Introduction

What is Analytical Chemistry (Instrumental Analysis) ?

“Analytical Chemistry deals with methods for

determining the chemical composition of
samples of matter. A qualitative method yields
information about the identity of atomic or
molecular species or the functional groups in the
sample: a quantitative method in contrast,
provides numerical information as to the relative
amount of one or more of these components.”
– Skoog, Holler, Nieman, Instrumental Analysis, 5th ed.

There are Analytical Methods and Classical

Methods

1
 Classical methods (wet chemistry)

– Crude separations: precipitation,

distillation
– Gravimetric analysis
– Titrimetric analysis
– Rely on chemical reactions and measured
quantities (e.g. mass, volume)
– …these are old methods,
nevertheless accurate

Instrumental methods
– Very efficient separations
– Exploits physical and chemical properties as
absorption of light; behavior in a magnetic
field; tendency to move across a membrane
– Analyte  electrical signal
– Computer control and data
acquisition
– Constantly evolving (recent Nobel
prizes)
–

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 …a “sneak peek” at instrumental methods
Chemical and Physical Properties are Employed
Characteristic Properties Instrumental Methods
Emission of radiation Emission spectroscopy (X-ray, UV,
visible, electron, Auger);
fluorescence, phosphorescence
Absorption of radiation X-ray, UV, Visible, IR;
photoacoustic spectroscopy,
Light NMR and Electron spin
resonance
Scattering of radiation turbidimetry; Raman
spectroscopy
Refraction of radiation refractometry; interferometry
Diffraction of radiation X-Ray and electron diffraction
methods
Rotation of radiation polarimetry; optical rotary
dispersion, circular dichroism
Electrical potential Potentiometry;
chronopotentiometry
Electrical charge Coulometry
Electrical current Amperometry; polarography
Electrical resistance conductometry
Mass gravimetry (quartz crystal
Mass microbalance)
Mass-to-charge ratio mass spectrometry
Rate of reaction kinetic methods
Thermal characteristics thermal gravimetry and
titrimetry; differential scanning
colorimetry; differential thermal
analyses ,
Electro-
magnetism

3
What is the general instrumental approach to
gathering information?

Data Domains refers to the various ways of

encoding information.
Instrument Energy Source Data Domain Readout
Any human sense light, pressure, electrical brain
organ chemical, currents via response…sense of
acoustic nerves sense
photometer UV-Vis, light from various electrical current current meter
IR, lamps
atomic emission flames voltage voltage meter
spectrometer

pH meter electrodes voltage voltage meter

other domains…
time, frequency, color, visual, digital

Electrical versus Non-electrical Domains

4
An example (fluorescence) of the progression
through other domains !

5
Selecting an Analytical Method: There is always
more than one way to ‘skin a cat’…but usually
one way is best!
1) What accuracy and precision is
required ?
2) How much sample is available ?
3) What is the concentration range
of the analyte ?
4) What interferences are/could be
present? (is my method selective ?)
5) What is the phase of the sample ?
6) How many samples need to be analyzed ?
(What time and money are available)

Performance Criteria impacts which

experimental technique to use…

Precision, Bias, Sensitivity, Detection Limits,

Dynamic Range, Selectivity, Recover, Speed,
Ease & Convenience, Skill required, Cost and
availability of equipment, Ruggedness

(Let’s deal with each of these in turn)

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 Precision

Given a normal distribution (Chem 350) of

measurements,

Mean value = u0 ‘true

value’ (no error!)
St. Dev. = σ measure
of the ‘spread’ of results

Precision only has meaning with respect to a number

of measurements…

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 Bias
Bias: (Systematic Error): The ‘direction’ of error
(magnitude and sign) remains the same if the
measurements are repeated under identical conditions

Imprecision (or Random Error): The sign and

magnitude of the error changes randomly between
measurements.

Bias = (measured value – true value)

Sensitivity

Broadly speaking, it is the ability to measure small

changes in concentration and is related to the
slope of a calibration curve and its associated
precision.

Analytical sensitivity = γ = slope/std. dev.

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 Detection Limits
Detection limits are also broadly defined. They are
usually quoted with respect to a given procedure.

LOD = Limit of detection

S = Signal response; N = Noise

a minimum concentration that can be

detected at a certain confidence level.

SLOD = Sblank + 3(St. Dev.)blank

Given a typical calibration curve, we know that…

S = slope × [C]

[C]min = (SLOD – Sblank)/slope

technically valid, but if you were an instrument

manufacturer how would you be sure what the
LOD was?

Limit of Quantification: Concentration at which signal is

provides either a linear response or statistically meaningful
response.

Usually, SLOQ = 10×(Std. Dev)blank

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 Is the Benzo(a) pyrene peak above the
LOD? LOQ? What would be the
estimated LOD of B(a)P in terms of mass?

10
 Dynamic Range of Instrument
The linear range of the calibration curve

Linearity usually stops at higher and

lower concentrations Why?
…depends on specific instrument (high
end reasons)

1) detector saturation
2) absorption of light (e.g. fluorescence)
3) space charge effects

What are the consequences of this?

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 Selectivity
The degree to which a method does/does not
respond to a given component in a sample.
Can be defined mathematically for ion-
selective electrodes.

Is Infrared (IR) Spectroscopy selective?

Is NMR selective ?
What is fluorescence selective for?

This parameter is strongly considered in gas

chromatographic methods !

GC detectors (more to come…)

PID (photoionizable) vs FID (combustable)
vs ECD vs XSD (Halogens) …

Let’s Quickly discuss each of these

– Speed
– Ease and convenience; Operator skill
– Cost and availability of the instrument
– Cost per sample (overhead)
– Ruggedness

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 Calibration (3 basic types)
The basic idea is to ‘train’ the instrument to
convert a measured signal to a concentration

"Honestly officer, battery-powered milk floats can't do 75

miles per hour.... even downhill."

A city police
department's radar
speed violation
tickets were legally
invalidated in court
after somebody
proved the
calibration process
for the radar guns
wasn't traceable to
national standards
1) External Calibration (most common)
•Standards are of known concentration …just like in
• Plot signal vs. concentration Gen Chem

•To find unknown concentration: interpolate

between known points
Calibration Example Plot
25
Instrument Response

y = 2x
20
R² = 1
15
10
5
0
0 2 4 6 8 10 12
X-Data (Arbitrary Units)

1 2
2 4
3 6
4 8
5 10
6 12
7 14
8 16
9 18
10 20
 2) Standard Addition Calibration

•Useful when interferences present in sample

•Samples are spiked with various amounts of
standard (known)

•All solutions in the same “matrix”

…accounts for artifacts from mixture

1.2
1
Absorbance

y = 0.0388x + 0.22
0.8
0.6
0.4
0.2
0
-10 0 10 20 30
Vs (mL)

Extrapolation from “zero” added

material determines how much
analyte was in original sample
 Internal Calibration
The beakers below have increasing amounts of
analyte as per a ‘standard’ calibration

1 2 3 4 5

How would the

calibration ideally
look?

Instrument
response

concentration
 Blue: ideal Response
Red: Actual Response
Instrument
response

concentration

The actual (red) response of the instrument

is hardly linear and is hence problematic!
What causes this?

For example, instruments that use

a flame (atomic absorption, FID)
have a high degree of variability
in their response. ..flames
naturally flicker and don’t have a
steady ‘condition’.

The way to overcome this

problem is to spike each of the
standards with a compound
similar (chemically speaking) to
the one under study
 Analyte of interest (e.g. Cocaine)
Spiked internal reference (e.g. morphine, codeine)

What can we say

Blue: ideal response about the ratio of
Red: actual response red:black as a
function of conc. ?
Black: internal
reference

concentration
 If we plot the ratio ‘Analyte/Reference’ to
the ‘Concentration of the Analyte’, we get a
linear response… (from your book)