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Chromatography

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”

Chromatography has application in every branch of the


physical and biological sciences
12 Nobel prizes were awarded between 1937 and 1972 alone
for work in which chromatography played a vital role
Chromatography
Web Dictionary:

Analytic technique to discover chemical components:


a method of finding out which components a gaseous
or liquid mixture contains that involves passing it
through or over something that absorbs the different
components at different rates
Chromatography
Chromatography is a physical method of separation in
which the components to be separated are distributed
between two phases
one of which is stationary (stationary phase) while the
other (the mobile phase) moves through it in a definite
direction.
The chromatographic process occurs due to differences
in the distribution constant of the individual sample
components.
Molecules that spend most of their time in the mobile
phase are carried along faster.
Classification
Stationary phase:
1- Thin layer chromatography (TLC): the stationary phase is a
thin layer supported on glass, plastic or aluminium plates.
2- Paper chromatography (PC): the stationary phase is a thin
film of liquid supported on an inert support.
3- Column chromatography (CC): stationary phase is packed in a
glass column.

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

[X]s is concentration in the stationary phase


[X]m is the concentration in the mobile phase
Partition Example:
Compound A: mass = 1 mg
Vol. Mobile Phase: 1 mL
Vol. Stationary Phase: 1 mL

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

If the mobile phase is moving, what would be?


Partitioning in a Mobile 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

0.16 mg 0.14 mg 0.12 mg 0.10 mg 0.08 mg 0.07 mg 0.06 mg

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

0.00 mg 0.00 mg 0.00 mg 0.03 mg 0.04 mg 0.07 mg 0.06 mg

1.0 mg
K=5

Two components are separated

How would you make this broad peak more narrow?


Analyte Peaks in the Mobile Phase
Theory of Column Efficiency
in Chromatography
1. Theoretical Plates

H : plate height Long column should


H =L/N L: column length be avoided→Small
N: number of plates H is required

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

Characteristics of a chromatographic peak. wb = 4σ.


Retention factor = k = (tR-tM)/tM = t’R/tM.
The width at half height, w1/2, equals 2.35σ, wb = 1.70w1/2.
The van Deemter Equation (Rate theory)

H = A + B/ū + Cū
Mobile
Phase

Stationary
Phase

interphase mass transfer


A Terms: Multipath (eddy diffusion)
The amount of spreading is affected by the
nature of the column material and how
Flow
Direction well the column is packed. This factor is
generally proportional to the particle size
of the packing material. This factor must be
2
1 taken into account for packed columns, but
for capillary columns, this term is not
needed since there are no particles.

Pathways of two molecules during elution. Distance


traveled by molecule 1 is longer than that traveled by
molecule 2, thus molecule 1 will take longer to
elute.
B Terms: Longitudinal Diffusion
(molecular diffusion)
At low velocities longitudinal diffusion has a negative effect
on resolution, but this effect is negligible at higher velocities.
This term is very important in gas chromatography as
diffusion coefficients in gasses are orders of magnitude
higher than in liquids. In liquid chromatography, this term is
typically close to zero relative to the other terms.

Molecules diffuse from areas of high


concentration to areas of low concentration.

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)

The faster the mobile phase moves, the


less time there is for equilibrium between
the phases and the mass transfer effect
on peak broadening is directly related to
mobile phase velocity.
van Deemter Plot

ū: liner velocity
Plate Height, H

A: eddy diffusion. Minimized


A + B/u + Cu with small, uniform
particles.
B: longitudinal (molecular)
diffusion. Decreases at
Mass Transfer (both), Cu faster flow.
Multipath Term, A
C: interphase mass transfer
Longitudinal diffusion, B/u Decreases at slower flow

Linear Velocity, u

→smaller particles give better resolution


2. Retention Factor

retention factor
k=(tR-tM)/tM=tR’/tM

Neff=N(k /(k+1))2

tR: retention time


tM: void volume
3. Resolution

Rs=(tR2-tR1)/((wb2-wb1)/2)
= 1/4√N[(α – 1)/α][(k2/(kave + 1)]

α=separation factor= tR2’/tR1’=k2/k1


k=retention factor=(tR-tM)/tM=tR’/tM
Increasing N increases resolution.
Increasing k (retention factor) increases retention
time, and broadens peaks.
Increasing the separation factor (a = k2/k1)
increases the resolution.
The number of plates required

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

Straight lengths of stainless steel columns are typical.


Usual i.d. is 3.9 or 4.6 mm, with 3- to 5-mm particles, 15-cm long.
These give plate counts of 9,000 (5-mm) to 15,000 (3-mm).
The fat column is a preparative column.
Columns
 Analytical column:
•length: 5 – 30 cm.
•ID: 1 – 5 mm.
•made of steel or plastic.
•expensive (>2000 L.E.),
easily damaged by dust or
particles in the sample or
solvent.
Protection:
1. by periodically renewed
guard columns containing
the same stat. phase like
main column.
2. by passing solvents and
samples through filters
(0.5 m). Preparative column  :
industrial scale (for 1 kg of material,
volume 300 L)
Reversed Phase Chromatography
Reversed phase HPLC Normal phase HPLC
column: polar
column: nonpolar solvent: nonpolar
solvent: polar
Endcapped C18 (ODS), C8, etc

Silanol groups on stationary phase silica particles are chemically


bonded with chlorosilanes (H3SiCl) to produce lipophilic organic.
Endcapping free SiOH groups with trimethyl silyl groups renders
them inert.
Detectors
• UV
• Single wavelength (filter)
• Variable wavelength (monochromator)
• Multiple wavelengths (PDA)
• Fluorescence
• Electrochemical
• Mass Spectrometric

The flow cell


Thin Layer Chromatography (TLC)

The eluent is drawn up the plate by capillary action.


The tank is covered to prevent evaporation of solvent from the plate.
The same stationary phases used in column chromatography are
used in TLC.
Adsorbents are most common.
Rf value

multiple development
two-dimensional
chromatography
two-dimensional
chromatography