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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

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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,

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

microbalance) Mass-to-charge ratio mass spectrometry

Rate of reaction kinetic methods Thermal characteristics thermal gravimetry and

titrimetry; differential scanning colorimetry; differential thermal

analyses ,

Light

Electro-magnetism

Mass

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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 organ

light, pressure, chemical, acoustic

electrical currents via

nerves

brain response…sense of

sensephotometer UV-Vis,

IR,light from various

lampselectrical current current meter

atomic emission spectrometer

flames voltage voltage meter

pH meter electrodes voltage voltage meter

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

Electrical versus Non-electrical Domains

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

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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

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

a minimum concentration that can be detected at a certain confidence level.

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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?

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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

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

Calibration (3 basic types)

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

miles per hour.... even downhill."

The basic idea is to ‘train’ the instrument to convert a measured signal to a concentration

…just like in Gen Chem

1) External Calibration (most common)

•Standards are of known concentration• Plot signal vs. concentration

•To find unknown concentration: interpolate between known points

y = 2xR² = 1

0

5

10

15

20

25

0 2 4 6 8 10 12

Inst

rum

en

t R

esp

on

se

X-Data (Arbitrary Units)

Calibration Example Plot

1 2

2 4

3 6

4 8

5 10

6 12

7 14

8 16

9 18

10 20

y = 0.0388x + 0.22

0

0.2

0.4

0.6

0.8

1

1.2

-10 0 10 20 30

Vs (mL)

Ab

so

rba

nc

e2) Standard Addition Calibration

•Useful when interferences present in sample

•Samples are spiked with various amounts ofstandard (known)

•All solutions in the same “matrix”…accounts for artifacts from mixture

Extrapolation from “zero” added material determines how much analyte was in original sample

The beakers below have increasing amounts of

analyte as per a ‘standard’ calibration

1 2 3 54

How would the calibration ideally

look?

concentration

Instrument response

Internal Calibration

concentration

Instrument response

Blue: ideal ResponseRed: Actual Response

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)

concentration

Blue: ideal response

Red: actual response

Black: internal

reference

What can we say about the ratio of

red:black as a function of conc. ?

If we plot the ratio ‘Analyte/Reference’ to

the ‘Concentration of the Analyte’, we get a

linear response… (from your book)