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History
Mikhail Tswett, Russian, 1872-1919
Botanist
In 1906 Tswett used to chromatography to
separate plant pigments
He called the new technique chromatography
because the result of the analysis was 'written
in color' along the length of the adsorbent
column
Chroma means “color” and graphein means to “write”
Mobile phase:
1- Liquid chromatography: mobile phase is a liquid. (LLC, LSC).
2- Gas chromatography : mobile phase is a gas. (GSC, GLC).
Various Chromatography
Injector
Port Detector
Flow Control Recorder
Column
Column Oven
Carrier Gas
Classification by separation
1- Partition chromatography.
2- Adsorption chromatography.
3- Gel filtration chromatography.
(Size Exclusion, Gel Permeation )
4- Ion exchange chromatography.
5- Affinity chromatography.
Classification by separation
Chromatography Theory
Partition Coefficient
𝐗𝐬
K=
𝐗𝐦
Mobile Mobile
Phase Phase
K = 0.2 K=2
grams in grams in
Stationary mobile phase = 0.83 Stationary mobile phase = 0.33
Phase Phase
1st batch
0.83 mg 0.69
0.83 mg 0.69 mg
0.58 0.58
0.48 mg 0.40
0.48 mg 0.33 mg
0.40 0.33 mg
0.28
1.0 mg
K = 0.2
Partitioning in a Mobile Phase
2nd batch
0.14 mg 0.14
0.23 mg 0.23 mg
0.29 0.29 mg
0.32 0.32 mg
0.33 0.34 mg 0.28 mg
0.16 mg
0.03 0.14
0.05 mg 0.12 mg
0.06 0.10 mg
0.06 0.07
0.08 mg 0.07
0.07 mg
mg 0.06 mg
mg
K = 0.2
Partitioning in a Mobile Phase
K=0.2
0.83
0.00 mg
mg 0.69
0.00 mg
0.83 mg 0.58
0.69
0.01 mg
mg 0.05 mg 0.17 mg 0.28 mg
0.34 0.28 mg
1.0 mg
K=5
HPLC: 0.01~0.03 mm
The efficiency of a column is described by the number of theoretical
plates. The narrower the peak, the greater the number of plates.
The more theoretical plates, the greater the resolving power.
N =L/H
=L・L/σ2=16L2/w1/22
=16(tR/wb)2
=(tR/σ)2
= 5.545(tR/w1/2)2
H = A + B/ū + Cū
Mobile
Phase
Stationary
Phase
Flow
Over time….
Flow
C Terms: Mass Transfer (Cs & Cm)
Equilibrium between the mobile and
stationary phases is never realized
It takes time for analytes to move from the
mobile phase into the stationary phase.
Because no equilibrium is reached, some of
the analytes are swept ahead of the of the
main band.
It also takes time for molecules to move back
out of the stationary phase, and some of the
analyte molecules will be left behind by the
rapidly moving mobile phase.
C Terms: Mass Transfer (Cs & Cm)
ū: liner velocity
Plate Height, H
Linear Velocity, u
retention factor
k=(tR-tM)/tM=tR’/tM
Neff=N(k /(k+1))2
Rs=(tR2-tR1)/((wb2-wb1)/2)
= 1/4√N[(α – 1)/α][(k2/(kave + 1)]
Nreq=16Rs2(α/α-1)2((kave + 1)/k2)2
Neff=N(k /(k+1))2
→Neff(req)=16Rs2(α/α-1)2
Q. Calculate the number of plates in the column resulting
following chromatographic peak
tR = 52.3 mm
wb= 9.0 mm
High Performance Liquid
Chromatography
• Mobile phase is liquid
• partition
• adsorption (liquid-solid)
• ion exchange
• size exclusion or gel
• Affinity
Typical HPLC columns
multiple development
two-dimensional
chromatography
two-dimensional
chromatography