Empower help: %Related compounds from high-low chromatograms

[cindy_ca]

Hello,

I need some help on creating custom fields for calculating % related compounds using hi-low chromatograms. The impurity peak areas are obtained from a 5xfold diluted solution (S1) and the main peak from a 50xfold diluted solution (S2). Multiple S2 solutions are injected before S1 solutions and different processing methods are used.

%Area=Area of Impurity/(Total Area of Impurities+10*Area of Main peak)*10

Thanks in advance.

Cindy

[michaelbarnes42]

cindy_ca,

I should be able to figure this out, just have some questions.

1st, do you process all of the S2 injections prior to the S1 injections? Or can you process an S2 injection followed by an S1 injection?

2nd,are there any other peaks in your chromatogram besides the impurity peaks and the main peak?

3rd, is this your formula?

%Area = (Area_of_Impurity_in_S1/(Total_Area_of_Impurities_in_S1 + (Area_of_Main_peak_in_S2 * 10)))*100

[cindy_ca]

Yes, the formula is right.

There are more than 10 peaks from S1 (known and unidentified) including over scaled main peak (double peaks). The known impurities also have different correction factors, which I think may require separate %area for known and unknowns.

S2 is processed first. S2 and S1 are not injected one after another. Sample sequence is as follows:

Spl1-S2
Spl2-S2
Spl3-S2
Blank
Spl1-S1
Spl2-S1
Spl3-S1

Will this help?

[michaelbarnes42]

I am going to think on this a bit more, but the only way I can think of doing this is if you process each injection like this.

Spl1-S2
Spl1-S1
Spl2-S2
Spl2-S1
Spl3-S2
Spl3-S1

I having trouble coming up with a single custom field that can match an S2 with its S1. I can easily make a formula that will match an S2 with a specific S1 or the previous S1.

How many samples do you have in a typical run. This could be accomplished by making a custom field for each sample in a sample set.

So, if you only have 3 samples normally, then you could have 3 custom fields, one for sample 1, one for sample 2 and 1 for sample 3.

If you have 50 samples, it would be a bit more work to have 50 custom fields, but it can be done.

[cindy_ca]

I normally have between 10 and 25 samples. How do you report average impurities of 10 peaks for 3 to 5 samples if each pair use different custom fields?

If S2 and S1 are raun in pairs, what the custom fields will look like?

[michaelbarnes42]

I normally have between 10 and 25 samples. How do you report average impurities of 10 peaks for 3 to 5 samples if each pair use different custom fields?

If S2 and S1 are raun in pairs, what the custom fields will look like?

You could create a custom field that averages the results of the other custom fields. This is getting quite complicated at this point, and is probably not worth it.

Now, if you were to process in pairs.

I would use the following custom field

to calculate % related compound for each peak in S1

In the sample set, under labels label S2 as S2
In your processing method label each impurity peak as ImpX or however you want to label them and Main Peak as Main_Peak or however you want to label it. You mention that there might be correction factors, in my formula I would use CFX, but you enter whatever correction factor you need.

%area = 100*Area/((Imp1[Area]*CF1)+(Imp2[Area]*CF2)+(Imp3[Area]*CF3)+(Imp4[Area]*CF4)+(Imp5[Area]*CF5)+(Imp6[Area]*CF6)+(Imp7[Area]*CF7)+(Imp8[Area]*CF8)+(Imp9[Area]*CF9)+(Imp10[Area]*CF10)+S2.1.%(Main_Peak[Area]))

To simplify this formulate you can group all the impurities under a group name and use that instead of each individual peak.

This will only work when you process S2 followed by S1 but it should work for all of your samples, unless I made a huge mistake somewhere. It uses the last result for S2, so if the last S2 processed is not the S2 for the S1 that you are processing, then it will not give the correct result.

I will try to test the calc when I get a chance, I have a lot of work today, so it may not be today.

[cindynli]

This is not something to be ready tomorrow. Currently we use Excel for all calculations.

In the equation:
%Area = (Area_of_Impurity_in_S1/(Total_Area_of_Impurities_in_S1 + (Area_of_Main_peak_in_S2 * 10)))*100

Only individual Area_of_Impurity_in_S1 needs to be corrected and the Total_Area_of_Impurities_in_S1 does not need correction. Do I still need to label each unknown impurity peak? As different processing method is used for S2 and S1, does this mean I should choose Run and Process. Can the processing be done at the end of the run?

[michaelbarnes42]

This is not something to be ready tomorrow. Currently we use Excel for all calculations.

In the equation:
%Area = (Area_of_Impurity_in_S1/(Total_Area_of_Impurities_in_S1 + (Area_of_Main_peak_in_S2 * 10)))*100

Only individual Area_of_Impurity_in_S1 needs to be corrected and the Total_Area_of_Impurities_in_S1 does not need correction. Do I still need to label each unknown impurity peak? As different processing method is used for S2 and S1, does this mean I should choose Run and Process. Can the processing be done at the end of the run?

Lol, yeah that makes sense, sometimes I focus too much on the math and forget what is actually happening.

Do you label the main peak in the S1? And are there any peaks besides the Impurity peaks and the main peak in S1?

If the only peaks are impurity peaks and the main peak, then you do not need to label your impurities. You either need to have all of your non-impurity peaks labeled or you will need to label your impurity peaks.

The easiest IMO is to label the non-impurity peaks.

Here is how you can do it if your main peak is your only non-impurity peak. Where I put Main_Peak you enter the label for your main peak.

100*Area/((Total Area - Main_Peak[Area])+((S2.%.%(Main_Peak[Area])*10))

This part of the formula "Total Area - Main_Peak[Area]" Takes the total area of the chromatogram and subtracts the area of your main peak, leaving you with only the impurity peaks.

This part of the formula "S2.%.%(Main_Peak[Area])" gives you the area of the main peak in the last S2 result.

Since the custom field uses the last S2 result, you would need to process the S2 chromatogram immediately before the S1.

[cindynli]

I could not get this part ((Total Area - Main_Peak[Area])+((S2.%.%(Main_Peak[Area])*10)) sum up correctly.

It either showed the first portion for both S2 and S1 or showed separately (Total Area - Main_Peak[Area]) for S1 and (S2.%.%(Main_Peak[Area])*10) for S2. They never added up.

This should follow the inter-sample summary rules, right?

[cindynli]

I am able to get the %Area for impurity now. The issue I had earlier on was due to I did not name the main peak in S1.

Thanks

[cindynli]

Hi michaelbarnes42,

I still have issues with the %Area for impurities. I could not get the %Area results from sample set processing. But when I open the processed result and click quantitate, the %Area shows up.

Any idea why this is happening?