I'm currently doing glucose standard curve graph. Do I need to plot zero absorbance for zero concentration in the standard curve graph or just start with the first concentration that I used?
Hello, the following link might help:
https://en.wikipedia.org/wiki/Standard_curve
To summarize:
1. Don't plot anything you did not measure
2. Absorption for zero concentration is not necessarily zero
3. Interpolation is only possible between measured values (and extrapolation is critical)
So: first make the zero concentration measurement, then you are free to plot it :)
Regards
Dear Nizzal Syafiq,
Based on Statistics and Chemometrics for Analytical Chemistry, Jane C. Miller, James N. Miller, "it is crucially important to include the value for a ‘blank’ sample in the calibration curve. The blank contains no deliberately added analyte, but does contain the same solvent, reagents, etc., as the other test samples, and is subjected to exactly the same sequence of analytical procedures. The instrument signal given by the blank sample will sometimes not be zero."
Thus, I think that the first point of the analytical curve must be the LOQ, Limit of Quantification or the lowest concentration you need since it is not below LOQ.
Best regards,
I think this link is very useful,
http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/
I'm not sure if Nizzal Syafiq's question is being answered. Perhaps if I re phrase the question, a more appropriate answer can be provided.
Lets say we have a 7 point standard curve and a "blank" (no analyte, but all other same reagents as standards).
When interpolated, is it best to subtract the "blank" value from the standards and base the interpolation off of standards 1-7, or should the interpolation be based off blank value through standard 7 (assuming standard 1 is low point and standard 7 is high point)
I think this may be what Nizzal is actually looking to get an answer for.
Dear all,
This is my way of dealing with it because it makes sense to me. I usually like to follow these steps
1) calculate average for all ODs (which are in triplicate)
2) substract average OD of blank sample from averages ODs of all samples including average OD for blank.
3) Make standard curve and include all values of standard (all average ODs - average blank OD) starting from blank value (blank average OD- blank average OD= 000).
There is one problem, if the solvent for included samples are different. Then, we shall use one more blank of that solvent and subtract average of that one from the average ODs of those samples to get actual OD for your target compound or molecule. See the attached file that help me to calculate all things within second. If you have any question about this calculator, please ask me.
The purpose of calibration is to calculate a measurement result for an unknown sample from a measured signal. This may be done in a variety of different ways, but only a few of them allow you to associate a reliable uncertainty to your result.
Such methods were applied to actual experimental data in my paper “Calibration uncertainty”[1]; a detailed comparison of different approaches showed that simple interpolation between the two nearest calibration points gave surprisingly good results; for the lowest signals the blank is a valid calibration point.
If you need to prepare a “standard curve” the actual analytical signals should be shown for all measurements including the blank.
[1] Accred. Qual. Assur. 7 (2002) 153-58
A standard 0 must be included in the calibration curve because the instrumental signal is subjected to the same kind of error for all points. The signal for the standard zero should not be subtracted from the response values for other standards before calculating the equation of the regression line because it can cause imprecision during the determination of the concentration values for unknown samples
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