Calibration methods

CHM 3204 Instrumental Analysis

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 (Webster’s)
 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 sensitivity—the 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

e*
Signal

rv
Cu
n
atio
li br
Ca
*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 m A c A  mB cB  mC cC

 No measurement is completely free of

interferences
 Selectivity coefficient: mB
kB, A 
mA
Calibration Curves:
Sensitivity and LOD
 For a given sample standard
deviation, s, steeper calibration

e
curve means better sensitivity

i ti v
ns Insensitive to amplification

Signal

Se
re
Mo

siti ve
S en
Less

S/N = 3
0
0
LOD LOD
Analyte Mass or Concentration
Dynamic range
 Themaximum 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
rve
Cu of analyte
n
Poor atio
Quant li br
Ca
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
 Determinethe 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)
 ExternalStandard – 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 Sample

A log  bc 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; can’t 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:
Vx Vx Vx Vx Vs = Volume of std. – variable

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.

𝑆 𝑡𝑜𝑡𝑎𝑙 =𝑆 𝑠𝑡𝑑 + 𝑆𝑥

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

𝑆 𝑡𝑜𝑡𝑎𝑙 =𝑆 𝑠𝑡𝑑 + 𝑆𝑥
k = slope or sensitivity

𝑘 𝑉 𝑠𝑡𝑑 𝑐 𝑠𝑡𝑑

Combined Signal
𝑆 𝑠𝑡𝑑 =𝑘∙ 𝑓 𝑑𝑖𝑙 ∙ 𝑐 𝑠𝑡𝑑 =
𝑉 𝑡𝑜𝑡𝑎𝑙
𝑘𝑉 𝑥 𝑐 𝑥
𝑆 𝑥 =𝑘∙ 𝑓 𝑑𝑖𝑙 ∙𝑐 𝑥 =
′
𝑉 𝑡𝑜𝑡𝑎𝑙 0
0 Concentration
How to use standard additions
𝑆 𝑡𝑜𝑡𝑎𝑙 =𝑆 𝑠𝑡𝑑 + 𝑆𝑥
𝑘 𝑉 𝑠𝑡𝑑 𝑐 𝑠𝑡𝑑 𝑘 𝑉 𝑥 𝑐 𝑥
𝑆 𝑡𝑜𝑡𝑎𝑙 = +
𝑉 𝑡𝑜𝑡𝑎𝑙 𝑉 𝑡𝑜𝑡𝑎𝑙
ard
and wn
st k no
f r om un
nal f r om
g al
Si n
g
Si
How to use standard additions
𝑘 𝑉 𝑠𝑡𝑑 𝑐 𝑠𝑡𝑑 𝑘 𝑉 𝑥 𝑐 𝑥
𝑆 𝑡𝑜𝑡𝑎𝑙 = +
𝑉 𝑡𝑜𝑡𝑎𝑙 𝑉 𝑡𝑜𝑡𝑎𝑙
Remember,
Vstd is the
𝑆 𝑡𝑜𝑡𝑎𝑙 =𝑚𝑉 𝑠𝑡𝑑 +𝑏 variable.

𝑘𝑐 𝑠𝑡𝑑 𝑘𝑉 𝑥 𝑐 𝑥 Knowns:
𝑚= 𝑏= cstd
𝑉 𝑡𝑜𝑡𝑎𝑙 𝑉 𝑡𝑜𝑡𝑎𝑙
Vtotal
Vx
 How to use standard additions
𝑆 𝑡𝑜𝑡𝑎𝑙 =𝑚𝑉 𝑠𝑡𝑑 +𝑏
𝑘 𝑐 𝑠𝑡𝑑

S, Combined Signal
𝑚=
𝑉 𝑡𝑜𝑡𝑎𝑙
Get m (slope) and
𝑘𝑉 𝑥 𝑐 𝑥 b (intercept) from
𝑏=
𝑉 𝑡𝑜𝑡𝑎𝑙 linear least squares

0
0 Vs
How do I handle k ?
 Determine cx via standard
curve extrapolation …
𝑘 𝑉 𝑠𝑡𝑑 𝑐 𝑠𝑡𝑑 𝑘 𝑉 𝑥 𝑐 𝑥
𝑆 𝑡𝑜𝑡𝑎𝑙 = +
𝑉 𝑡𝑜𝑡𝑎𝑙 𝑉 𝑡𝑜𝑡𝑎𝑙

At the x-intercept, S = 0

𝑘𝑉 𝑠𝑡𝑑 𝑐 𝑠𝑡𝑑 𝑘 𝑉 𝑥 𝑐𝑥
=−
𝑉 𝑡𝑜𝑡𝑎𝑙 𝑉 𝑡𝑜𝑡𝑎𝑙
𝑉 𝑠𝑡𝑑 𝑐 𝑠𝑡𝑑 =−𝑉 𝑥 𝑐 𝑥
Skoog, Fig. 1-10
( 𝑉 𝑠𝑡𝑑 ) 0 𝑐 𝑠𝑡𝑑 known
𝑐 𝑥 =−
𝑉𝑥

Seeking known
[analyte] Vstd when S = 0
 … or determine cx by directly
using fit parameters
𝑉 𝑠𝑡𝑑 𝑐 𝑠𝑡𝑑
𝑐 𝑥 =−
𝑉𝑥
𝑆 𝑡𝑜𝑡𝑎𝑙 =𝑚𝑉 𝑠𝑡𝑑 +𝑏=0
𝑏
𝑚 𝑉 𝑠𝑡𝑑 =−𝑏 𝑉 𝑠𝑡𝑑 =−
𝑚
Final calculation: All knowns

𝑐 =−
( −
𝑏
𝑚 )
𝑐 𝑠𝑡𝑑
=
𝑏 𝑐 𝑠𝑡𝑑
𝑥
𝑉𝑥 𝑚𝑉 𝑥
… 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