Chapter 21

Principles of Chromatography

Chromatography is the most powerful tool for separating & measuring the components of a complex mixture.

Quantitative & qualitative analysis

21.1 What is Chromatography?-1

1) Solvent Extraction :

transfer of a solute from phase 1 phase 2

S (in phase1) S (in phase 2)

Partition coefficient

1

2

s

sK

2) Chromatography : same as extractiona) One phase: held in place stationary phase. solid material (packing material)

Another phase : fluid phase mobile phase. sample: gas (GC) liquid (LC)

21.1 What is Chromatography?-2

21.1 What is Chromatography?-3b) A solute equilibrates between a mobile

and a stationary phase. The more it interacts with the stationary phase,

the slower it is moved along a column.

Xm Xs Ks =

Solutes with a large Ks value will be retained more strongly by the stationary phase.

s

m

X

X

21.1 What is Chromatography?-4

c) The science & art of separation

d) Originator : adsorption chromatography by M.Tswett in 1903

e) Eluent, eluate, elution.

21.1 What is Chromatography?-5

Elution : always (100%) dilution

21.1 What is Chromatography?-6

sam plein

eluentin

CaCO 3

(adsorption)

colum n

eluantout

detector

chrom atogram(m ass spect. IR

spect. etc)

3) Types of Chromatography

Is divided into categories on the basis

of the mechanism of interaction of

the solute v.s. the stationary phase.

21.1 What is Chromatography?-7

polar s.p.

21.1 What is Chromatography?-7

for GC & LC for GC

21.1 What is Chromatography?-8

resin-SO3- gel filtration

resin-N(CH3)3+ by size

21.1 What is Chromatography?-9

Ask Yourself 20-A p.432pH, and ionic strength

Most selective one

21.2 How do we describe a chromatogram -1

1) Chromatogram :

A graph showing the detectors

response as a function of elution

time :

band’s shapes, position, resolution.

2) For individual band :

a) Retention time (tr) :The time needed after injection for an individual solute to reach detector.

b) An ideal chromatographic peak Gaussian shape. w½ = 2.35σ, w = 4σ

21.2 How do we describe a chromatogram -2

21.2 How do we describe a chromatogram -3

21.2 How do we describe a chromatogram -3

3) For pairs of bands

a) Efficiency : two factors contribute to how well components are separated :

the widths of the peaks :

the wider the peak, the poorer separation.

the spacing in time :

the further apart, the better separation.

21.2 How do we describe a chromatogram -4

b) Theoretical plates (N): (from distillation)the more plates on a column, the more

equilibration steps, and the better the

separation.

Number of plates on column :

N = 5.55(tr/w½)2

Plate height : H = L/N

The smaller plate height

narrower peaks better separation

21.2 How do we describe a chromatogram -5

c)Resolution (Rs)

Rs2 s.p.the of length2

1.5Rs analysis,ve quantitati For

Lww21

tt

w

tΔRs

21

rr

av

r 12

21.2 How do we describe a chromatogram -6

21.2 How do we describe a chromatogram -7

Qualitative: • Co-chromatography• Mass spectrometer• IR spectrophotometer

Quantitative:• The area of peak

Internal standard

d) Qualitative & Quantitative analysis

e) Scaling up (rule at p.452)

1.Analytical chromatography: long & thin column. For a small scale: separate, identify, or measure.

2.Preparative chromatography: short, fat column. For large scale : purify

1 flow

2 flow

2

1

2

V

V

r

r

1 mass

2 mass :eqn Scaling

21.2 How do we describe a chromatogram -8

21.3 Why do bands spread ? -1

1) Why broadening?a) diffusionb) slow equilibration of solute between the

m.p and s.p.c) irregular flow paths.

21.3 Why do bands spread ? -2

2) Longitudinal diffusion :

the faster the flow

the less a band spends in column.

the less time for diffusion.

broadeningu

1

3) solute requires time to equilibrate between phases.

(s.p.m.p.) with temp. broadening u

Can’t equilibrate rapidly enough.

21.3 Why do bands spread ? -3

m.p.

s.p.

21.3 Why do bands spread ? -4

21.3 Why do bands spread ? -54) An optimum rate : flow rate for the best

separation.

21.3 Why do bands spread ? -6

5) Multiple paths

21.3 Why do bands spread ? -6

6) Plate height equation

Plate height equation

21.3 Why do bands spread ? -7

21.3 Why do bands spread ? -8

7) open tubular columns

Packed column (A, B, C 0 in van Deemter’s eqn.)

Open tubular column (A = 0 in van Deemter’s eqn.) resolution (∵ H & column length) sample capacity (∵ less s.p.)

21.3 Why do bands spread ? -98) Funny shapes

polarsolute

OH OH

SiSi S i S iOO

OSi(CH 3)3(CH3)3SiO

s.p. silanization

20.4 Chemical Analysis by Chromatography -2

21.4 Mass Spectrometry

Transmission Quadrupole Mass Spectrometer

• Ionization: 1) Electron ionization

2) Chemical ionization

21.4 Mass Spectrometry

1) Electron ionization

M + e- M+ + e- + e-

70 eV -55 eV 0.1eV

Molecular ion break into fragments.

Base peak:

most intense peak.

2) Chemical ionization

CH4 + e- CH4+ + 2e-

CH4+ + CH4 CH5

+ + CH3

CH5+ + M CH4 + MH+

CH4+ CH3

+ + H

CH3+ + CH4 C2H5

+ + H2

• Total ion Chromatograms

• Selected ion Chromatograms:– Simplify analysis – improve S/N

21.5 Information in a mass spectrum

Rxn : CH3(CH2)2CH2–OH + Br- CH3(CH2)2CH2–Br

1–Butanol 1–Bromobutane

CH3 15

CH2 14

Br 79

C4H979Br+ 50.0%

C4H981Br+

21.5 Information in a mass spectrumFragmentation Patterns

21.5 Information in a mass spectrum

21.5 Information in a mass spectrumIsotope PatternsCnHxOyNz

12C/13C

Intensity = n x 1.1%

Ex: C6H6

(M+1)/M+ = 6 x 1.1 %

Nitrogen Rule: A compound: odd nominal mass / odd number of N at

oms; even nominal mass/ even number of N atoms