1

5

Choice of method

1. Accuracy: the best method is not always the most accurate

• frequently, we do not know what the "right" answer is, so we can't actually know this

2. Precision

• in most cases, we rely on precision as our guide

3. Sensitivity

• related to the detection limit, or Minimum Detectable Quantity (MDQ)

4. Selectivity

is the method subject to interferences from other species besides the analyte?

5. Speed

• faster is always better (equipment time, analysts time, etc. - how many samples can be analyzed per day?)

6. Cost and Legal acceptance

6

The General Analytical Problem

Select sample

Extract analyte(s) from matrix

Detect, identify and

quantify analytes

Determine reliability and

significance of results

Separate analytes

7

Impossible to eliminate errors.

How reliable are our data?

Data of unknown quality are useless!

•Carry out replicate measurements

•Analyse accurately known standards

•Perform statistical tests on data

Errors in Chemical Analysis

8

Mean Defined as follows:

x

x

N

i

N

= i = 1

∑

Where xi = individual values of x and N = number of replicate

measurements

Median

The middle result when data are arranged in order of size (for even

numbers the mean of middle two). Median can be preferred when

there is an “outlier” - one reading very different from rest. Median

less affected by outlier than is mean.

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9

Illustration of “Mean” and “Median”

Results of 6 determinations of the Fe(III) content of a solution, known to

contain 20 ppm:

Note: The mean value is 19.78 ppm (i.e. 19.8ppm) - the median value is 19.7 ppm

10

Precision

Relates to reproducibility of results..

How similar are values obtained in exactly the same way?

Useful for measuring this:

Deviation from the mean:

d x xi i= −

individual value mean value

modulus

11

Accuracy

Measurement of agreement between experimental mean and

true value (which may not be known!).

Measures of accuracy:

Absolute error: E = xi - xt (where xt = true or accepted value)

Relative error: Er

xi

xt

xt

=−

×100%

(latter is more useful in practice)

12

Illustrating the difference between

“accuracy” and “precision”

Low accuracy, low precision Low accuracy, high precision

High accuracy, low precision High accuracy, high precision

3

13

Types of Error in Experimental Data

Three types:

(1) Random (indeterminate) Error

Data scattered approx. symmetrically about a mean value.

Affects precision - dealt with statistically (see later).

(2) Systematic (determinate) Error

Several possible sources - later. Readings all too high

or too low. Affects accuracy.

(3) Gross Errors

Usually obvious - give “outlier” readings.

Detectable by carrying out sufficient replicate

measurements.

14

Error Sources of Systematic

1. Instrument Error

Need frequent calibration - both for apparatus such as

volumetric flasks, burettes etc., but also for electronic

devices such as spectrometers.

2. Method Error

Due to inadequacies in physical or chemical behaviour

of reagents or reactions (e.g. slow or incomplete reactions)

3. Personal Error

e.g. insensitivity to colour changes; tendency to estimate

scale readings to improve precision; preconceived idea of

“true” value.

15

Systematic errors can be

constant (e.g. error in burette reading -

less important for larger values of reading) or

proportional (e.g. presence of given proportion of

interfering impurity in sample; equally significant

for all values of measurement)

Minimise instrument errors by careful recalibration and good

maintenance of equipment.

Minimise personal errors by care and self-discipline

Method errors - most difficult. “True” value may not be known.

Three approaches to minimise:

• analysis of certified standards

• use 2 or more independent methods

• analysis of blanks 16

SEPARATION of what may be a large number of components

IDENTIFICATION of these components (often called SPECIATION)

QUANTITATIVE MEASUREMENT of the amount of each of them

Principles of Separation Science

4

17

Chromatography Most general and common

separation technique

Discovered 1905 - Mikhail Tswett - Russian botanist

Developed 1940’s-50’s - Martin and Synge (U.K.)

General Principle:-

Sample contained in a mobile phase, which is carried

through or over a stationary phase. The components of

the mixture are partitioned between the phases.

Separation because of differing affinities of components

for the stationary phase.

Some components stay longer in the stationary phase - and hence move more slowly. 18

General Classification Specific Method Stationary Phase Type of

EquilibriumLiquid chromatography

(LC). Mobile phase: liquidLiquid/liquid Liquid adsorbed Partition

on solid (immiscible liquids)

Liquid/bonded phase Organic species Partition (liquid/

bonded to solid surface bonded surface)

Liquid/solid Solid Adsorption

Size exclusion Liquid in polymeric Partition/sieving

solid

Ion exchange Ion-exchange resin Ion exchange

Gas chromatography

(GC). Mobile phase : gas Gas/liquid Liquid adsorbed Partition

on solid (gas/liquid)

Gas/solid Solid Adsorption

Supercritical fluid

chromatography

Mobile phase: SF

Organic species Partition

bonded to solid (supercritical fluid/

surface bonded surface)

19

Column Chromatography

Routinely used in synthetic labs for cleaning up reaction products

Solid phase: silica

Mobile phase: organic solvent20

TLC Thin Layer Chromatography

UV detection of spots Coloured spots

For qualitative determination of reaction, and used in conjunction with column

chromatography for identification of fractions

5

21

A typical column chromatographic experiment

Mixture of A and B at top of column.

Carried down by mobile phase

Affinity for stationary phase: B > A

Detector at end of column records

nothing until time t3, when A emerges,

and t4, when B emerges.

Plot is called a chromatogram.

Peak positions used to identify

components; peak areas to determine

amounts of each analyte.22

Process of flushing mixture down the column = ELUTION

Some useful terms:

Mobile phase described as ELUENT

Material leaving column is ELUATE

Plot of detector response versus time

is called a CHROMATOGRAM

23

This shows the concentration profiles of A and B at times

t1 and t3 in the column separation shown earlier.

Separation increases with time, but so do peak widths.

Simply increasing column length does not necessarily

give better separation (resolution).

BAND BROADENING AND RESOLUTION

24

This shows two ways of

improving resolution -

(b) increased separation or

(c) decreased band width.

Details later on influencing

band widths.

Note: If there was always an equilibrium distribution between

mobile and stationary phases, there would be much less

band broadening - but this would take excessively long times.

Usually competition between speed and resolution.

6

25

Non-retained species: time to pass

down column = tM (dead time - time

for mobile phase to pass down column)

Time for analyte to pass down

column = tR = RETENTION TIME

Average linear velocity of mobile phase: u Lt M

=

(where L = length of column packing)

Average linear velocity of analyte molecules: ν = LtR

From these we can deduce the relationship between

migration rate and partition ratio

Retention Time

26

Usually gas-liquid chromatography (GLC) - but shorter abbreviation preferred

Mobile phase - carrier gas + vapour of analyte

Stationary phase - (usually) involatile liquid on inert solid support

Carrier gas - must be inert to analytes and stationary phase - usually He, H2, N2 or CO2

Types of column - Capillary (or “open tubular”) - fused silica tube (i.d. ~0.3 mm), with

inside wall coated with stationary phase. Length of column 10 - 100 m. High

resolution, but slow, and can only inject small samples on to column.

Packed - shorter (~1 m), wider (i.d.. 2 - 5 mm), with stationary phase

supported on small particles (~0.1 - 0.2 mm in diameter). Less resolving power

but quicker, and can cope with larger samples.

Gas Chromatography (GC)

27

Schematic diagram of a gas chromatograph

28

Stationary Phases

Must have: (1) low volatility (2) thermal stability (4) chemical inertness

(5) solvation properties giving suitable values for resolution of components

Commonest are polysiloxanes:

O Si

R

R

O SiR3n

R3Si

Nature of R varied to give different polarities.

e.g. All R = Me : non-polar column. Best for non-polar analytes (hydrocarbons, PAH’s etc.)

or R = 50% Me, 50% cyanopropyl - increased polarity - best for alcohols, acids etc.

Greater polarity from polyethylene glycols:

CH2

O CH2

CH2

OHCH2

OHn

Detectors will discuss these later (and applications of GC in real

analytical problems).

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29

Choice of stationary phase

• In general, polarity of stationary phase should match

that of sample components.

• In this case, order of elution is determined by boiling

points of eluents.

General polarity series: aliphatic hydrocarbons < olefins < aromatic hydrocarbons < halides < sulphides < ethers < nitro

cpds. < esters / aldehydes / ketones < alcholols / amines <

sulphones < sulphoxides < amides < carboxylic acids < water

30

General Elution Problem in GC

(a) - low temperature (450C) - good resolution

initially - but too slow later.

(b) - higher temperature (145oC) - much faster

but poor resolution for early-eluting species.

In general - best results for temperatures near

boiling point of analyte.

If there is a wide range of boiling points in the

sample - then the best results \re obtained by

temperature programming as shown in (c),

for the same mixture, where the temperature

steps are as shown.

31

Especially high-performance liquid chromatography (HPLC).

The term “high-performance” refers to the

use of packed columns with very small

packing particles (diam. 5-10 µm) -

giving greatly enhance resolution.

Note: several types of LC. In

addition to partition (as described

so far) - there are also ion-

exchange and size-exclusion

chromatography using liquid

mobile phases. We will concentrate

only on partition.

Liquid Chromatography (LC)

32

Schematic HPLC Apparatus

Rather complicated! High pressures needed to push mobile phase through finely-

packed column. “Sparging” = sweeping dissolved gases out of mobile phase using a

stream of inert gas.

8

33

Simple HPLC uses mobile phase of constant composition -

isocratic elution.

For more complex mixtures - programme a changing (stepwise or continuous)

mobile phase composition during the run - gradient elution. In HPLC this is

the usual solution to the general elution problem (solved in GC by temperature

programming

34

HPLC resources

http://kerouac.pharm.uky.edu/ASRG/HPLC/hplcmytry.html

35

Pumps

Needed because HPLC performed at high pressure.

Ideally need a steady flow - achieved using syringe pump. This has limited capacity.

Often need a reciprocating pump:

Unlimited capacity -

but pulsed flow. Therefore

need to include a ballast

volume (pulse damper) to

even this out.

36

Columns

Usually stainless steel (to withstand pressure), 1 - 5 mm diameter, ~5 µm packing.

Very efficient but limited length (cf. GC) because of pressure drop.

Packing - usually silica

Stationary phase - could be involatile liquid (like those in packed-column GC).

More usual now to use similar chemical species actually bonded to the silica

(longer column life), e.g.

Si O Si

Me

Me

R

R can be non-polar (C8 or C18hydrocarbon chain) or polar (amine,

nitrile etc.).

9

37

Stationary phase polar, mobile phase non-polar

= NORMAL PHASE CHROMATOGRAPHY.

Stationary phase non-polar, mobile phase polar

= REVERSED PHASE CHROMATOGRAPHY.

In normal phase, least polar analyte elutes first.

In reversed phase, most polar analyte elutes first.

38

Detectors

In both GC, HPLC – great effort to separate analytes.

When the separated analytes leave the column, we need to detect them.

What are the criteria for an ideal detector?

It should be:

UNIVERSAL (i.e. detects everything)

SENSITIVE (i.e. detects a very small amount of everything)

It should have a LINEAR RESPONSE (i.e. linear relationship between

intensity of response and amount of analyte).

It should give STRUCTURAL INFORMATION (i.e. tell you what the

analyte is, even if you didn’t know beforehand).

39

Selective Detectors

Very many possibilities - some of the more common:-

- thermionic detection (mainly for N, P)

- fluorescence

- light-scattering

- electron-capture detection (ECD) - especially for elements with high

electron affinities (e.g. halogens)

- UV - single wavelength or scanning

- FTIR

- mass spectrometry

40

Spectroscopic methods used particularly

where structural information is important.

Need to be careful not to use large detector

cells (causes loss of chromatographic

resolution) - especially when linking

successive detectors.

10

41

Detector Parameters

Sensitivity - defined in terms of

minimum detectable quantity (MDQ) -

the amount of material giving a

signal/noise ratio of ~3.

Linear dynamic range -need

linearity of response over at

least the range of amounts to be

analysed.

Detectors should have an MDQ

of <1 ng. Many do much better,

e.g. 270 fg of 2,3’,4’-trichloro-

biphenyl using ECD. On-line FTIR

gives a value of 95 pg for

caffeine.Linear dynamic range should cover at

least 4 - 5 orders of magnitude.

N

N N

N

O

CH3

O

CH3

CH3

42

Some Real-Life

Examples

of

Chromatographic

Analyses

43

2. An Environmental Example

(C.Aguilar, F.Borrull and R.M.Marcé, J. Chromatography, A, 1997, 771, pp. 221-231)

Pesticides - persistent pollutants - highly toxic.

Increasingly strict regulation (EU directive - <0.1 µg/l for drinking

water, <1-3 µg/l for surface water).

Report on analysis of water samples taken from delta of River Ebro

(northern Spain). Concentrate samples by passing 500 ml samples

through adsorbent material – then wash off with small amount of

organic solvent (hexane/ethyl acetate).Samples then subjected to

GC, with MS detection, and also using electron capture detection

(ECD).

ECD: irradiate carrier gas with β-electrons (from 63Ni). This

generates a large number of low-energy electrons, which give

a current on applying a voltage. In presence of analytes which

can capture electrons (e.g. halogen-containing compounds) the

current is reduced. A very sensitive method for such compounds.

44

Many pesticides contain halogens, e.g. lindane (hexachlorocyclohexane),

aldrin:

Cl

Cl

Cl

Cl

Cl2

and heptachlor:Cl

Cl

Cl

Cl

Cl2

Cl

These are particularly suitable for ECD detection.

The GC-MS and GC-ECD chromatograms, and the mass spectrum of lindane

are shown on the next slides

11

45

GC-MS chromatogram of water from delta of

River Ebro (N. Spain)

standard

malathion

aldrin

heptachlor

lindane

Time (min) 46

GC-ECD chromatogram of water from delta

of River Ebro (N. Spain)

Time (min)

standard

aldrin

heptachlor

lindane

47

Mass spectra of lindane: standard (a) and sample (b)

(a)

(b)

181219

145

111

181219

145

111

48

The GC-ECD chromatogram shows greatly increased sensitivity for chlorinated

species, but no structural information (assignments by comparison with standards).

N.B. Malathion (in the GC-MS) is a non-chlorinated species.

Concentrations (in µg/l) and relative standard deviations (%) are as follows

MS ECD

Conc RSD Conc RSD

Lindane 2.1 9 2.1 8

Heptachlor 1.7 13 1.7 11

Aldrin 1.5 11 1.5 9

Malathion 4.3 10 N/A N/A

Note that all are at the top end of, or above, the EU recommended levels for surface

water – and would require extensive treatment to bring down to the levels for

drinking water.

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49

Case Studies in HPLC

1. Heterocyclic amines in beef extracts.

(P.Pais et al., J. Chromatography, A, 778 (1997), pp 207 – 218)

Evidence for the formation of carcinogenic heterocyclic amines on

pyrolysis (cooking!) of protein-rich foods, e.g. meats. Recent HPLC

study of a beef extract, with MS detection.

The total ion chromatogram of the extract shown on next overhead:

50

HPLC-MS chromatogram of a beef extract

The peaks were identified as follows from the accompanying mass spectra:

1. TriMeIQx

N

N

N

NMe

Me

NH2

Me

Me

2. Glu-P-1

NN NH

2

Me

3. Harman

NN NH

2

Me

4. Norharman

NN

H

5. AαC

N N NH2

51

The concentrations of the largest components were:

3. (Harman) 129.5 ± 16.8 ng/g

4. (Norharman) 74.0 ± 7.4 ng/g

These are small amounts - but large enough to be a cause for concern

with these potent carcinogens.

52

Gramivimetric Analysis

• A quantitative method for determining concentration of a species in

solution.

• React solution species with (usually excess) of a soluble

compound and obtain a non-soluble precipitate.

Good for metals e.g.

Ag+aq + Cl-aq→ AgClsolid

The precipitate needs to be washed and dried, them carefully

weighed. Allows determination of Ag+ conc. in original solution.

Very Classical Method – as old as Chemistry itself!

13

53

Since silver chlorides are insoluble, can use Ag to determine chloride salt conc.

A common method for determining the amount of chloride in a sample is to

precipitate out the chloride with a solution of silver nitrate (gravimetric

analysis), according to the following reaction

20.00 ml of a solution of magnesium chloride was treated with excess silver

nitrate. Once filtered, dried, and weighed the mass of silver chloride found

was 0.2212 g. What was the original concentration of the magnesium

chloride solution?

MgCl2(aq) + 2 AgNO3(aq)→ 2AgCl(s) + Mg(NO3)2(aq)

54

Molecular weight of AgCl = 148.3 g mol-1

Therefore 0.2212 g of AgCl = 1.5 x 10-3 moles

MgCl2(aq) + 2 AgNO3(aq)→ 2AgCl(s) + Mg(NO3)2(aq)

Keep in mind reaction stochiometry, we can clearly see that 1 mole of insoluble

product requires 1 mole of MgCl2

So, providing silver nitrate is in excess, and reaction goes to completion, we can

see that original solution contained 1.5 x 10-3 moles of MgCl2 / 20 ml.

Concentrations are generally (not always expressed in mol dm-3

1 dm-3 = 1 litre

Therefore, 0.75 x 10-3 moles of MgCl2 in 20 ml, will be 50 x (1000 / 20) greater in

1 dm-3:

Original concentration of MgCl2 = 0.0375 dm-3

Please, NOT M

55

Limiting Reagents

Cu2S reacts with O2 to form Cu2O and SO2,

2 2 2 22Cu S(s) + 3O (g) 2SO (g) + 2Cu O(s)→

Suppose you have 3 moles each of Cu2S and O2: How much SO2 is produced?

e.g. A fuel mixture used in the early days of rocketry is composed of two liquids,

hydrazine (N2H4) and dinitrogen tetroxide (N2O4), which ignite on contact to form

nitrogen gas and water vapour. How many grams of nitrogen gas form when

1.00x102 g N2H4 and 2.00x102 g N2O4 are mixed?

Solution: The first step is to write down an equation and balance it.

2 4 2 4 2 2N H (l) + N O (l) N (g) + H O(g)→

2 4 2 4 2 22N H (l) + N O (l) 3N (g) + 4H O(g)→ balanced

• 2 moles of Cu2S will react with the 3 moles of O2 leaving 1 mole of Cu2S

• with no more oxygen left absolutely no more Cu2S will react

• O2 is called the limiting reagent because it is used up in the reaction before any of

the other reactants are used up allowing no further reaction

56

Next, determine the number of moles each of N2H4 and N2O4 which have molar

masses of 32.04524 and 92.01108 g mol-1, respectively.

2 4

2 4

2 4

2N H

N H -1

N H

m 1.00 10 gn = = = 3.12 mol

MM 32.04524 g mol

×

2 4

2 4

2 4

2N O

N O -1

N O

m 2.00 10 gn = = = 2.17 mol

MM 92.01108 g mol

×

From the balanced equation it is readily seen that:

2 4 2 42 mol N H reacts with 1 mol N O

2 4 2 4

11 mol N H reacts with mol N O

2

2 4 2 4

13.12 mol N H reacts with 3.12 = 1.56 mol N O

2×

So once all of the N2H4 reacts, there is still some N2O4 left over

ie. 2.17-1.56=0.61 mol excess N2O4.

Therefore N2H4 is the limiting reagent.

14

57

From the balanced equation

2 4 22 mol N H yields 3 mol N

2 4 2

31 mol N H yields mol N

2

2 4 2

33.12 mol N H yields 3.12 = 4.68 mol N

2×

So 4.68 mol N2 is produced from our mixture of rocket fuel.

2 2 2

-1

N N Nm = n MM = 4.68 mol 28.01348 g mol = 131 g× ×

131 g of N2 is produced from the mixture of rocket fuel.

2 4 2

3 mol N H yields mol N

2x x