HPLCmethodlogies_III-1

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Ion Exchange Chromatography
Principles: Retention is based on electrostatic interaction between solute ions and
fixed ion charge on the stationary phase (S.P)
Cations needs a cation exchanger and anions needs anion exchanger
How a stationary phase (S.P) is built?
Crosslink
between
polymers
Styrene divinylbenzene resin
Cation
Exchanger
Anion
Exchanger
(aka. backbone)
Polymeric
Silica-based
Ion-Exchange Groups (A group that carries a separation capacity)
Strong Cation Exchanger
Weak Cation Exchanger
At low pH CM loses cation
exchange capacity due to
protonated S.P
Strong Anion Exchanger
Weak Anion Exchanger
At high pH DEAE loses
anion exchange
capacity due to
deprotonated S.P
What mobile phase characteristics will affect the retention of ionic compounds
(e.g., cations) if weakly acidic cation exchanger is used?
*pH of the M.P. The M.P affects retention of cations. For example, at low pH
the ionogenic groups of the resin is protonated and cation exchanger loses its
ion-exchange capacity -------- tR of cations decreases (case a)
4<pH<8
pH <4
pH>8
COOH
COO-
COO-
COOH
COOH
COO-
COOH
COO-
COO-
COOH
COOH
COO-
a) Undissociated
weak cation exchanger
b) Partially dissociated
weak cation exchanger
c) Dissociated
weak cation exchanger
Mechanism of Retention in Ion-Exchange Chromatography (IEC)
Taken from Weston
All M.P
counterions
except one
has been
displaced
frome the IE
sites
Some sample
ions have
already being
displaced by
M.P
counterions
1. The column is equilibrated with the M.P and the counterions have occupied ion-exchange
sites on the S.P. The sample ions are injected and are about to enter the first segment of S.P
2. As the sample cations enters the column they start displacing the M.P counterions from the ionexchange sites on the S.P (process called adsorption of solute ions on the S.P).
3. Because this is a flowing system(M.P is continously being flushed into the column).
Therefore,the M.P counterions will start displacing the sample ions from the IE sites.
4. After all the sample ions are eluted off the column the S.P is regenerated (ie., flushed with
M.P counterions) to clean off extra sample ions that have retained on the S.P
Synthesis of polymeric-based ion-exchange columns
I.
Copolymerization of styrene with divinylbenzene
II.
Sulfonation or amination of copolymer (PSDVB)
Type of Ion-Exchange packing material
Strongly acidic cation-exchange resin prepared from sulfonation of PSDVB
Ion Exchange Resin
Strongly basic anion exchanger is prepared from amination of PSDVB
Polymeric resins are made in 3-D networks by cross-linking hydrocarbon
chains. The resulting resin is insoluble, inert and relatively rigid. Ionic
functional groups are attached to this framework.
Crosslinking is a measure of
polymer strength.
--Heavily crosslink resin have
more IE sites-----tR increases
--Liqhtly crosslink resin have
less IE sites ----tR decreases
--Resin crosslinking varies from
2—10%. Resin <2% crosslink
are soft gels
Lightly crosslinked rapid
Equilibrium Between S.P and M.P,
which Increase N but also tR
resins sells with organic solvents (if lightly
crosslinked)
Heavily crosslinked- More rigid and less
Porous. Slow equilibration of solutes. Higher
IE capacity, tR increases, N increases)
Resins do not swell with organic solvents
due to greater mechanical strength
Ion-Exchange Equilibria:
Due to the competition between M.P counterions and sample ions an
equilibrium is established between the counterions (C+) and the sample ions (S+)
F-C+
(S.P)
+
S+(M.P)
F-S+
(S.P)
+
C+
(M.P)
S+ = sample ions
C+ = counter ions of the M.P
“Ions of same charge are reversibly exchange between the two phases”
Ion-Exchange Selectivity
Selectivity Coefficients
Preference for ions of particular resins is often expressed through an
equilibrium relationship using the selectivity coefficient. The
coefficient is described below.
For the exchange of Li+ in solution for Na+ on the resin:
R-
+
LiS.P
+
+
+ Na R Na
M.P
K
=
S.P
+
+ LiM.P
[R-Na+]S.P [Li+]M.P
[R-Li+]S.P [Li+] S.P
K describes the relative selectivity of resin for Li+ and Na+ ions.
Greater the value of K means forward reactions is more favorable than reverse
reactions. Thus, selectivity of Na+ for the ion-exchange resin is higher than
for Li+ but why?
Why Li+ has lower selectivity (retained less) compared to Na+ on ion-exchange
columns?
H20
H20
H20
H20
H20
H20
H20
H20
Li+
H20
H20
H20
H20
Na+
H20
H20
H20
H20
Ionic radius = 1.16 0A
Hydrated radius = 2.760A
Ionic Radius = 0.90 0A
Hydrated radius = 3.40 0A http://www.bbc.co.uk/dna/h2g2/A1002709
Li+ ions is small cation compared to Na+. However, it has large hydrated radius.
The larger hydrated radius of the Li+ ion do not have much access to the negative
charge on the S.P, compared to Na+ cation.
Effectively, these selectivity coefficients are a measurement of a resins
preference for an ion. The greater the selectivity coefficient, the greater the
preference for the ion. For example, a strong acid cation resin with 8%
crosslink has a selectivity coefficient for sodium vs. hydrogen of 1.56, while
the selectivity coefficient for calcium vs. hydrogen is 4.06. As a result,
calcium is strongly retained by the ion exchange over the sodium
K value is measured with
respect to H+
--*Increasing the percentage of crosslinking of the resin usually increases the
retention on polymer-based columns
Polymeric resins are made in 3-D networks by cross-linking hydrocarbon chains.
The resulting resin is insoluble, inert and relatively rigid. Ionic functional groups
are attached to this framework.
Donnan Equilibrium in Ion Exchange Chromatography
Nice animation at --
www.physioviva.com/movies/gibbs-donnan/index.html
When an ion-exchanger (S.P) is placed in an electrolyte solution:
“the concentration of electrolyte is higher outside the resin than inside it.”
K+
The equilibrium between the M.P ions in
R+ClR+Clsolution and the M.P ions inside the
K+
K
resin
+
resin (S.P) is called Donnan Equilibrium
K+ClK+K+
R+ClR+ClK+
Consider a quaternary ammonium [N(CH3)4]+,
K+Cl+
K+Clanion-exchange resin = R in its chloride (Cl )
K+K+
K+Clform immersed in a solution of KCl
K+
R+ClR+ClIt can be shown from the thermodynamic that
the ion-product inside the resin is approximately equal to the ion product outside
the resin:
[K+]i [Cl-] i
= [K+]0 [Cl-]0
-----------(1)
i= concentration of electrolyte (M.P) ions inside the resin
o= concentration of electrolyte (M.P) ions outside the resin
--From consideration of charge balance we know that:
[K+]0
= [Cl-]0
---------------------(2)
--Inside the resin there are three charged species, and the charge balance is
[R+]i + [K+]i = [Cl-]i
-----------------------------(3)
Substituting (eq.2) and (eq 3) into eq(1) we get :
[K+]i ([R+]i +[K+]i)
= [K+]02
-----------------------------(4)
What does the above equation tell us?
The concentration of [K+]0 must be higher than [K+]i, which is true because K+
has the same charge as the R+ and the electrostatic repulsion will decrease its
concentration inside the resin.
Example calculation showing the exclusion of cations by the anion exchanger
Suppose that the concentraton of cationic sites in the resin (in anion exchange
resin) is 6.0 M. When the Cl- form of the resin is immersed in 0.050M KCl, what
will be the ratio of [K+]0/[K+]i?
We know that from the above equation:
[K+]i ([R+]i +[K+]i) = [K+]02
-----------------------------(4)
[K+]i 2 + [R+] [K+]i)
2
= [0.050]0
2
[K+]i + (6.0 [K+]i - 0.025 = 0
ax2 +
bx
-c
=0
b2 - 4ac
x = -b +
2a
[K+]i =
-6.0 +/- (6)2 - 4 (1)(-0.0025)
2(1)
= -6.0 +/2
36.01
= -6.0 +/- 6.000833
2.0
[K+]i = 0.000833
2.0
[K+]0
[K+]I
=
0.0500
0.0042
= 0.0004166
~ 1.2 x 102
~ 0.00042
0.00042 X 100 = 0.84%
0.050
conc of K+ inside the resin
is less than 1%!
The counterion (e.g., Cl-) is not excluded from the resin. There is no electrostatic
barrier to penetration of a solute anion into the anion exchange resin
“Cations with the same charge as the resin are excluded.”
Solute anions competes for the anion exchange sites with the counter ions
Silica-Based Ion Exchange Materials
Functional group is covalently attached
to the silica surface and fixed ions are
formed as a part of this group
H
+
What are the advantages/disadvantages of silica-based over polymer based IEC?
Silica-based
Advantages: (1) Have lower capacity than polymer-based. Due to low IE capacity
N is better. (2) Do not swell with organic solvents.
Disadvantages: (1)If not fully functionalized or bonded, unreacted silanols itself
can act as a cation exchanger (peak tailing and adsorption becomes a problems
for the separation of cations e.g., quaternary amines).
(2) Limited pH range to work with
Therefore, it appears that polymeric ion-exchange columns are best for the
separation of small inorganic and organic anions and cations. For large organic
cations/anions silica-based columns would provide better separation capability
due to limitation of the use of organic solvents on majority of polymeric based
columns
I.
A)
B)
C)
Major Factor affecting retention in ion-exchange chromatography
Influence of Solvent (M.P)
Eluent or M.P pH
Nature and Charge of the competing ions (i.e., M.P counter ions)
Concentration of the competing ions
II. Influence of Solute (Injected sample ions)
A) Charge on the solute ion
B) Solvated size of the solute ion
C) Polarizibility of the solute ion
III. Influence of Stationary Phase
A) The ion-exchange capacity of the ion-exchanger
B) The functional group of the ion-exchanger
I.
Influence of Solvent (M.P)
pH>9
pH <4
+
N(CH3)2
+
N(CH3)2
+
N(CH3)2
+
N(CH3)2
a) Undissociated
weak anion exchanger
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
c) Dissociated
weak anion exchanger
A) pH of the M.P
If we increase in pH of the mobile phase
For example, use of sodium borate at pH 10 (instead of using HCl at
pH 2.0) will make the weak anion exchange resin neutral at high pH and
retention of anions will decrease
B) Nature and Charge of the mobile phase counterions
+N(CH3)3
+N(CH3)3
CO32- (Na2CO3) is a much stronger mobile
phase counter-anion for the separation
anions (F-, Cl- Br-, I-, SO42-) compared to
the use of HCO3- (NaHCO3)
NO3-(NaNO3) is stronger eluent anion
compared to Br-(NaBr)
+N(CH3)3
+N(CH3)3
a) Strong anion exchanger
SO3SO3SO3-
Ca2+ (CaCl2) will be a much stronger
mobile phase counter-cation, i.e., decrease the
retention time for the separation of alkali metal
cations (Li+, Na+, K+, Rb+, Cs+) compared to
the use of H+ (HCl) as M.P
SO3a) Strong cation exchanger
Ba2+ (BaCl2) is a much stronger
mobile phase compared to Ca2+(CaCl2)
(C) Concentration of the Competing Ions
Increasing the concentration of counter ions (e.g., H+) in the mobile phase
For example, the use of 1M HCl is much stronger mobile phase compared
to 0.1 M HCl (because the former concentration have more mobile phase
counter cations (shown on the right). Hence the retention of solute cations
will decrease.
II. Influence of solute injected
A) Ions of weak acids (phenols) or bases (aniline) may loose charge and are
retained less on ion-exchange columns.
For example phenol (pKa =9.1) is weakly retained if the mobile phase pH is
less than 9.0, but will be retained at high pH values between 10-12
Similarly, aniline (pka = 3.2) is weakly retained on cation exchange columns at
pH 7.0 mobile phase compared to pH 2.0 mobile phase
(B) The charge on the solute cations and anions.
--inreasing the charge of the solute cation or anion increases its affinity for the
IE sites on the column. The decreasing retention order will be:
Pu4+>>La3+>>Ba2+>Tl+
PO43->SO42->Br(C ) The solvated size of the solute cation or anion. Ions with the smaller degree of
solvation exhibit greater binding affinity and retained longer (e.g., Cs+>Rb+>K+>
Na+> Li+) or (I->Br->Cl->F-)
H20 H 0
2
H20
H20
H20
Li+
H20
H20
H20 H20
H20
H20
H20 H 0
2
H20 H 0
2
Cs+
H20
H20
H20
H20
H20
H20
F-
H20
H20
H20 H20
H20
H20
H20 H 0
2
IH20
H20
H20
D) Polarizibility of the solute anions
--Ability of an ion’s electron cloud to be deformed by nearby charges.
Deformation of the electron cloud induces a dipole in the solute ion. Thus,
attraction between induced dipole and the nearby charges (resin charge) and
increase the binding fo the ion. For example R-SO3- resin shows greater
Affinity for polarizable cations (e.g., Ag+, Tl+) compared to alkali metals (Li+,
CS+)
Affinity of
Affinity of
SCN-
Ag+
>
SO42-
>
Li+
III. Influence of Stationary Phase Charge
The functional group of the ion exchanger. A sulfated stronger cation exchanger
exhibit higher retention compared to a carboxylated weak cation exchanger.
Similarly, a quaternary ammonium cation will be a strong cation exchanger than
Secondary or tertiary amine.
SO3SO3-
SO3SO3a) Strong cation exchanger
COOCOOCOOCOOc) Weak cation exchanger
+N(CH3)3
+NH(CH3)2
+N(CH3)3
+NH(CH3)2
+N(CH3)3
+NH(CH3)2
+N(CH3)3
+NH(CH3)2
(B) Ion-exhange capacity of ion exchanger
Increasing the ion-exchange capacity increases the retention, but selectivity
coefficient remains constants
Application of IEC in the preparation of deionized water
We need two ion-exchange columns (cation exchange RSO3- and anion exchange
R-N+(CH3)3) to remove cations and anions, respectively. Suppose we need
remove Cu(NO3)3 from the faucet water.
Cation Exchange
Cu2+  2H+
Anion Exchange
2NO3- - 2OH-
Anion exchanger
will bind NO3And OH- ions
are knocked off
Cation exchanger
will bind Cu2+
and H+ ions
are knocked off
2OH-
2H+
2H+ + 2OH-
2H2O
Applications of IEC (Contd)
(b) Convert one counterion of the salt to another
(CH3-CH2-CH2)4---N+I-
(CH3-CH2-CH2)4---N+-OH-
Tetrapropyl ammonium iodide
Tetrapropyl ammonium hydroxide
I- is a UV absorbing counterion
OH- is a non-UV absorbing counterion
(c) Preconcentration of trace components
--For example we can pass large volume of fresh lake water through a cation
exchange resin in the H+ form (using HNO3) to concentrate metal ions onto the
Resin.
--Chelex 100, S-DVB resin containing iminodiacetate groups binds transition
metals (e.g., Fe3+, Ni2+)
Fe3+
1 pptr
CH2-COOH
Resin --------N:
Fe2+
CH2-COOH
Metals can be eluted using HNO3
from the column in small volume
followed by column regeneration in H+ form
Resin Fe3+ 1ppm
ION CHROMATOGRAPHY
--abbreviated as IC is a high pressure version of
ion-exchange chromatography, has become the
method of choice for anion analysis
Recent IC applications in rainwater, groundwater, surface
water, wastewater, drinking water, fog samples, ice, snow,
soil, sediments, sludge, plants, air, exhaust, aerosols, flue
dust, fly ash, fuel oil and coal are reviewed with a major
emphasis on speciation of ions. IC shows great promise
for the sequential determination of ionic species in a wide
variety of water samples
Types of Ion Chromatography
A) Non-Suppressed IC
aka. IEC
with direct or indirect
conductivity and
indirect-UV detection
B) Suppressed IC with
direct conductivity
Detection employs two
columns
a) ion-exchange columns -- separate solute ions
b) suppressor column --- decreases the background conductivity of the eluent
Suppression
Suppression
Cation IC
Pump
Inj
Resin—SO3
-Y+
H+
+
Cl-
+ H20
Resin—N+ OH- + Y+Cl-
Suppressor
Column
Resin—N+ OH- + H+ClResin—N+
Eluent
NaHCO3
Resin—N+HCO3- + X-
Analytical
Column
+ Y+
Analytical
Column
Resin—SO3
Pump
X- = F-, Cl-, NO2-, PO43-
Y+ = Na+, K+, Rb+, Cs+
-H+
Inj
…....
…....
……
……
……
Suppressor
Column
Eluent
(HCl)
Anion IC
Resin—N+X…....
…....
……
……
……
+ HCO3-
Resin—SO3-H+ + Na+HCO3Resin—SO3-Na+ + H2CO3
Resin—SO3-H+ + Na+XResin—SO3-Na+ + H+X-
Resin—N+ Cl- + Y+OH
Conductivity
Conductivity
Conductivity
cell
meter
Cell
Counterions associated with active
eluent species are replaced in the
Recorder
M.P by either H+ or OH- to form H20 and
H2CO3
Conductivity
meter
Recorder
Key to suppression
Supppression
Comparison of Non-suppressed vs. Suppressed Conductivity Detection
Problems with Suppressor Column
--Need to regenerate the suppressor column periodically (typically after every
8-10 hrs) to convert the suppressor column back to acid/base form
-- Increase dead volume (extra column band broadening) and decreases the
Overall efficiency for analysis
Eluent and suppressor solution flows in opposite direction on either side of the
Ion-exchange membrane
For analysis of cations - membrane is a cation exchanger
For analysis of anaion - membrance is an anion exchanger
List of Mobile Phases Used in Non-Suppressed IC
M.P used in non-suppressed IC
(direct conductivity)
-O O C
COO-O O C
-O O C
OH
P hthalate
p-hydroxybenzoate
B enzoate
M.P used in non-suppressed IC
(Indirect conductivity)
Sodium Hydroxide (anion exchange)
Nitric Acid (cation exchange)
Why non-suppressed IC is less sensitive than suppressed IC?
We are measuring the change in conductance of the eluted solute. This
is contrast to IC where eluent conductivity is suppressed to zero before
detection
M.P used in suppressed IC
TYPICAL APPLICATIONS OF IC WITH SUPPRESSED CONDUCTIVITY DETECTION
The two chromatogram shown to the left
shows the application of IC with a suppressor
column. In each of these cases analytes are
detected in ppb (mg/L) are getting popular
these days.
Chromatogram shows separation of doubly
charged species (Mg2+------.Ba2+) but no
resolution of singly charged species (Li+--Cs+)
all coeluting as single band.
a) 28 mM NaHCO3/23 mM Na2CO3
b) 25 mM phenylenediamine dihydrochoride/
25 mM HCl
(Taken from Dionex, Inc, Sunnyvale, CA.)
Simultaneous separation of both singly and doubly charged species is difficult in
IC because differences in selectivity and interaction with S.P for 2 classes of
cations is wider
Non-Suppressed IC with Indirect Photometric Detection
Principle: UV absorbing anions/cations (depending on mode of IEC) are added to
the M.P. So detector has a high UV-absorbance in the baseline
What happens when we inject a non-UV absorbing
anions (e.g., Cl-)?
A
Baseline noise
-O O C
+N(CH3)3
-O O C
-O O C
+N(CH3)3
2Cl-
-O O C
+N(CH
3 )3
+N(CH3)3
+N(CH
3 )3
+N(CH3)3
-O O C
Cl-
+N(CH3)3
-O O C
+N(CH3)3
-O O C
+N(CH3)3
-O O C
+N(CH
3 )3
+N(CH
3 )3
-O O C
-O O C
+N(CH3)3
Cl- elutes
off the column
Original condition
is restored
A
Cl-

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