11/18/2024

Chromatography
 Is an experimental technique used to separate and
identify the components of a mixture.
probably
 Works by allowing the molecules present in the
mixture to distribute themselves between 2 phases
 one of which is stationary (stationary phase)
while the other (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.
molecules that wants to interact more to stationary phase moves slower .

the
Chemical
sampleMix
-

*
a
Distributitanton
The analytes interacting most
strongly with the stationary
phase will take longer time to
KA
·

pass through the system than

KB
those with weaker interactions.
·

These interactions are usually

chemical
interaction
in nature, but in some
cases physical interactions can
also be used. 4 size exclusion (SEC)

V(retention + or V)
peak A ·
Report as + or
peak B
volume occupied by the sample
baseline in the column
-

or Volume
CHROMATOGRAM

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separation
Types of Chromatography (according to mobile phase)

1.) The primary division of chromatographic techniques is based on the type of mobile
phase used in the system:

Type of Chromatography Type of Mobile Phase

① Gas chromatography (GC) gas
② Liquid chromatography (LC) liquid

2.) Further divisions can be made based on the type of stationary phase used in the
system:

Mobile phase = Gas Mobile phase = Liquid

Gas Chromatography Liquid Chromatography

Stationary Phase = Liquid Stationary Phase = Solid Stationary Phase = Liquid Stationary Phase = Solid
Gas-Liquid Gas-Solid Liquid-Liquid Liquid-Solid
Chromatography Chromatography Chromatography Chromatography
GLC GSC LLC LSC

mobile phase-stationary phase chromatography

Types of Chromatography
(according to the packing of the stationary phase)
> solid film

1- Thin layer chromatography (TLC): the

stationary phase is a thin layer supported on
glass, plastic or aluminium plates.

2- Paper chromatography: the stationary phase

is filter paper strips. fit Filter - solvent for paper

> widely used

3- Column chromatography (CC): stationary

phase is packed in a glass column.

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Thin Layer Chromatography (TLC)

as

- coatstations

(mobile phase) I solvent depth

↑
samples,
here , pure components

*
Stop when solvent mobile phase
reached the endline .

Thin Layer Chromatography (TLC)

As the solvent moves past the spot that was applied, an
equilibrium is established for each component of the mixture
between the molecules of that component which are
adsorbed on the solid and the molecules which are in
solution.

In principle, the components will differ in solubility and in the

strength of their adsorption to the adsorbent and some
components will be carried farther up the plate than others.

When the solvent has reached the top of the plate, the plate is
removed from the developing chamber, dried, and the
separated components of the mixture are visualized.

If the compounds are colored, visualization is straightforward.

Usually the compounds are not colored, so a UV lamp is
used to visualize the plates.

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Column Chromatography
analyte
mixture
↓
Column
chromatography
Stationary phase is
held in a narrow
tube through which
the mobile phase is
forced under
pressure or under
the effect of gravity

·
LiquidPhaseph should be dissolved in mobile phase

But , polymer :

Gas Chromatography ~
cut in smaller pieces,
sample is in phase .
gas
but not polymer anymore.
ex .
propylene and ethylene

Name of GC Method Type of Stationary Phase

Gas-solid chromatography solid, underivatized support
Gas-liquid chromatography liquid-coated support
Bonded-phase gas chromatography chemically-derivatized support

- sample

=>
observes the separation .

>
- mobile phase

stationary phase

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Liquid Chromatography
Name of LC Method Type of Stationary Phase
Adsorption chromatography solid, underivatized support (Lsc)
t
&
Separation involves
interaction between
samples and Partition chromatography liquid-coated or derivatized support (4c)
stationary phase. 3) Ion-exchange chromatography support containing fixed charges
4) Size exclusion chromatography porous support
no interaction b/w sample and S P.
.

> separates purely by the size of molecules .

Types of Mechanism in the

Chromatography
Adsorption Chromatography
• Adsorption chromatography is probably one of the oldest
types of chromatography around.
• It utilizes a mobile liquid or gaseous phase that is adsorbed
onto the surface of a stationary solid phase. The equilibration
between the mobile and stationary phase accounts for the
separation of different solutes.
stationary
phaseactiets
mobile
X


◌ ✹
sample ↓
polarity is

✹ the same...

so
,
this component
will move slower

Liquid-Solid than others.

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Types of Mechanism in the

Chromatography
Partition Chromatography
• This form of chromatography is based on a thin film
formed on the surface of a solid support by a liquid
stationary phase. Solute equilibriates between the
mobile phase and the stationary liquid.

 ◌
Liquid-Liquid

Types of Mechanism in the

Chromatography
Partition Chromatography
low polar S .
P.
>
-

in comparison to
stationary phase polarity in comparison to
stationary phase polarity

St w
StrongestPolarity comes outfirstsinc
highly polar aastate

>
-

separation is not good separation time

,

b/w other components are

quite close .

m
sample mixture of A B C
,
.
,

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Types of Mechanism in the

Chromatography
Ion Exchange Chromatography >
-
ionic interaction

• In this type of chromatography, the use of a resin (the

stationary solid phase) is used to covalently attach
anions or cations onto it. Solute ions of the opposite
charge in the mobile liquid phase are attracted to the
resin by electrostatic forces.
counterior y
in .


+ Fixed charge

◌ + -

◌
·
If O OF
solute ions
can replace
Counter ion the counterior
Of S P.
o
.

Ion-Exchange
- To remove

elute it to
another M P. .

components analy attaches to it.

# CC can decrease its efficiency when used very long ... some of

Types of Mechanism in the

Chromatography
Size Exclusion Chromatography
• Also known as gel permeation or gel filtration,
• this type of chromatography lacks an attractive interaction
between the stationary phase and solute. The liquid or
gaseous phase passes through a porous gel which separates
the molecules according to its size. The pores are normally
small and exclude the larger solute molecules, but allows
smaller molecules to enter the gel, causing them to flow
through a larger volume. This causes the larger molecules to
pass through the column at a faster rate than the smaller
ones.




Exclusion

large see

(D
*

>
-
Fastest to more out
the column
Santanary * smaller size
=> can enter pores ,
so

moves relatively slower.

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Theory of Chromatography
1.) Typical response obtained by chromatography (i.e., a chromatogram):

chromatogram - concentration versus elution time

occurswheample
-
- peak

Spy shouldharrais
to each other
Wh

-peakFromcase

SELINE only flow of mobile phase

Wb
baseline width of peak

Inject

Where:
tR = retention time

.
time that mobile phase out the column
tM = void time along the voids .
moves

W b = baseline width of the peak in time units

W h = half-height width of the peak in time units
Volume - > VR

Note: The separation of solutes in chromatography depends on two factors:

(a) a difference in the retention of solutes (i.e., a difference in their time or volume of
elution
(b) a sufficiently narrow width of the solute peaks (i.e, good efficiency for the separation
system)

Peak width & peak position

determine separation of peaks

·
peak width
-
relates to
of column

Cretention)
A similar plot can be made in terms of elution volume instead of elution time. If volumes
are used, the volume of the mobile phase that it takes to elute a peak off of the column is
referred to as the retention volume (VR) and the amount of mobile phase that it takes to
elute a non-retained component is referred to as the void volume (VM).

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2.) Measures of Solute Separation:

①
resolution (RS) – resolution between two peaks is a measure of how well two peaks are separated:
tr2 – tr1
RS =
(W b2 + W b1)/2
where: tr1, W b1 = retention time and baseline width for the first eluting peak
tr2, W b2 = retention time and baseline width for the second eluting peak

Icould notseparatas
Rs  1.5 represents baseline
resolution, or complete separation of
two neighboring solutes  ideal case.
the
! , Rs  1.0 considered adequate for
tre most separations.

I
best resolution

highest column
efficiency

2.) Solute Retention:

A solute’s retention time (tR) or retention volume (VR) in chromatography is directly

related to the strength of the solute’s interaction with the mobile and stationary phases.
parameters
Retention on a given column pertain to the particulars of that system:
- size of the column
- flow rate of the mobile phase

VR = F tR

F : mobile phase flowrate =

[m/min]

Response I
VR,
1
= 10 m So , VR is independent of flow rate !

VR z = 10 ml · void volume is the same in the column

.

VR don't change

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The interactions of a solute in a column is illustrated for partition chromatography:

KD
A (mobile phase) A (stationary phase)
making decision ofA :
if it prefers to
stay in mobile or stationary phase .
>
-

where: KD = equilibrium constant for the distribution of A between the mobile

phase and stationary phase

Assuming local equilibrium at the center of the chromatographic peak:

&
[A]stationary phase >
-
amount of A that stays with SP IFKD < 1 +

stays
KD =
[A]mobile phase >
-
amount of A that stays with MP

As KD increases, interaction of the solute with the stationary phase becomes more
favorable and the solute’s retention increases

Separation between two solutes requires different KD’s for their interactions
with the mobile and stationary phases

3.) Efficiency: can use Resolution

Efficiency is related experimentally to a solute’s peak width.

- an efficient system will produce narrow peaks
- narrow peaks  smaller difference in interactions in order to separate two solutes

Efficiency is related theoretically to the various kinetic processes that are involved in
solute retention and transport in the column
- determine the width or standard deviation () of peaks

Estimate  from peak widths,

assuming Gaussian shaped peak:
Wh
W b = 4

W h = 2.354

EFFICIENCY Dependent on the amount of time that a solute spends in the column (k’ or tR)

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Number of theoretical plates (N): compare efficiencies of a system for solutes that have
N
different retention times
&
efficiency

I
-
N = (tR/)2
-

VS .

or for a Gaussian shaped peak

↓ N = 16 (tR/W b)2
more efficient

N = 5.54 (tR/W h)2

The larger the value of N is for a column, the better the column will be able to separate
two compounds.
- the better the ability to resolve solutes that have small differences in retention
- N is independent of solute retention (tr UR)
or

- N is dependent on the length of the column

Plate height or height equivalent of a theoretical plate (H or HETP): compare efficiencies of

columns with different lengths:

width of peak a H H = L/N

where: L = column length

N = number of theoretical plates for the column

Note: H simply gives the length of the column that corresponds to one theoretical plate

H can be also used to relate various chromatographic parameters (e.g., flow rate, particle size,
etc.) to the kinetic processes that give rise to peak broadening:

Why Do Bands Spread?

a. Eddy diffusion
b. Mobile phase mass transfer
c. Stagnant mobile phase mass transfer
d. Stationary phase mass transfer
e. Longitudinal diffusion
Bands

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Why Do Bands Spread? 1

-

Fast (wider way

↓ ↓ <
slower (narrower way

stationarye

some
-
molecules

stilwester

a.) Eddy diffusion – a process that leads to peak (band) broadening due to the presence
1 1 1
of multiple flow paths through a packed column.

As solute molecules travel through the column,

some arrive at the end sooner than others simply
due to the different path traveled around the
support particles in the column that result in
different travel distances.

Longer path arrives at end of column after (1).

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b.) Mobile phase mass transfer – a process of peak broadening caused by the
presence of different flow profile within channels or
between particles of the support in the column.

A solute in the center of the channel

moves more quickly than solute at the
edges, it will tend to reach the end of
the channel first leading to band-
broadening

The degree of band-broadening due to eddy diffusion and mobile phase

mass transfer depends mainly on:

1) the size of the packing material

2) the diffusion rate of the solute

c.) Stagnant mobile phase mass transfer – band-broadening due to differences in the
rate of diffusion of the solute molecules between the
mobile phase outside the pores of the support
(flowing mobile phase) to the mobile phase within
the pores of the support (stagnant mobile phase).

Since a solute does not travel down

the column when it is in the stagnant
mobile phase, it spends a longer time
in the column than solute that
remains in the flowing mobile phase.

The degree of band-broadening due to stagnant mobile phase mass

transfer depends on:

1) the size, shape and pore structure of the packing material

2) the diffusion and retention of the solute
3) the flow-rate of the solute through the column

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d.) Stationary phase mass transfer – band-broadening due to the movement of solute
between the stagnant phase and the stationary phase.

Since different solute molecules

spend different lengths of time in the
stationary phase, they also spend
different amounts of time on the
column, giving rise to band-
broadening.

The degree of band-broadening due to stationary phase mass transfer

depends on:

1) the retention and diffusion of the solute

2) the flow-rate of the solute through the column
3) the kinetics of interaction between the solute and the
stationary phase

e.) Longitudinal diffusion – band-broadening due to the diffusion of the solute along the
length of the column in the flowing mobile phase.

The degree of band-broadening due

to longitudinal diffusion depends on:

1) the diffusion of the solute

2) the flow-rate of the solute through
the column

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5. Chromatographic compromise

Peak resolution

very high speed

pour Resolution

Speed of Sample
Mobile Phase capacity

-
can not increase altogether if wants
best result .

Resolution Example
• A peak with a retention time of 407 s has a
base width of 13 s. A neighboring peak is
eluted at 424 s with a width of 16 s. Find
the resolution.

T
tre - tr 2
Re
,
=

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Example
• A solute with a retention time of 407 s has
a width at the base of 13 s on a column
12.2 m long. Find the number of plates
and plate height.

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Example
• A solute with a retention time of 407 s has
a width at the base of 13 s on a column
12.2 m long. Find the number of plates
and plate height.

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Types of Liquid Chromatography

Separation is related to the interaction blw sample and stationary phase.
>
-

① Interactive technique - adsorption, partition, ion exchange, etc

② Non-interactive technique - GPC, SEC, GFC
main
subsets of SEC

High-performance
Liquid Chromatography
(Luc)

Nomenclature

Size Exclusion Chromatography SEC

(
1
Gel Permeation Chromatography GPC organic mobile phase

Gel Filtration Chromatography GFC Water mobile phase

that
used in polymers are

dissolvable in water

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Size Exclusion Chromatography

needs to be filtrated,
>
-
use degasser. . .
mobile
phase
reservoir W

1. Reciprocating piston pump delivers eluent from

controls the reservoir at a constant volumetric flow rate.
Flow
SEC is always an isocratic separation, i.e.
composition of the mobile phase remains
constant throughout the experiment.

2. injection valve permits introduction of sample

separationa solution without interrupting solvent flow.

3. SEC columns perform a separation based on

detect the
molecules .
separated the molecular size of polymer molecules in
solution. Resolution and/or resolving range is
increased by use of multiple column systems

4. Detector responds to concentration of polymer

molecules eluting from the SEC columns

Additional Components Used in SEC

Concentration detectors >
signal is related to [sample]

 Differential refractometer (RI)

 Ultraviolet absorbance (UV)
 Evaporative light scattering or mass detector (ELS, EMD)
 Infra-red (IR)
&
Molecular weight sensitive detectors > detection is based on the

difference of molecular weight.

 Viscometry
 Light scattering
Additional systems
 Online degasser , treatment of mobile phase

 Autosampler
 Column oven
 Additional specific detectors

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Polymer Molecules in Solution

- Polymer
solutiona
-
solvent [a
 SEC is based on the behaviour of polymer molecules in solution
 In the solid state polymers can be considered like spaghetti – a confusing mass of
intertwined chains
 In solution, polymer molecules are discrete entities
 Due to entrophic effects, polymer chains ideally curls up in solution to form a ball
like shape.

so

SEC Column

stationary phase

*
Columns are packed with porous particles, controlled pore
size and particle size
 Columns are produced by slurry packing technique, packed
at pressures in excess of 2000psi
 Column dimensions typically 7-8 mm and 250-600 mm in
length

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Permeation of Polymer Molecules

* Separation could not occur

if 100 % Exclusion + molsize por size

or
100 % Total permeation

PolymNation
I
can not enter
in the pores

>
-

enters big pores

-
enters every pores
4

 Polymer coils in solution can

permeate the pores on SEC packing
materials
 Exclusion,partial permeation and
total permeation are possible

SEC Separation Mechanism

 Polymer is prepared as a dilute solution in the

eluent and injected into the system
 The SEC column is packed with porous
beads of controlled porosity and particle size
 Large molecules are not able to permeate all
of the pores and have a shorter residence
"Stay"
time in the column
 Small molecules permeate deep into the
porous matrix and have a long residence time
in the column
 Polymer molecules are separated according
to molecular size, eluting largest first,
smallest last

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Elution Profile – Nomenclature

Exclusion volume (V0) - Upper MW (size) limit / molecules are too

large to get trapped in the stationary phase. They will elute at
the same retention volume/time (also known as void ↓
If all molecules
volume/time). constant this in
too large
case .
are .

Total permeation volume (Vt) – Lower MW limit / all molecules

below this molecular limit are so small that they penetrate all
pores of the stationary phase and elute as a single band.

Pore volume (Vp) – Working resolving range of MW

&
Vt = V0 + Vp

·
Do
O
couldn't enter the pars

por size size a sample mol.

stays in 000 8
void volume >

#A
Size Exclusion Chromatography
M

e
M

- If scrph selectivity range (pousize)

>

- con't evites pore sizes ,

corus out together fast
size, Sample size are

If sample par
=
>

enough to enter

eventamai
or exit the
pures

Total permeation Exclusion

volume/time Selectivity
lower limit Upper limit volume/time
lated
to above upper limit

size
pore
Molecular
Size : V big
small
Selective
permeation region

Small molecules : large molecules :

high permeation volume low permeation volume

* Separation occurs in S P.
.

* S P's
.
particle should
not vary widely in size
and must have poses !

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Size Exclusion Chromatography

Vo : volume of
the void

Total permeation volume; Vt = V0 + Vp Up : total volume g

total volume when samples could stay ports

Where

Separationa Retention volume; Vr = V0 + KsecVp

- Vo : exclusion volume
:
KsecVp :
possibility they
could enter pores
diffusion constant
of sample that could

[X]s
enter the pores .

stay
Xs ; Ksec =
X could
Xm
Some in
mobile phase and some could
a
[X] :
sample molecule
concentration
stay in stationary phase [X]m
where: KSEC = KD = equilibrium constant for the distribution of X between
the mobile phase and stationary phase

Size Exclusion Chromatography

[X]s = [X]m
Therefore
Ksec = 1 Vt = V0 + Vp
X
all
could enter
the port sizes
Total permeation volume

0 < Ksec < 1 Selectivity or separation occurs

[X]s = 0 >
-
X could not enter any pores

Ksec = 0 V = V0 exclusion limit

X can't spend a

diffuse in stationary
phase's ports .

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 18/11/67
quantitative t prepar sample , you want
to know
quartitative
definition of each sample (MW amount) etc.
-
...

SEC
Y qualitative -
you know sample contains A B C
, ,
and
could be separated using SEC.

quantitative approach :

① What is sample polymer A ? Elution Profiles

② Calibration Curve of standard polymer A (know MW and size

needs standard polymer


·

As a result of the SEC separation calibration of different

molecular weights.
mechanism, polymer molecules elute 10 000
from the column in order of size in ↓ V

solution 60000


Largest elute first, smallest elute last
The separation is purely a physical
partitioning, there is no interaction or m
o
binding should have different sizes
when diluted.
 The separation is isocratic
10000

X ↓ This range
-
60 , 000

If polymer molecules have the same -

X can only be used ;
numbers
not
molecular dimensions, they will co-elute X more or less can use

calibration
-

this curve.
by SEC and may not be separated by Y

this technique
 The calibration curve describes how
Y
different size molecules elute from the D

column

Combination of Individual Pore Size Columns

Traditional approach to increasing MW operating range of column set

10 10
A
Log MW

Log MW

A+ B columns

5 B 5

can use this range

10 25 50 10 50 100
Retention Vol. Retention Vol.

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sample mixtures/component
e
Retention of solutes in column

 Separation and retention of solutes in SEC column

depend on:
 pore vol., pore shape and pore size distribution of
stationary phase
hydrodynamic vol. (Vh) and radius of gyration (Rg) of
ata tra


polymer largerthanusual in goodsola Un Rg

 solvent power ---
good solvent/ theta solvent/ poor solvent
 concentration of the sample

consider  polymer branching, copolymer composition

*** Separation by SEC – NO surface interaction/
agglomerates
NO polymer aggregation/
NO polymer degradation

Polymer Calibrants for SEC

standard polymer to use as calibration curves

Most commonly used polymer calibrants

Polystyrene - THF, toluene, chloroform, TCB
mobile phases

Polymethyl methacrylate - MEK, ethyl acetate,

acetone, DMF
Polyethylene oxide/glycol - aqueous eluents, DMF,
DMSO

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Calibration of SEC Columns

Using Narrow Standards

 Chromatograph a series of well

High MW Low MW
characterized, narrow polydispersity
polymer standards

 Plot peak retention time (RT) versus

peak log molecular weight (log M)

 Fit the data using a mathematical

function

 The calibration curve will be

characteristic of the SEC column set
used W

notexamis Peak Integration in SEC

The sample peak when integrated can be assumed to be a histogram consisting of
a number of individual “slices”
What is polymer ?
~

Hi = kci = kni Mi ; Ci = MiMi

Mn = ∑Hi / ( ∑Hi/Mi )
#

meaningthe section Mw = ∑Hi Mi / ∑ Hi

tr = 15

Itr =
10

Mw
MWD =
Mn

For each slice, i, the molecular

weight (Mi) can be derived from the
RT and the number of molecules (ni)
from the detector response

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TRIVIA !
Limitations with Conventional SEC
canSeparaa size
 Column separates on basis of molecular size
NOT molecular weight
each polymer
 two different polymers will interact differently can have diff.
with solvent size in the
same solvent .
 At any molecular weight, the two polymers
will have different sizes in solution

 Molecular weights from conventional SEC are

dependent on a comparison in size between the
standards and the sample

Viscosity of Polymers

 All polymers increase the viscosity of solutions by increasing the resistance to flow
 Different types of polymers have differing viscosities depending on the interactions
with the solvent
 Viscometers are used to determine intrinsic viscosity, IV or [ŋ]

 Intrinsic viscosity can be

though of as the inverse of
the molar density
 At any given MW, a high
IV means the sample is a
large diffuse molecule, a
small IV means a compact,
dense molecule

larger [n] -o large diffuse molecule

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Accurate Molecular Weights

 As a result of using the viscometer, a universal calibration can be set up that gives
the same calibration line regardless of the type of standards employed

 The chemistry of the sample is also unimportant – the column is separating on

size and that is the parameter we have calibrated

 Therefore the SEC/viscometer experiment will give accurate molecular weights

for any samples regardless of their or the standard’s chemistry assuming that pure
SEC takes place

 Still, the column must be calibrated.

exam

tin
n

So Why do Viscometry? – The Universal Calibration

 If a calibration of size versus
retention time could be generated
then one true calibration would hold
for all sample types

Hydrodynamic volume = [] M if know log (n]

of
that polymer,

A
can Find MW

Universal Calibration plot of at specific

retention time .
log[]M versus RT holds true for all
polymer types Universal
Calibration
curve

 Can use measured intrinsic viscosity

and retention time to get accurate
molecular weights

Ref : Grubisic, Rempp, Benoit, J. Polym. Sci., Part B, Polym.

Lett., 5:753 (1967)

[η ] ∝ Vh  log [η] MW ≈ KVh

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Detectors Used in SEC

Concentration detectors (gives a response proportional to
concentration of material eluting from the column)
 Differential refractometer (RI)
S
 Ultraviolet absorbance (UV)
 Evaporative light scattering or mass detector (ELS, EMD)
 Infra-red (IR)

Molecular weight sensitive detectors

 Viscometry
&  Light scattering

① Differential Refractive Index Detector

 The most commonly used detector in SEC, "Universal" detector

 Monitors difference in refractive index of eluent stream as solutes
emerge from column with respect to a static reference cell filled with the
pure solvent
 Can give positive and negative peaks
 Must have sizeable difference in refractive index of solvent and solutes
 Extremely sensitive to pressure and temperature fluctuations
 Modest sensitivity, unsuitable for low solute concentrations
 Non-destructive to sample
 Easy to use
 Approximately linear response with concentration

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Differential Refractive Index Detector

R = reference cell (usually static)
S = sample cell, eluent flowing through

RIsignal = KRI (dn/dc) C

Where K is a constant, (dn/dc) is the refractive index increment and C is concentration

signal ∝ nsolute- nmobile phase

∝ n
∝ conc. of solute

Low MW dn/dc Effects

Columns 2 x PLgel 5um 50Å 300x7.5mm

Eluent THF
Flow rate 1.0ml/min
Solutes Linear hydrocarbons, all prepared at equal concentration
Linear Hydrocarbons
4 Peak HC MW RI
3 O
1 C12H26 170 1.4216
even have
concentrations,
same
2 C16H34 226 1.4340
have diff.
response 3 C22H46 310 1.4420
2
4 C32H66 450 1.4550

O
1 Refractive index of a
lowest MW,
homologous series changes
canes outsa rapidly below a MW of
around 1000.

13
 18/11/67

samplePS : every
small
② UV Detectors
only polymers containing
* Set specific X according to the sample chromophore
 Relies on UV absorbing groups being present in solute
 Very sensitive detector with small cell volumes and therefore low system
dispersion
 Good linearity
 Insensitive to temperature and pressure fluctuations
 Many polymers do not have chromaphores
 Many solvents or solvent additives absorb UV and either prevent use or cause
decrease in sensitivity.
 Sometimes used in conjunction with RI for copolymer analysis when only one
of the monomers has UV chromaphore.

7 -

S
③
Light Scattering Detector
light scattering detection
 use simple light scattering mechanism to detect solute particles
 can offer some significant advantages in SEC applications when compared
to the more widely used differential refractometer or alternative UV detector :

 Responds to compounds with no UV chromaphore

 Positive response for all non-volatile solutes
 Stable baseline, no drift with eluent or ambient temperature changes
 High sensitivity, ideal for low dn/dc polymer/solvent combinations
 No interference from spurious peaks around total permeation
 Fast setup and equilibration

14
 18/11/67

⑧
Molecular-mass detectors
(MW sensitive detectors)

Low angle laser light scattering detector (LALLS)

Signal CMW

LALLS signal
C
MW

Signal a MW

·
Molecular-mass detectors
(MW sensitive detectors)

Viscometric detector
M [η] = KVh ; [η] = Intrinsic Viscosity
1 ηi
-1 = [ηi]
Ci ηs

[η] ∝ CVh
M

15
 18/11/67

SEC Applications

① ➲ MWD information : purity of compounds,

First elution

MW = 400,000 MW
toluene
Detector Response

Internal marker
2nd elution

MW = 5,000
PS400000
W THE M

Considerto e
· a

* If consider Toluene as internal

marker , smallest molecule ; if
eluted can mark the end of
,

elution.

Vr

SEC Applications

② Two PE Samples with same MWD (Mw/Mn) exhibit

different mechanical properties and flow properties
* SEC can detect properties of polymer .

PE
[ S
broad MWD

* two peaks

PE1
[ S
broad MWD

16
 18/11/67

l b
③
EX. linear polymer and branched polymer

·
signal

v
detects sameI

I
b l same Vh same si,
same conc.
concentration
conc. detector >
-

response
and He =
Hb I same conc

Vr
same Vr
signal

scatter b same Vh,

intensity
= l signal ∝ CMW higher siga
,
as a

V
MWb > MWl
r
same Vr
signal ((n2) branched chain
viscosity detector -
l Lower [η]
response -
b [η] ∝ CVh ·
Thesignald
Vr M
same Vr MWb > MWl

17
1. Explain the characteristic that differentiates SEC from other LC techniques.
2. In an LC experiment, explain how band spreading of a pure component can occur.
3. What should be concerned while preparing a solvent (mobile phase) for an SEC Instrument?
4. What should be considered while selecting a LC detector?

5. From a given cross section of a stationary phase particle, draw molecules of various sizes
and explain according to the given terms.

·
·

.......

........

total permeation volume or time

·
·

exclusion limit

-"
selectivity
6. In a free radical polymerization of polystyrene, GPC is used to follow conversion of styrene
monomers at various reaction time.
- Solvent used to dissolve reaction mixture is carbon disulfide (refractive index = 1.6)
- Refractive index of styrene = 1.52
- Refractive index of polystyrene = 1.61
- Initiator is 2, 2 – Azobisisobutyronitrile (AIBN) (the amount is very small)
6.1 Explain characteristics of these two concentration detectors
6.1.1 UV-visible detector
6.1.2 Differential Refractometer detector
6.2 From 6.1 which detector you prefer to use in this experiment? Why?
6.3 Draw expected GPC chromatograms of the samples obtained from 0% conversion and
50% conversion.
7. Draw GPC chromatograms of a mixture of PS (MW = 5,000), PS (MW = 10,000) and PMMA
(MW = 5,000). RI is used as the instrument detector, THF is the mobile phase and toluene is
used as the marker. While dissolving the polymer mixture in THF, PMMA has smaller size
than PS of the identical MW.
Advices: - refractive index (RI) of PS = 1.0 - RI of Toluene = 0.9
- RI of THF = 0.8 - RI of PMMA = 1.4
 *
Chromatography
separate components in a mixtur
according to distribution
of components in M .

P/S P.
.

·
interaction

size
·

· The two phases M .

P/SP should not dissolve to one another.

* GC
checks
purity of monome

- EFFICIENCY OF LC COLUMN :

① peak column
broadening a
efficiency
② resolution blu 2
peaks -

③ #
o theoretical plates related

① the longe the column

&

totalpenation & charge colum at

to a
 on = 0 6
.

On

On =
= 0 52

0 61
.
.

it
THE-+ RI =
1 0
.
(mobile phase)
I assume

sameincentration

6 .

3) O UV-visible detector

l
PS styrene

-1
·
*
characteristic of free-radical chain
poly
mo mixture has polymer
Response 750 % + unreacted monur at 50 % canr .

- -
>
only monomer

< 0%

VR

Realistically 50 %
*
,
height of styrenea conversion must decrease.

① IR detector

1
/PS
-

Response
 1. Explain the characteristic that differentiates SEC from other LC techniques.
2. In an LC experiment, explain how band spreading of a pure component can occur.
3. What should be concerned while preparing a solvent (mobile phase) foru/an SEC Instrument?
>
-
M P should hav no interaction S p
. .

4. What should be considered while selecting a LC detector?

.

accordingtotypesms set
and what information you

5. From a given cross section of a stationary phase particle, draw molecules of various sizes
and explain according to the given terms.

total permeation volume or time

exclusion limit

selectivity
* 6. In a free radical polymerization of polystyrene, GPC is used to follow conversion of styrene
monomers at various reaction time.
- Solvent used to dissolve reaction mixture is carbon disulfide (refractive index = 1.6)
- Refractive index of styrene = 1.52
- Refractive index of polystyrene = 1.61
- Initiator is 2, 2 – Azobisisobutyronitrile (AIBN) (the amount is very small)
6.1 Explain characteristics of these two concentration detectors
hav chromophon
6.1.1 UV-visible detector >
-

6.1.2 Differential Refractometer detector Fblw solute M P e ON

.
. -
*

6.2 From 6.1 which detector you prefer to use in thisUV-

>
experiment? Why?
have chromophone RI make or
whether they
to sun
defect resports
-

6.3 Draw expected GPC chromatograms of the samples obtained from 0% conversion anddetects , on sample

RI enything but
50% conversion. make RI P R saph
sure M
.
e
,

.
I
* 7. Draw GPC chromatograms of a mixture of PS (MW = 5,000), PS (MW = 10,000) and PMMA ar direct .

(MW = 5,000). RI is used as the instrument detector, THF is the mobile phase and toluene is
used as the marker. While dissolving the polymer mixture in THF, PMMA has smaller size
than PS of the identical MW.
Advices: - refractive index (RI) of PS = 1.0 - RI of Toluene = 0.9
- RI of THF = 0.8 - RI of PMMA = 1.4
 Un = 0 2
.

On =
0 1 .

basis
un = 0 6
-

mobile phase

·
PMMA is the smallest so the last one
for sample.
,

·
Toluene last to elute as the marker.
,

For IR
i
,
M

signal PMMA5000
a un

000
PS10 ,
PSsurr

Toluere

&
VR

*
In MV
,
when X-
**
M

Response

000
PS10 ,
PSsurr

Toluere

&
VR
 - In LLASC
signal <CMW
; cone are the same !
,

but
M
PMMA has smaller
size ,
so PMMA has smallsignal
/

Response

000
PS10 ,
PSsurr

PMMA sa

↑
Tolvern

&
VR