Calibration: Constructing A Calibration Curve
Calibration: Constructing A Calibration Curve
Calibration: Constructing A Calibration Curve
In this unit, we will review how to construct a calibration curve. Each calibration has its own limits. A proper understanding of those limits will help you develop the best possible calibration and avoid many problems.
You typically have two (or more) variables to work with. One (or more) is set at known values. Your analyte Other experimental conditions One is a measured response. Absorbance, current, area, ....
The simplest approach to developing our model is to: Select a series of known analyte standards. Hold other factors constant - or as many as possible Measure the response. Develop a model (calibration curve).
Most methods have a fixed range where the relationship between response and analyte amount is valid.
response
LOD
lin
LOQ
ar
n ra
ge
LOD LOD limit limitof ofdetection detection LOQ LOQ limit limitof ofquantitation quantitation LOL LOL limit limitof oflinearity linearity
LOL
analyte amount
Linear modeling
General unweighted least squares Assumptions Standards are correct and all errors come from the measurement of response. Variances are independent of analyte concentration.
Linear model
R =b 1 X + b 0 + e
R =response b 1 =slope parameter X =Standard value b 0 = intercept parameter e =residual error
Linear model
Weve already showed how to calculate the slope and intercept.
Linear model
2 sY !X 2 2 N !^X i - X h
2 sb =
0
b1
X Y !XY - ! N! = ` !X j !X - N
2 2
slope
2 sb =
1
2 sY 2 !^X i - X h
2
We can estimate the variances via propagation of errors - produced during ANOVA analysis.
b0 =
!Y - b !X
1
intercept
2 e
!^Y =
2 -Y h -b1 !^X i - X h ^N - 2h
slope range = b 1 ! t s b
intercept range = b 0 ! t s b
You are just plotting out the condence limits for each data point.
relative error
LOD Point below which we can no longer be confident that our analyte is present. LOQ Point below which we can no longer confidently report a quantitative value.
-100
1%
The calibration curve and regression band can determine{ Detection limit. Smallest amount we can see with a known level of condence. The upper CL at Y=0, then converted to concentration (X). Sensitivity. Smallest change in amount we can see with a known level of condence. Often based on smallest change that an instrument can display.
10 20 30 40 number of measurements
50
Linear range. Range where we can quantify with a known level of condence. (lower = DL, upper = where curve intersects upper/lower CL.
Example
sensitivity
linear range
detection limit
Example
12000 10000
Example
8000
6000
4000
2000
0 0 20 40 60 80 100 120
-2000
Concentration
200
Example
Noise +/- 100
180 160 140 120 100 80 60 40 20 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
Concentration
A plot of the residuals can give you an idea of how well your model ts the data. Saved time by using upper 95% condence limit value that XLStat provided for [ ] = 0 Residual = measured - predicted
error
This This residual residual plot plot indicates indicates a a reasonable reasonable fit fitof ofthe thedata data to tothe themodel. model.
concentration analyte
+/-2000 example
2 1.5
Standardized residuals
OK, these are reasonable ts of a linear model but we should check the residuals to be sure
Regression of Noise +/- 2000 by Concentration (R!=0.965)
12000 12000
10000
10000
8000
8000
6000
6000
4000
4000
-2
Concentration
0 20 40 60 80 100 120
Concentration 60 80
100
120
Concentration
+/-100 example
2 1.5 1 0.5 0 0 -0.5 -1 -1.5 -2 20 40 60 80 100 120
Standardized residual
Residuals are normalized by the standard error. This results in both of our plots looking pretty much the same since the error introduced was random (even if it varied by a large factor. Typically, expect to see residual values to be randomly distributed around + 2 units for a good model.
Concentration
Standardized residuals
Standardized residual.
2 1.5 1 0.5 0 0 -0.5 -1 -1.5 -2 20 40 60 80 100 120
Standardized residuals
Standardized residuals
-0.5 -1 -1.5 -2
Concentration
Concentration
So, by using standardized residuals, we can directly compare the two data sets - even though the actual noise level is signicantly different.
40 35 30 25 20 15 10 5 1.5
Here is an example that demonstrates a bad model. Were going to try and model MPG vs. car weight.
MPG
2.5
3.5
4.5
Weight, tons
Now what?
The model clearly has a problem. Researchers attempted different models and found that log(weight) gave a better t.
Standardized residuals
2.5
3.5
4.5
Also, MPG is determined differently in other countries - USA uses miles traveled/gallon. It is often full consumed per xed distance (100 miles). So, they used log(wt) vs 100/MPG
Weight, tons
Regression of 100/mpg by
3
8 7 6
2
Standardized residuals
100/mpg
5 4 3 2 1 0.2
0 0.2 -1
0.3
0.4
0.5
0.6
0.7
-2
0.3
0.4
0.5
0.6
0.7
-3
Log(Wt)
Log(Wt)
Residual range is excessive. Ultimately, the best model had to include other factors. Including Drive Ratio turned out to be the key in developing the best model.
Pred(100/mpg) / 100/mpg
7
2 1.5
Standardized residuals
100/mpg
2 2 3 4 5 6 7
-2
Pred(100/mpg)
100/mpg
Standardized residuals
Its clear that there are two different types of samples so a simple calibration model wont work.
0.36 0.38 0.4 0.42
This residual plot indicates that there is some sort of response dependency based on the sample used. Consider using Analysis of Covariance to conrm.
a1360
Pred(Octane #) / Octane #
93
91
Octane #
89
87
Standardized residuals
Pred(Octane #)
Standardized residuals
ANCOVA can then be used to build a model that includes the season factor as a way of merging what are clearly two different (but related) models.
1.5 1
83 83 85 87 89
2 91 1.5 1 0.5 0 -0.5 -1 -1.5 83 85 87 89 91 93
93
Octane #
-2
Octane #
Standard addition
Standard addition
Initial response. No standard addition. R 0 = k C 0 =k c n 0 m V0 Response when a standard is added. R T = R 0 +R S =k c n 0 + n S m V 0 +V S We can then set Q as:
A calibration method where standards are added to replicates of your sample. You then measure the total response. The matrix is near identical for all samples. You can measure the response away from LOD and LOQ values.
Q = R T ^V 0 + V S h = k n 0 + k n S
We can then plot Q vs. ns added using different amounts of the standard.
Sample
With an approach like this,Vn+Vs would be the same for all standards.
no
n1
n2
n3
Standard addition
By extrapolating to the x intercept, where Q=0, we have !
k n 0 =- k n 1
and
n 0 =- n i
The primary advantage of this method is that you can move your measurement from an area of high relative error.