Linearity of Response

[gclc]

Hi
we have a typical situation here, we are tring to do linearity of an Active substance from 15µg/mL to 630µg/mL, surprisingly the linearity is not passing , the square of correlation coeffcient is around 0.9966( Limit NLT 0.999), we have repeated the experiment and ending up with almost same result ,iam giving response against Concentration


Conc( µg/mL) Response
15.738 97063
23.607 144072
47.213 281772
94.426 544904
118.033 670788
236.066 1236063
393.443 1908790
472.132 2213992
550.82 2510956
629.509 2811985
we can not change the column as we are verfying this method at our Lab,
Chromatogrphic Conditions :-
Flow :- 1.5ml/min
Temp: 30°C
Column : Symmtry C18 150x 3.9 mm , 5µ
Wavelength : 220 nm
the Sample is Dextroamphetamine sulfate,
Surprisingly linearity is fine when you consider from 15.738 to 236 and it is fine from 236 to 629 , why it is not linear from 15 to 630µg, the peak is not saturated at 220 nm , the height of peak at 220nm for 630µg/ml is only about 0.4AU., please somebody help me in this issue.
thanks

[gclc]

Additional detalis

Chromatographic parameters:--

Flow : 1.2 ml/min
Wavelength : 212nm & 220 nm
Inj Volume : 20 µL
Temperature : 35°C
Run time : 23 min
Column : Symmetry C18, 3.9x150mm, 5µ

Preparation of Buffer for Mobile Phase:

Dissolve 10.44 g of 1- Pentane sulfonic acid sodium salt and 27.2 g of Potassium phosphate monobasic in 3500 mL of purified water. Adjust pH to 2.50+ 0.10 using concentrated Phosphoric acid and dilute to 4000 mL with water and mix well.

Preparation of Mobile Phase:

Transfer 650 mL of buffer and 350 mL of Methanol to suitable container. Mix well and degas.

[danko]

the peak is not saturated at 220 nm

What do you mean by â€

[gclc]

What do you mean by â€

[danko]

Is 220 nm the absorbance max. for your analyte?
What about 212 nm – are the highest peaks off-scale at this wavelength? And what is the reason for using both 212 and 220 nm wavelengths?
Maybe 220 nm is on a steep slope of the analyte's spectrum.

Best Regards

[gclc]

Is 220 nm the absorbance max. for your analyte?
What about 212 nm – are the highest peaks off-scale at this wavelength? And what is the reason for using both 212 and 220 nm wavelengths?
Maybe 220 nm is on a steep slope of the analyte's spectrum.

Best Regards

Thanks for your quick replies , @212 highest peaks off-scale at this wavelength, @212 we are monitoring impurities and @220 Assay, 205 nm is the Lambda max

[danko]

The mystery solve then

Best Regards

[Butter]

The mystery solve then

Best Regards

Please can you explain more?

[Bruce Hamilton]

The problem appears to be that you are not reading at the top of a peak, but on the side of a slope as suggested by Danko, and your absorbance is also quite high.

Also, the 205nm "peak" may just be the appearance of stray light, as the peak should be about 215nm.

However, your method has presumably worked for others ( you use "surprisingly" ), so I suggest that you look very carefully at the spectrum of your mobile phase ( against water - to ensure it is not strongly absorbing at any of the wavelengths you are using ), and also a dilute solution of you standard, to ascertain the peak shape.

If you have to stay with the wavelength and concentrations, then you may have to play with the spectral bandwidth settings of your detector to try and obtain the desired linearity. If you can't, then choose the peak wavelength ( 215nm? ), and dilute accordingly, as the method doesn't work on your instrument..

Please keep having fun,

Bruce Hamilton

[gclc]

The problem appears to be that you are not reading at the top of a peak, but on the side of a slope as suggested by Danko, and your absorbance is also quite high.

Also, the 205nm "peak" may just be the appearance of stray light, as the peak should be about 215nm.

However, your method has presumably worked for others ( you use "surprisingly" ), so I suggest that you look very carefully at the spectrum of your mobile phase ( against water - to ensure it is not strongly absorbing at any of the wavelengths you are using ), and also a dilute solution of you standard, to ascertain the peak shape.

If you have to stay with the wavelength and concentrations, then you may have to play with the spectral bandwidth settings of your detector to try and obtain the desired linearity. If you can't, then choose the peak wavelength ( 215nm? ), and dilute accordingly, as the method doesn't work on your instrument..

Please keep having fun,

Bruce Hamilton

Thanks for reply, how to change Spectral Bandwidth settings for a 2487 detector from Waters, please explain

[danko]

Hi GCLC,

The orthodox solution here is utilizing the lambda max. (working wavelength = top of the spectrum plus/minus 1 – 2 nm) which might as well be 212 – 215 or whatever you determine it to be by a scan. That will of course force you to reduce the injection volume or dilute your samples/standards. It will secure you a linear range for your assay.
As for the impurity test, you can inject a larger volume or more concentrated solutions. In this case your main peak will “flyâ€