5.
2 Method Validation
•selectivity
•selectivity
•linearity
•linearity
•accuracy
•accuracy
•precision
•precision
•sensitivity
•sensitivity
•range
•range
•limitof
•limit ofdetection
detection
•limitof
•limit ofquantitation
quantitation
•ruggedness/robustness
•ruggedness/robustness
Standard
Standardreference
reference
materials
materials (SRMs)best
(SRMs) bestfor
for
determining accuracy.
determining accuracy.
General process for evaluation/validation of methodology.
5.2 Method Validation
Accuracy: How close are the results
measured by your method to true?
Student t value
Precision: How reproducible are the results
measured by your method?
F test
5.2 Method Validation
• Specificity (Selectivity) : the ability to
distinguish the analyte from other
species in the sample
Electropherogram of the drug cefotaxime (peak 4) spiked with known 3
impurities (peaks 2, 3, 5–9) from synthesis of the drug.
Method Validation
• Linearity: A measure of how well data
in a graph follow a straight line.
R2 must be very close to 1 to represent a
linear fit.
A good fit:
R2 >= 0.995 or 0.999
R2 =
0.985.
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Method Validation
• Detection Limits
• 1. Prepare a sample whose concentration is ∼1 to 5 times the detection limit.
• 2. Measure the signal from n replicate samples (n ≥7).
• 3. Compute the standard deviation (s) of the n measurements.
• 4. Measure the signal from n blanks (containing no analyte) and find the mean
value, yblank.
• 5. The minimum detectable signal, ydl is defined as
Lower limit of quantitation
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5.2 Method Validation
• Robustness :
• Ability of an analytical method to be
unaffected by small, deliberate
changes in operating parameters.
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Method Validation
• Range: the concentration interval over
which linearity, accuracy, and precision
are all acceptable.
c.f.
Linear range: Concentration range
over which calibration curve is linear.
Dynamic range: Concentration range
over which there is a measurable
response .
Calibration curve illustrating
linear and dynamic ranges.
5.3 Calibration Methods
• (1). Calibration Curves : A graph of the
response versus concentration of
analyte.
ch4 Calibration curve.xlsx
4-G In the Bradford protein determination, the
color of a dye changes from brown to blue
when it binds to protein. Absorbance of light is
measured. An unknown protein sample gave an
absorbance of 0.973. Calculate the number of
micrograms of protein in the unknown.
Protein, A595
μg
0.00 0.466
9.36 0.676
18.72 0.883
28.08 1.086
37.44 1.280
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Method Validation
• Accuracy: nearness to the truth
Spike Recovery:
Spike: Addition of a known compound
(usually at a known concentration) to an
unknown.
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Spike
• An unknown was found to contain 10.0
μg of analyte per liter. A spike of 5.0
μg/L was added to a replicate portion
of unknown. Analysis of the spiked
sample gave a concentration of 14.6
μg/L. Find the percent recovery of the
spike.
Good selectivity has a spike recovery in the range of 100 ± 2%
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Example: An unknown sample of Ni2+ gave a current of 2.36
μA in an electrochemical analysis. When 0.500 mL of
solution containing 0.028 7 M Ni2+ was added to 25.0 mL of
unknown, the current increased to 3.79 μA. Find [Ni2+]i in the
unknown.
C1 C 2
=
A1 A2 C1 C2
=
C1 is the unknown 2.36 3.79
A1=2.36
A2=3.79 C2 is the mixture of the 25C1 + 0.5 × 0.0287
0.500ml known solution C2 =
with 25 ml unknown 25 + 0.5
3.79
C1 =
25C1 + 0.5 × 0.0287
2.36 25 + 0.5
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5.3 Standard Addition
• (2). Standard Addition
CClO - >=18 μg/L in drinking water
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is of concern because it can reduce
thyroid hormone production!
Matrix effect: A change in
analytical signal caused by
anything in the sample other than
analyte.
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Standard Addition: A technique
in which an analytical signal due
to an unknown is first measured.
Then a known quantity of analyte
is added, and the increase in
signal is recorded. From the
response, it is possible to
calculate what quantity of analyte
was in the unknown.
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5.3 Internal Standard
• (3). Internal Standard
An internal standard is a known amount
of a compound, different from analyte, that
is added to the unknown.
F is response factor.
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Example Example Using an Internal Standard
In a preliminary experiment, a solution containing 0.083 7 M X and 0.066 6 M S
gave peak areas of AX = 423 and AS = 347. (Areas are measured in arbitrary
units by the instrument's computer.) To analyze the unknown, 10.0 mL of 0.146
M S were added to 10.0 mL of unknown, and the mixture was diluted to 25.0
mL in a volumetric flask. This mixture gave the chromatogram in Figure 5-7, for
which AX = 553 and AS = 582. Find the concentration of X in the unknown.
Figure 5-7 Chromatographic separation of
unknown (X) and internal standard (S). A known
amount of S was added to the unknown. The
relative areas of the signals from X and S allow
us to find out how much X is in the mixture. It is
necessary first to measure the relative response
of the detector to each compound.
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