and I was trying to obtain my results (I can put this Excel later so you can see). I try 300 mg because if I would have unknown sample I would put exactly amount I weighted to ChemStation Sample Amount and operated on this number. 1000 mg it is not what I weighted.

[Consumer Products Guy]

See https://www.agilent.com/cs/library/supp ... faq205.pdf

https://www.agilent.com/cs/library/Supp ... a10007.pdf

[Paul_1002]

Hi
As vmu said, I left calibration table in mg/ml and I set up Sample Amount as 1000 and it works perfect for all kinds of sample I ran during method developing.

Thank you for solving my problem with ESTD%