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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