Calibration methods

Chemistry 243
Figures of merit: Performance
characteristics of instruments
Precision
Accuracy
Selectivity
Sensitivity
Limit of Detection
Limit of Quantitation
Dynamic Range
Precision vs. Accuracy in the
common verbiage (Websters)
Precision:
The quality or state of being precise; exactness;
accuracy; strict conformity to a rule or a standard;
definiteness.
Accuracy:
The state of being accurate; exact conformity to
truth, or to a rule or model; precision.

These are not synonymous when describing

instrumental measurements!
Precision and accuracy in this
course
Precision: Degree of mutual agreement among data
obtained in the same way.
Absolute and relative standard deviation, standard error of
the mean, coefficient of variation, variance.
Accuracy: Measure of closeness to accepted value
Extends in between various methods of measuring the
same value
Absolute or relative error
Not known for unknown samples

Can be precise without being accurate!!!

Precisely wrong!
Precision - Metrics

Most important

Often seen as %

Handy, common
Sensitivity vs.
Limit of Detection
NOT THE SAME THING!!!!!
Sensitivity: Ability to discriminate between small
differences in analyte concentration at a
particular concentration.
calibration sensitivitythe slope of the calibration
curve at the concentration of interest
Limit of detection: Minimum concentration that
can be detected at a known confidence limit
Typically three times the standard deviation of the
noise from the blank measurement (3s or 3s is
equivalent to 99.7% confidence limit)
Such a signal is very probably not merely noise
Calibration Curve, Limit of
Detection, Sensitivity

Sensitivity* = Slope
Signal

*Same as Working Curve

**Not improved by amplification alone

S/N = 3
0
0 LOD
Analyte Mass or Concentration
Selectivity
Degree to which a method is free from
interference from other contaminating signals
in matrix
S mAcA mB cB mC cC

No measurement is completely free of

interferences
mB
Selectivity coefficient: kB, A
mA
Calibration Curves:
Sensitivity and LOD
For a given sample standard
deviation, s, steeper calibration
curve means better sensitivity
Insensitive to amplification
Signal

S/N = 3
0
0
LOD LOD
Analyte Mass or Concentration
Dynamic range
The maximum range over which an accurate
measurement can be made
From limit of quantitation to limit of linearity
LOQ: 10 s of blank
LOL: 5% deviation from linear
Ideally a few logs
Absorbance: 1-2
MS, Fluorescence: 4-5
NMR: 6
Calibration Curves:
Dynamic Range and Noise
Regions
Calibration
Curve
becomes
poor above
Signal

this amount
of analyte

Poor
Quant
Noise
Region
Dynamic Range
S/N = 3
0
0 LOD LOQ LOL

Analyte Mass or Concentration

Types of Errors
Random or indeterminate errors
Handled with statistical probability as already shown
Systematic errors
Instrumental errors
Personal errors
Method errors
Gross errors
Human error
Careless mistake, or mistake in understanding
Often seen as an outlier in the statistical distribution
Exactly backwards error quite common
Systematic errors
Present in all measurements made in the same way
and introduce bias.
Instrumental errors
Wacky instrument behavior, bad calibrations, poor
conditions for use
Electronic drift, temperature effects, 60Hz line noise,
batteries dying, problems with calibration equipment.
Personal errors
Originate from judgment calls
Reading a scale or graduated pipette, titration end points

Method errors
Non-ideal chemical or physical behavior
Evaporation, adsorption to surfaces, reagent degradation,
chemical interferences
 Instrument calibration
Determine the relationship between response
and concentration
Calibration curve or working curve
Calibration methods typically involve standards
Comparison techniques
External standard*
Standard addition*
Internal standard*
* calibration curve is required
 External standard calibration
(ideal)
External Standard standards are not in the
sample and are run separately
Generate calibration curve (like PS1, #1)
Run known standards and measure signals
Plot vs. known standard amount (conc., mass, or mol)
Linear regression via least squares analysis
Compare response of sample unknown and
solve for unknown concentration
All well and good if the standards are just like the
sample unknown
 External standard calibration
(ideal)

Sample
Unknown

External
Signal

Calibration
Standards
including
a blank
Sample
Unknown
Amount
S/N = 3
0
0 LOD
Analyte Mass or Concentration
In class example of external
standard calibration

Skoog, Fig. 13-13

P0
bc
Sample

A log Unknown

P Signal
External
Calibration

molar absorptivity
Standards
including
a blank
Sample

b pathlength S/N = 3
Unknown
Amount

c concentration 0
0 LOD
Analyte Mass or Concentration
 Real-life calibration
Subject to matrix interferences
Matrix = what the real sample is in
pH, salts, contaminants, particulates
Glucose in blood, oil in shrimp
Concomitant species in real sample lead to different
detector or sensor responses for standards at same
concentration or mass (or moles)
Several clever schemes are typically employed
to solve real-world calibration problems:
Internal Standard
Standard Additions
 Internal standard
A substance different from the analyte added in a
constant amount to all samples, blanks, and
standards or a major component of a sample at
sufficiently high concentration so that it can be
assumed to be constant.
Plotting the ratio of analyte to internal-standard as a
function of analyte concentration gives the
calibration curve.
Accounts for random and systematic errors.
Difficult to apply because of challenges associated
with identifying and introducing an appropriate
internal standard substance.
Similar but not identical; cant be present in sample
Lithium good for sodium and potassium in blood; not in blood
Standard additions
Classic method for reducing (or simply
accommodating) matrix effects
Especially for complex samples; biosamples

Often the only way to do it right

You spike the sample by adding known amounts of

standard solution to the sample
Have to know your analyte in advance

Assumes that matrix is nearly identical after standard

addition (you add a small amount of standard to the
actual sample)
As with Internal Standard this approach accounts for
random and systematic errors; more widely applicable
Must have a linear calibration curve
How to use standard additions
To multiple sample volumes of an unknown,
different volumes of a standard are added and
diluted to the same volume.
Fixed parameters:
Calibration Standard cs = Conc. of std. fixed
(Fixed cs) Vt = Total volume fixed
Vx = Volume of unk. fixed
cx = Conc. of unk. - seeking

Non-Fixed Parameter:
Vs = Volume of std. variable
Vx Vx Vx Vx
Vs1 Vs2 Vs3 Vs4
Volume top-off step:
Vt Vt Vt Vt Vx diluted to Vt
Vs diluted to Vt
How to use standard additions
To multiple sample volumes of an unknown,
different volumes of a standard are added
and diluted to the same volume.

= +

Combined Signal
S1 S2 S3 S4 0
0 Concentration
How to use standard additions

= +
k = slope or sensitivity

Combined Signal
= =


= =
0
0 Concentration
How to use standard additions

= +

= +

How to use standard additions

= +

Remember,
Vstd is the
= + variable.

Knowns:
= = cstd

Vtotal
Vx
 How to use standard additions
= +

S, Combined Signal
=

Get m (slope) and

b (intercept) from
= linear least squares

0
0 Vs
How do I handle k ?
 Determine cx via standard
curve extrapolation

= +

At the x-intercept, S = 0

Skoog, Fig. 1-10

0 known
=

Seeking known
[analyte] Vstd when S = 0
 or determine cx by directly
using fit parameters

=

= + = 0

= =

Final calculation: All knowns



= =

in conclusion, an easy procedure to perform and interpret;
you take values you know and do a linear least squares fit to get m and b