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Calibration

A Calibration Curve shows the response of an analytical method to known quantities of analyte. A spectroscopic analysis of a protein sample is an example of a calibrated curve. The Calibration Curve can be used to determine the sensitivity and specificity of a method.

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Eka Sulistyaningsih
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141 views25 pages

Calibration

A Calibration Curve shows the response of an analytical method to known quantities of analyte. A spectroscopic analysis of a protein sample is an example of a calibrated curve. The Calibration Curve can be used to determine the sensitivity and specificity of a method.

Uploaded by

Eka Sulistyaningsih
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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Calibration

Chemistry 215 Copyright D Sharma

Finding the Best Fit


Finding the best straight-line fit Usually by computer: 1. Excel 2. Origin 3. Sigmaplot Most computer methods based on the method of least squares
Chemistry 215 Copyright D Sharma 2

Method of Least Squares

y = mxi + b

Chemistry 215 Copyright D Sharma

Find best line by minimizing vertical deviation between the points and the line. 3

Vertical Deviation
If your uncertainty in y is greater than the uncertainty in x, the calculation is relatively simple:

d i = y i y = y i ( mx i + b )
and to minimize the magnitude of the deviations irrespective of their signs:

d i = ( y i y ) = ( y i mx i b )
2 2
Chemistry 215 Copyright D Sharma

Calculating the Residual

Chemistry 215 Copyright D Sharma

Least Squares Parameters


Slope m= Intercept N ( xi y i ) xi y i N x ( xi )
2 i 2 i i i

( )

( x ) y ( x y ) x b= N ( x ) ( x )
i 2 i 2 i

D = N x ( xi )
2 i

( )

Chemistry 215 Copyright D Sharma

Least Squares Uncertainty


y ( s y ) = m ( sm ) x ( s x ) + b( sb )
Standard deviation of response (sy): Standard deviation of slope (sm): Standard deviation of intercept (sb):
In Excel, use LINEST function to determine uncertainties
Chemistry 215 Copyright D Sharma

sy =

(d )
2 i

N 2
N D

sm = s y sb = s y

(x )
2 i

D = N x ( xi )
2 i

( )

Calibration Curves
A calibration curve shows the response of an analytical method to known quantities of analyte.
For example, a spectroscopic analysis of a protein sample Necessary solutions: 1. Standard solutions 2. Blank solution 3. Sample solution(s)
Chemistry 215 8 Copyright D Sharma Protein from the cancer-causing oncogene called ras (Credit: Sung-Hou Kim/UC Berkeley)

A = bc

Constructing a Calibration Curve


Spectroscopic analysis of a protein sample

Chemistry 215 Copyright D Sharma

Constructing a Calibration Curve


1. Prepare standards covering a range inclusive of the estimate sample concentration 2. Measure the response (e.g., absorbance at a specific wavelength) of each standard solution 3. Plot the results and find the best straight line using the least-squares method, and derive the equation to express this fit 4. Measure the signal of the unknown solution 5. Use the equation to solve for the unknown concentration
Chemistry 215 Copyright D Sharma 10

Constructing a Calibration Curve


Spectroscopic analysis of a protein samplecont.
Determination of an unknown value (x) based on its response (y)

Equation of linear response y = m (x) + b Abs = m (g protein) + b y = 0.0163 (x) +0.004 where y is the corrected abs. Determine the unknown concentration based on its absorbance

Chemistry 215 Copyright D Sharma

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Standard Addition
a) Add known quantities of analyte to the unknown b) Deduce the amount of initial analyte from the increase in signal c) A linear response is required
Concentration of analyte at initial volume Concentration of analyte plus standard in final volume = Signal from initial solution Signal from final solution

Chemistry 215 Copyright D Sharma

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Standard Addition
For example: An aqueous solution containing a trace amount of benzene has an absorbance of 0.005 at 263 nm. 10.0 mL of 100 ppm benzene solution was added to 90.0 mL of the unknown solution, which gave a signal of 0.010. Calculate the original concentration of benzene.

[X] [S ] + [ X ]
i f
Chemistry 215 Copyright D Sharma

Ix = Is + x
13

Standard Addition

Chemistry 215 Copyright D Sharma

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Standard Addition

Chemistry 215 Copyright D Sharma

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Standard Addition

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Internal Standards
To the unknown add a known amount of a compound that is different from the analyte (e.g., a different metal ion or small molecule, but chemically and physically similar to the analyte). Signal from the internal standard is compared with that from analyte to determine how much analyte is present.

Chemistry 215 Copyright D Sharma

Useful when You have a non-reproducible instrumental response from run to run Sample loss can occur during sample preparation 18

Internal Standards
Area of analyte signal Area of standard signal = F Concentrat ion of analyte Concentrat ion of standard

Ax As = F [ X ] [S ]
F = response factor

Chemistry 215 Copyright D Sharma

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Internal Standards

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Tips for Calibrating Instruments


Know the limitations of your instrument
Limits of detection (or LOD) Range of linearity

Watch-out for interferences


Overlapping spectral responses (e.g., from impurities) Unwanted sample precipitation Matrix effects Internal standards can help determine if this is the case

Use serial dilutions where possible


Less error than preparing individual samples
Chemistry 215 Copyright D Sharma 21

Serial Dilution (A Review)

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Chemistry 215 Copyright D Sharma

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Chemistry 215 Copyright D Sharma

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Homework
Suggested Readings: Chs. 4 and 5 Suggested Problems: Ch. 4, #23, 28, 29, 30, 32, 35, and 36 Ch. 5, #5-B, 5-C, 15, 24, 26, 30, and 31

Chemistry 215 Copyright D Sharma

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