F12.

Regression Analysis

KEY POINTS

1. Calibration importance
2. Least Square Regression
3. Prediction
4. LC, LD and LQ

Assoc. Prof. Gaston A. Crespo

gacp@kth.se

https://crespo-groupkth.com @crespolab
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Calibration

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Issues related to the calibration

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y (𝑥" , 𝑦" ) 𝑒$ 𝑦! = 𝑎 + 𝑏𝑥! + 𝑒!
(𝑥$ , 𝑦$ )
𝑒" 𝑒# 𝑦!!
𝑒! 𝑦! = 𝑦(! + 𝑒!
(𝑥# , 𝑦# )
(𝑥! , 𝑦! )
𝑦
'! ; 𝑦' 𝑒! = 𝑦! − 𝑦(!
"; 𝑦
'# ; 𝑦
'$ (𝑒𝑠𝑡𝑖𝑚𝑎𝑡𝑜𝑟)

x
x1 x2 x3 x4
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Least square regression
We want to find the best line:

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Homoscedasticity condition

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Time for exercising (~30 min)

Example 5.3.1

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Note: You find the EXCEL solution in “Solutions to Exercises_Miller.xls” CANVAS
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Errors in slope and intercept

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 General form of confidence limits
Calculation
Confidence of afor
limits concentration and its random
a concentration error
determined 123
by using
regression lines

y0
Note that (𝑥$, 𝑦)
' is the center
Signal

– y– )
(x,

x0
Concentration
al form of the confidence limits for a concentration determined by using an
ssion line.

n: number of calibrants ; m: number of readings y0

Time for exercising (~15 min)

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13
Correlation coefficient

−1 ≤ 𝑟 ≤ 1

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Correlation

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Misinterpretation of r

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Residual analysis

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 Residual analysis (~30 min)

Below you find 4 calibrations performed in different conditions. Calculate

and plot the residuals for each calibration. What can it be concluded?

Concentration Fluorescence readings

pg/mL-1 signal_1 signal_2 signal_3 signal_4
0 1.6 1.3 2 1.2
2 5 5.1 5 5.3
4 9 9.3 8.5 9
6 12.6 12.8 12.2 13.1
8 17.3 17.3 16.4 18
10 21 20.5 21 21
12 24.7 25.4 25.4 24.7

b (slope) 1.957 1.983 1.966 1.98

a (intercept) 1.285 1.196 1.132 1.303
 Time for exercising (~30 min)

Exercise 5.9

Tips: (i) plot the data, (ii) build a linear model, (iii) check the
residuals, (iv) remove data, (v) calculate new residuals

Note: you can use Excel, but it is recommended to use your calculator and write down the
solution in a sheet. The general solution is provided in Excel file format for simplicity in
CANVAS (solutions to exercises_Miller.xls)

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Typical calibration

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 SSPE a component due to the variability within each
group of replicate measurements {𝑦!" 𝑏𝑙𝑢𝑒 𝑎𝑛𝑑 𝑦$! (𝑟𝑒𝑑)}
SSLOF a component due to the variability of group averages
about the regression line {(𝑦!! 𝑔𝑟𝑒𝑛 𝑎𝑛𝑑 𝑦$! (𝑟𝑒𝑑)}

𝑦
!!

x
x1 x2 x3 x4
 𝑆𝑆𝑇 = 𝑆𝑆#$% + 𝑆𝑆&$ + 𝑆𝑆'()

df MS
#
𝑆𝑆𝑇 = , , 𝑦!" − 𝑦- , 𝑛! − 1
! " !

#
𝑆𝑆$% = , , 𝑦!" − 𝑦.! , 𝑛! − 𝑘 𝑀𝑆&$
! " !

𝑆𝑆&'( = , 𝑛! 𝑦.! − 𝑦(! #

𝑘−2 𝑀𝑆'()
!

𝑛! : Number of replicates per concentration

, 𝑛! : Total number of replicates for all concentrations

!
𝑘: Number of concentrations
 𝑀𝑆'() 𝐹*1!.!*+, = 𝐹234,637,∑! 9!36
𝐹*+,*-,+./0 = vs.
𝑀𝑆&$

H0: the set of data can be explained by a linear model

 Time for exercising (~35 min)

Xi (conc) Yi (signal)
replicate 1 replicate 2 replicate 3
2 89 82 70
4 137 142 128
6 215 201 205
8 245 242 243
10 302 317 290
12 335 325 327
14 366 375 370
16 413 405 400
18 434 441 420
20 442 450 441

Tips: run ANOVA for regression, MLOF and MPE must be

calculated.
 Time for exercising
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component 2 measures the variation of the group means jji about the line. The
ANOVA table is given in Table 1.
Exercise 5.X
The linearity test is performed by calculating F = MSLOF/MS,,.
Example. The results of a calibration experiment are given in Table 2.
Given the following data, demonstrate whether a linear model is suitable or not.
TABLE 2
Results of a calibration experiment
xi 1 2 3 5 10
Yij 16.6 21.8 31.0 49.3 82.4
13.7 22.2 30.3 51.4 85.9
14.8 21.8 32.3 51.6 85.6
14.3 21.8 31.2 51.6 84.0

CYij 59.4 87.6 124.8 203.9 337.9

i

k
Tips:n =run
E n i - ANOVA
20 for regression, MS_LOF and MS_PE must
I
be calculated.
k=S
Calculation of the total sum of squares, corrected for the mean gives

ss,= C C<n,- 8)’- C C Y ; -

(7 p j ) ’

i j i i
Note: you can use Excel, but it is recommended to use your calculator and write down the
solution in a sheet....
=16.6’+13.7’+
The+84.0’- ==12553.41
general20solution is provided in Excel file format for simplicity in
Calculation(solutions
CANVAS of the sum of
tosquares due to pure error gives
exercises_Miller.xls)
Decision limit, limit of detection
and limit of quantification

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Distribution of the blank

CRITICAL VALUE or CRITICAL LIMIT

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Retain null hypothesis when it is false (type II, area)
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Limit of detection

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 IUPAC recommends a value of
𝐿𝑂𝐷 = 𝑠𝑖𝑔𝑛𝑎𝑙 𝑏𝑙𝑎𝑛𝑘 + 3 𝑠:!;9+, <,+96

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