principles of chromatography. chromatography is the most powerful tool for separating &...
TRANSCRIPT
Principles of Chromatography
Chromatography is the most powerful tool for separating & measuring the components of a complex mixture.
Quantitative & qualitative analysis
What is Chromatography?
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)
What is Chromatography?
What is Chromatography?
b) 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 = [X]s / [X]mSolutes with a large Ks value will be retained more strongly by the stationary phase.
What is Chromatography?
c) The science & art of separation
d) Originator : adsorption chromatography by M.Tswett in 1903
e) Eluent, eluate, elution.
What is Chromatography?
elution : always (100%) dilution
What is Chromatography?
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.
What is Chromatography?
polar s.p.
What is Chromatography?
for GC & LC for GC
21.1 What is Chromatography?
resin-SO3- gel filtration
resin-N(CH3)3+ by size
What is Chromatography?
Ask Yourself 20-A p.461pH, and ionic strength
the most selective one
How do we describe a chromatogram
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
How do we describe a chromatogram
How do we describe a chromatogram
How do we describe a chromatogram
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.
How do we describe a chromatogram
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
How do we describe a chromatogram
c) Resolution (Rs)
How do we describe a chromatogram
Rs2 s.p. theoflength 2
1.5Rs analysis, vequantitatiFor
Lw
0.589t
ww21
tt
w
ΔtRs
1/2av
r
21
rr
av
r 12
Qualitative: • Co-chromatography• Detector:
– Mass spectrometer– IR, UV-VIS spectrophotometer
d) Qualitative & Quantitative analysis
How do we describe a chromatogram
• Figure illustrates the point that computers and humans may not choose the same baseline for measuring area.
P.464
Qualitative and Quantitative Qualitative and Quantitative AnalysisAnalysis
Internal Standards
• An internal standard is known amount of a compound, different from analyte, that is added to an unknown.
• To use an internal standard, we prepare a known mixture of standard and analyte and measure the relative response of the detector to the two species. In Figure 5-6, the area under each peak is proportional to the concentration of each compound injected into the column.
P.119
• [X] and [S] are the concentrations of analyte and standard after they have been mixed together.
P.119
ExampleExample : : Using an Internal StandardUsing an Internal Standard• In a chromatography experiment, a solution containing
0.083 7 M X and 0.066 6 M S gave peak areas of Ax=423 and AS=347.
• To analyze the unknown, 10.0 mL of 0.146 M S were added to 10.0 mL of unknown, and the mixture was diluted to 25.0 mL in a volumetric flask.
• This mixture gave the chromatogram in Figure 5-6, with peak areas Ax=533 and AS=582. Find the concentration of X in the unknown.
P.119
SOLUTIONSOLUTION ::
P.120
Because X was diluted from 10.0 to 25.0 mL when the mixture with S was prepared, the original concentration of X in the unknown was (25.0/10.0)(0.057 21 M)=0.143 M.
Why do bands spread ?
1) Why broadening?a) diffusionb) slow equilibration of solute between the m.p and s.p.c) irregular flow paths.
Why do bands spread ?
2) Longitudinal diffusion :
the faster the flow
the less a band spends in column.
the less time for diffusion.
broadeningu
1
Why do bands spread ?
3) solute requires time to equilibrate between phases.
(s.p.m.p.) with temp. broadening u
Can’t equilibrate rapidly enough.
m.p.
s.p.
Why do bands spread ?
Solute requires a finite time to equilibrate between the mobile and stationary phases.
P.466
4) A Separation Has an Optimum Flow Rate4) A Separation Has an Optimum Flow Rate
• The rate of mass transfer between phases increases with temperature.
Optimum resolution (minimum plate height) occurs at an intermediate flow rate. Curves show measured plate height in gaschromatography of n-C17H36 at 175°C, using N2, He, or H2 mobile phase.
Why do bands spread ?
Why do bands spread ?
5) Multiple paths
Band spreading from multiple flow paths. The smaller the stationary-phase particles, the less serious is this problem. This process is absent in an open tubular column.
Why do bands spread ?
6) Plate height equation
Plate height equation
Why do bands spread ?
Why do bands spread ?
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.)
Why do bands spread ? 8) Funny shapes
polarsolute
OH OH
SiSi S i S iOO
OSi(CH 3)3(CH3)3SiO
s.p. silanization
Mass Spectrometry
P.470
Mass Spectrometry• Mass spectrometry measures the masses and
abundances of ions in the gas phase.
A Mass SpectrometerA Mass Spectrometer
• Figure next page shows a transmission quadrupole mass spectrometer, which is the most common mass separator in use today.
• The mass separator consists of four parallel metal rods to which a constant voltage and a radio-frequency oscillating voltage are applied.
Transmission quadrupole mass spectrometer.
P.470
Figure 21-13
• Ionization: 1) Electron ionization
2) Chemical ionization
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 is a reconstructed total ion chromatogram showing all ions from seven opium alkaloids found in street heroin.
• Selected ion Chromatograms:– Simplify analysis – improve S/N
P.473
Information in a Mass Spectrum
Nominal Mass : CNominal Mass : C44HH99Br is 136 Br is 136
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+
Information in a mass spectrumFragmentation Patterns
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 atoms;
even nominal mass/ even number of N atoms