# Calibration Curve

Purpose: To determine specific analyte concentration (amount) from signal, detector response, intensity, A, etc. Step 1: Measure response to a set of known concentrations B equation or plot Step 2: Measure response to sample B concentration (amount) from plot or eqn

0 1.5 2 1.5 4 3.5 0 0.0 3.5 3 2. A 4.5 1 0.Calibration Curve 5 Signal.0 Concentration or Amount . Detector Response.0 2.

Calibration Process  Standards prepared by serial dilution  For each analyte of interest  Multiple measurements (either a different concentration or at selected concentrations)  Blank (typically reagents alone) can give a ³blank correction´ .

5 2 1.5 3 2. A 4.5 4 3.0 3. Detector Response.5 0 0.Characteristics Range: lowest concentration to highest concentration (validity) Linear Range: data fit by y = mx + b Calibration Curve 5 Signal.0 2.5 1 0.0 Concentration or A ount .0 1.

Uncertainty in analyte value . Analyte value 2.Objective To obtain sample(s) concentration from measured signal 1.

Absorbance = Signal Transmittance = T = I/I0 Light Source h I0 I  Monochromator  Sample cuvet  Detector .

c = M . b A = log10 (I0/I) = .log T A = bc (Beer¶s Law) = molar absorptivity.

Least Squares Fit Set of n yi and xi values di = yi ± mxi ± b D= | §(xi2 ) §xiyi | | §xi n | .

Because average di = 0 (§di = 0). sy = (di ± d)2 _________ }½ { deg of freedom = { (di)2 / (n-2) ½ } sm2 = nsy2 / D and sb2 = sy2 (xi2) _________ D (y ± sy) = (m ± sm) x + (b ± sb) .

0 d^2 917.2 1512.97326 .8 26.3382 -y = 36.3 986.1 -31.3 14.6 7111.99756 b = 112.6 26.4 d -30.9 1063.3 812.4 218.9 -32.4 0.4 53.6 723.= 3.4 14109.9 16909.1 = 11702300 = 13.1 4311.02547 IC Chl ide 20000 15000 al 10000 i 5000 0 0 200 400 600 800 1000 1200 1400 M la i y x 10^6 .8 2820.1 722.8 749.9 -27.1 11310.2 8202.057E-02 -b = 20.i al ( ) 10^6 [Cl-] Corr 785 20 362 1250 50 827 1962 100 1539 4702 300 4279 7561 500 7138 11706 800 11283 14586 1000 14163 17301 1200 16878 3970 423 Bla k x^2 400 2500 10000 90000 250000 640000 1000000 1440000 3432900 y-calc 392.

02)]/ (13.997 0.34 20.997 .(112.Linear Equation S = signal (corrected for blank) M = molarity x 106 S 37.0 = (13.34 20.02) or M = [S .031)M +(112.031) .

what is sample M and uncertainty? x = (y .Uncertainty in Sample M If sample is measured and signal is 8888.b)/m = [(8465 37) (112 20)]/(14.03) so M = 567 x 10-6 and %sM = 0.00 0.549 or sM = 3 x 10-6 .

2 x 10-6 (compared with 3 x 10-6 before) .Correction ± m and b dependent sx = __ sy _ |m| { 1 + x2n + (xi2) .2x xi _ ____________________ k D ½ } (k is the number of unknown replicates) = 1.

Calculate analyte value and uncertainty .Summary of Process 1. Prepare/calculate graph/equation 4. Measure response to standards (corrected for blank) 3. Prepare standard solutions and blank 2. Measure response to sample(s) 5.