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C A Libratio N (Thegood Curve) : Greg Hudson Envirocompliance Labs, Inc

This document discusses calibration curves and how to evaluate the quality of a calibration curve. It provides an overview of calibration curves and what makes a good curve. It then discusses tools for evaluating curve quality, such as the correlation coefficient, response factor variance, linear calibration range study, intercept, and visual inspection. Potential problems that can affect curve quality are outlined, such as bias, tilt, higher order curves, and scatter. Corrective actions for improving curve quality include performing maintenance, cleaning glassware, checking dilutions, remaking standards and reagents, and rerunning standards.

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Laverne Marcelo
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Download as PDF, TXT or read online on Scribd
56 views

C A Libratio N (Thegood Curve) : Greg Hudson Envirocompliance Labs, Inc

This document discusses calibration curves and how to evaluate the quality of a calibration curve. It provides an overview of calibration curves and what makes a good curve. It then discusses tools for evaluating curve quality, such as the correlation coefficient, response factor variance, linear calibration range study, intercept, and visual inspection. Potential problems that can affect curve quality are outlined, such as bias, tilt, higher order curves, and scatter. Corrective actions for improving curve quality include performing maintenance, cleaning glassware, checking dilutions, remaking standards and reagents, and rerunning standards.

Uploaded by

Laverne Marcelo
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 16

C a libratio n

( T h e G o o d C u rv e )

Greg Hudson
EnviroCompliance Labs, Inc.
[email protected]
www.envirocompliance.com
A b s tra c t
n It should come as no surprise that the
correlation coefficient is only one way to
judge the quality of a calibration curve.
n The fact is that there are numerous tools
available to evaluate how good a
calibration curve represents a given data
set.
n With a basic understanding of how to use
regression analysis, some simple methods
can be applied to determine both the
quality of necessary to improve calibration
technique.
O v erv iew

n The black box versus thinking


outside the box.

n The black box is the regression


algorithm.

n Thinking outside the box is


evaluating analytical processes or
biases which may effect the quality
of the curve.
W h at is a C u rv e ?

n STANDARD CURVE: A plot of


concentrations of known analyte
standards versus the instrument
response to the analyte. Calibration
standards are prepared by
successively diluting a standard
solution to produce working
standards which cover the working
range of the instrument. Standards
should be prepared at the frequency
specified in the appropriate
section.... SW-846
OK, what do we really
mean by curve?
n The ability to predict
behavior by establishing
known outcome.
n Simply put, getting a
result with reasonable
certainty.
W h at’ so important about a
g o o d C u rv e ?

n Easier to correct problems if the


calibration is bad
n Higher confidence in analytical
results
A Peek inside the Black Box
n Basic linear regression by least squares

n LINEAR Eq.: x=(y-


x=(y-b)/m or y=mx+b

n where m = slope or rise over run

n b = intercept (curve crosses the y axis)

n (Σxy - ((Σ
(Σ Σx * Σy /n))
n Slope = ---------------------------
n Σx^2 - ((
((Σ Σ x)^2 / n))
((Σ

n (Σy/n) - (m *Σ
intercept = (Σ * Σx / n)
T h e C o rrelatio n C o e ffic i e n t
n An estimate of the remainders derived from the
regression.

F How do we calculate it?

n (Σxy - ((Σ
(Σ Σx * Σy / n))^2
n r = ----------------------------------------------
n Σx^2 - ((
((Σ
(( ((ΣΣx)^2 / n)))*(Σ
n)))*( Σy^2 - ((Σy)^2 / n))
((Σ

u What does it mean?

u For our purpose it must be >0.995

u The correlation coefficient only indicates


variance from the averages; therefore it is not
the best indicator of curve quality.
r = 0.997
45
40
35
30
25
20
15
10
5
0
0 10 20 30 40 50

Std 1 3 1
Std 2 2 2
Std 3 1 3
Std 4 10 10
Std 5 20 20
Std 6 40 40
r = 0.9999
0.1
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
0 5 10 15 20 25

Std 1 1 0.011 90.90909


Std 2 2 0.015 133.3333
Std 3 5 0.028 178.5714
Std 4 10 0.048 208.3333
Std 5 20 0.091 219.7802
O u ts i d e t h e B o x

n Beer's Law states that the absorbance of a


solution is directly proportional to the
concentration. (Ideally linear)

n What effects Beer?


F Noise is most often the function of the detector

F Quenching - At high concentration, Beer's law will


not continue to behave

F Limiting factors of method or reagents. Most methods


are intended to operate at specific trace ranges.
Dilution after reaction or extraction beyond trace
levels may bias final results. Capture
E v a lu a t i o n o f C u r v e Q u a lity
n Slope vs. RF
F Response Factor is the ratio of Concentration/Response
and represents the instantaneous slope for a given
point.
n Variance of RF
F The average of the RFs should be close to the slope.
The variance of the RFs is an excellent indication of
curve quality. RSD <10%
n Linear Calibration Range Study - LCR
F LCR is simply plugging the response of each standard
into the line function and determining the variance from
predicted value (conc.). These should be <20% and
ideally <10%
n Intercept - Should not be greater than the lowest standard.
n Visual
F Date should appear to be linear and fall along the curve.

n Equal Data points on either side


F Generally, data points should be evenly distributed
around the curve. If most of the points fall to one side
of the line or are not evenly divided by the line, there
may be a problem with the curve.
Pro b lem s E ffecting
C u rv e Q u a lity
n Bias
u Bias is any part of the analytical process which may cause unequal
treatment from one part of the procedure to another- such as using
that "special" beaker for blanks.
n Intercept
u High intercepts imply bias for instrument background or
contamination.
n Tilt
u Tilt or skewed sloped is often the result of poor instrument
performance.
n Higher order curves
u Calibration curves which are not linear may imply problems in the
analytical process such as saturation or degradation.
n Bimodal data
u Bimodal data may appear to have two distinct 'lines' and is most
often related to dilution technique like using a eppendorf for the
lower part of a curve and a pipet for the upper part of the curve.
n Scatter
u Random divergence in a curve can result from numerous factors
including extraction/digestion, poor technique, or dirty or
malfunctioning instrumentation among others.
Pro b lem s E ffecting
C u rv e Q u a lity
n Forcing through zero
u Linear regression should not be forced through zero for two
reasons:
u 1) Good information can be gained from the natural intercept.
u 2) The regression formula is skewed by passing through zero.
n Span
u The entire range of the analytical curve can be problematic if it
goes beyond the linear range of the instrument or system.
n Spacing of stds
u Standards should represent the analytical range evenly. Keep in
mind that the Least Squares algorithm can be biases by using higher
concentrations. Standards should not exceed five (5) fold changes
in concentration at most.
n Dilution and general pipetting technique
u It is critical to use consistent and precise dilution techniques
for standards and all spiking or QC functions. Use of varying
means for dilutions can add significant variance for each standard.
C o rrectiv e A c tio n s
n Perform Maintenance
n Clean glassware
n Check dilutions and be consistent
n Remake standard and reagents (INCLUDING STOCKS)
n Re-run standards
n Re-zero

n Avoid:
u Deleting standards
u Running extra standards
u Excessive re-zeroing
u "Special" Behavior (i.e. using one cuvette for standards
and another for samples)
C a libratio n
( T h e G o o d C u rv e )

Discussion/Questions

Greg Hudson
EnviroCompliance Labs, Inc.
[email protected]
www.envirocompliance.com

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