EFFECT OF ORGANIC IONIC LIQUID (BMIM)

(BF4) AS MOBILE PHASE ADDITIVE ON THE

ADSORPTION OF AMINO ACID (TRYPTOPHAN)
BY HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY

Presented by:
SATHISH
Chemistry Department.
.

INTRODUCTION

• IONIC LIQUIDS

• COMPOSITION OF IONIC LIQUIDS

Figure 1: structure of ionic liquids[ BMIM]

 • ATTRACTION OF IONIC LIQUIDS
 Their application in analytical chemistry, is merited because of
properties such as
 negligible vapor pressure
 Good thermal stability
 Miscibility with water and organic solvents

PROPERTIES OF IONIC LIQUIDS

 Densities
 Solubility and miscibility
 Thermal stability
 Viscosity
 Melting point
 Surface tension
 APPLICATIONS OF IONIC LIQUIDS
Ionic liquids as stationary phases in gas chromatography

Ionic liquids in capillary electrophoresis

 Ionic liquids as background electrolyte additives in non-

aqueous media

Ionic liquids as electrolyte additives in aqueous media

 N N N
N N N N
N N
N R
RCH3 RCH3
RCH3 R CH3
CH4

OH

OH
OH O

N
N
CH3 R
OH

EOF
OH O

CH
R3 CH4
R CH3
R CH
R3 CH
R3
N N
N N N N
N N N
N

Figure 2: Mechanism of polyphenols separation using 1-alkyl-3-methylimidazolium based ionic

liquid
 APPLICATIONS OF IONIC LIQUIDS CONT.
Analytical applications of ionic liquids as a micelle-forming
surfactant

Application of ionic liquids to the electrodeposition of metals

Ionic liquids in spectrometry

Ionic liquids as extractions

Extraction of bioactive compounds in natural plant

 IONIC LIQUIDS IN LIQUID CHROMATOGRAPHY

Reversed-phase liquid chromatographic analysis of ionic liquids

O
Si
OH
OH N

H N
Si
O
OH
OH

Figure 3: Scheme illustrating potential interactions between methylimidazolium cation and phenyl-based
reversed-phase stationary phase.
APPLICATIONS OF IONIC LIQUIDS IN NORMAL-
PHASE LIQUID CHROMATOGRAPHY
• Ionic liquids as mobile-phase additives in liquid
chromatography
• Ionic liquids as stationary phases in liquid chromatography
• A Surface –confined ionic liquids as reversed phase stationary
phases in liquid
R

MeO R
N
Si N
OMe
MeO H H
Si
OH O OH O
OH O
O

Si Si Si Si
Si Si Si
O O O O
O
O O

Figure 4: Scheme illustrating potential reorientation of bonded imidazolium ligands in response to deprotonation of
residual silanols
DRAWBACKS FOR INDUSTIAL APPLICATION

•Lack of physical parameters such as conductivity, viscosity is

serious drawbacks for industrial application of ionic liquids.

• Hydrophobic ionic liquids although they are stable and allow

easiest recovery from biphasic processes bur never designed to
dissolve carbohydrate-based macromolecules because their
solubilisation depends on the competitive replacement of
intermolecular hydrogen bonding.

•One of the potential problems with ionic liquids is the possible

pathway into environment through waste water but this problem is
common with all solvents
 THEORY

ADSORPTION

Adsorption is the process in which matter is

extracted from one phase and concentrated at the
surface of a second phase. (Interface accumulation).
This is a surface phenomenon as opposed to
absorption where matter changes solution phase,
e.g. gas transfer. This is demonstrated in the
following schematic.
.
If we have to remove soluble material from the solution phase,
but the material is neither volatile nor biodegradable, we often
employ adsorption processes. Also adsorption has application
elsewhere, as we will discuss later.

Adsorbate: material being adsorbed

Adsorbent: material doing the adsorbing. (examples are

activated carbon or ion exchange resin).
 TYPES OF ADSORPTION

Exchange adsorption (ion exchange)

Electrostatic due to charged sites on the surface. Adsorption

goes up as ionic charge goes up and as hydrated radius goes
down.
Physical adsorption

Van der Waals attraction between adsorbate and adsorbent. The

attraction is not fixed to a specific site and the adsorbate is
relatively free to move on the surface. This is relatively weak,
reversible, adsorption capable of multilayer adsorption
 CHEMICAL ADSORPTION

Some degree of chemical bonding between adsorbate and

adsorbent characterized by strong attractiveness. Adsorbed
molecules are not free to move on the surface. There is a high
degree of specificity and typically a monolayer is formed. The
process is seldom reversible.

Generally some combination of physical and chemical

adsorption is responsible for activated carbon adsorption in
water and wastewater
 ADSORPTION EQUILIBRIA

If the adsorbent and adsorbate are contacted long enough an

equilibrium will be established between the amount of
adsorbate adsorbed and the amount of adsorbate in solution.
The equilibrium relationship is described by isotherms.

qe = mass of material adsorbed (at equilibrium) per mass of

adsorbent.

Ce = equilibrium concentration in solution when amount

adsorbed equals qe.

qe/Ce relationships depend on the type of adsorption that

occurs, multi-layer, chemical, physical adsorption, etc.
 ISOTHERM MODELS
The figures below show that there are four common models for
isotherms.
 LANGMUIR ISOTHERM

This model assumes monolayer coverage and constant binding

energy between surface and adsorbate.
The model is:

0
K  Qa
 Ce
qe 
1  K  Ce
Q0a represents the maximum adsorption capacity (monolayer
coverage) (g solute/g adsorbent).
Ce has units of mg/L.
K has units of L/mg
 BET (BRUNAUER,EMMETT AND TELLER)
ISOTHERM
This is a more general, multi-layer model. It assumes that a
Langmuir isotherm applies to each layer and that no transmigration
occurs between layers. It also assumes that there is equal energy of
adsorption for each layer except for the first layer.

K B  C e  Q 0a
qe 
(C S  C e ){ 1  ( K B  1 )( C e / C S )}

CS =saturation (solubility limit) concentration of the solute. (mg/liter)

KB = a parameter related to the binding intensity for all layers.

Note: when Ce << CS and KB >> 1 and K = KB/Cs BET isotherm

approaches Langmuir isotherm.
 FREUNDLICH ISOTHERM
For the special case of heterogeneous surface energies
(particularly good for mixed wastes) in which the energy term,
“KF”, varies as a function of surface coverage we use the
Freundlich model.

1
qe  KFC e
n

n and KF are system specific constants.

 RESEARCH OBJECTIVE
The objective is to use ionic liquids as mobile phases along
with acetic acid and methanol to study their effect as mobile
phase additives in reversed phase liquid chromatography.

Here tryptophan was used as solute and an aqueous solution

of methanol along with ionic liquid was used as mobile phase.

Study involved comparision of calibration curves, profile,

calibbration data, adsorption behaviour and peak shapes with
two different columns (prevail, Xterra)
 Experimental
Commercially available tryptophan was purchased from
Spectrum (Gardena, CA 90248 and Newburnswick, NJ,
08901).

Thio urea was purchased from Sigma Aldrich.

Methanol, water and acetic acid used are all HPLC grade and
were purchased from Fischer Scientific Fair Law, NJ.
 APPARATUS
Apparatus used is Shimadzu liquid chromatography, HPLC-model 20A,
which is equipped with auto sampler (SIL-20A/20AC) and online
degasser (DGU-20 A3/ DGU-20 A5) was used.

UV-VIS (SPD-20A/SPD-20AV) was used as a detector.

XTERRA-C18 (150mm×4.6mm, Particle size-5μ) was used as a

stationary phase that was supplied from (Waters, 20 Liberty way,
Franklin, MA 02038).

PREVAIL-C18(250mm×4.6mm, Particle size-5μ) was used as a

stationary phase.
 SOFTWARE PROGRAMS
ORIGIN
SR6 Origin 7.5, Origin Lab Corporation, One round house
plaza, Northampton, MA 01060 USA, 1991-2006 was used

 Preparation of mobile phase

Aqueous solutions 10% methanol, 1%acetic acid Without ILs

Aqueous solutions of 1%acetic acid, No methanol With ILs

5Mm Ionic liquid with No methanol and 1% acetic acid

Preparation of 0.1 g/L, 1g/L, 10g/L of tryptophan
10 Mm Ionic liquid 1% acetic acid, No methanol
Preparation of 0.1 g/L, 1g/L, 10g/L of tryptophan

Aqueous solutions of 1%acetic acid, 10% methanol With Ils

5Mm Ionic liquid with 10% methanol and 1% acetic acid

Preparation of 0.1 g/L, 1g/L, 10g/L of tryptophan
0Mm Ionic liquid with 10% methanol and 1% acetic acid
Preparation of 0.1 g/L, 1g/L, 10g/L of tryptophan
MECHANISM

Interaction of [BMIM]BF4 on modified silica surface.

CH3
N CH3 C4H9
C18 C18
N
N
O C18
OH
N
N
C4H9 OH N
C4H9
H3C
C4H9

N
H3C

C4H9

N
C18
H3C

H3C
CH3
N N

O
N N
H3C
C4H9
RESULTS AND DISCUSSION
NO MEOH NO IL 1% HAC TRYPTOPHAN 10G/L
1000 NO MEOH NO IL 1% HAC TRYPTOPHAN 1g/L

1000
800
800
600
600
400
400
200 Abs (mau)
200
Abs (mau) 0
0 2 4 6 8 10 12
0
t (min) (A)
0 5 10 15
t (min) (B)

BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 1G/L

BMIM 5MM NO MEOH 1% HAC TRYTOPHAN 10G/L
600
1000
500
800
400

300 600
Abs (mau)
200 400
Abs (mau)
100 200

0 0
10 12 14 0 2 4 6 8 10 12 14 16
t (min) (c) t (min) (D)

BMIM 10MM NO MEOH 1% HAC TRYTOPHAN 1G/L

600 BMIM 10MM NO MEOH 1% HAC TRYTOPHAN 10G/L

500 1050

900
400 750

300 600

450
200
300
Abs (mau)
100 150
Abs (mau)
0
0 0 4 8 12 16
10 12 14 t (time) (F)
t (min) (E)

Figure 5: Effect of the concentration of BMIM in mobile phase on breakthrough curves of tryptophan A-B: No MeOH NO IL; C-D: No
MeOH, 5 mM BMIM; E-F: No MeOH, 10 mM BMIM; left 1 g/L Tryptophan and right 10 g/L Tryptophan. Column: C18 X-terra, Flowrate:
1.0ml/min, Wavelength: 305 and 310 for tryptophan 1 g and10 g/L respectively.
 NO MEOH NO IL 1% HAC TRYPTOPHAN 1G/L
600

400

200
Abs (mau)

0
0 2 4 6 8 10
(A)
t (min)

BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 1G/L BMIM 5MM NO MEOH 1% HAC TRYTOPHAN 10G/L
600
500
500
400
400
300 300

200 200

100 Abs (mau)

100
Abs (mau)
0
0 0 2 4 6 8 10 12 14 16
0 2 4 6 8 10 12 14 16 t (min) (D)
t (min) (C)

BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 10G/L

BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 1G/L

500
400
400

300

200
Abs (mau)
200
100 Abs (mau)

0
0 2 4 6 8 10 12 0
t (time) (E) 0 3 6 9 12
t (time)
(F)
Figure 6: The effect of concentration of BMIM in mobile phase on the overloaded band profiles of tryptophan A-B: No MeOH No IL; C-D:
No MeOH, 5 mM BMIM; E-F: No MeOH, 10 mM BMIM; left 1g/L Tryptophan and right 10g/L Tryptophan. Column: C18 X-terra, Flow
rate: 1.0 ml/min, Wavelength: 305 and 310 for tryptophan 1g and 10g/L respectively
 CALIBRATION GRAPHS

Table 4: Parameters of the polynomial fit for the different

concentrations of BMIM in the mobile phase.

Mobile phase C(g/L) Parameters of polynomifit : y=a+bx+cx2+dx3

a b c
d

No IL No MeOH 1 TRYP
0.02225 0.00103 8.28317×
1%HAC
10 TRYP
0.06058 0.00347 4.94741×
5 mM BMIM 1%HAC 1 TRYP 0.00616 0.001 8.98003×
No MeOH 10 TRYP 0.06884 0.00357 4.68651*
10 mM BMIM 1%HAC 1 TRYP 0.01923 9.35613×10-4 9.1786×
No MeOH
10 TRYP -0.09964 0.00652 5.019×
 NO MEOH NO IL 1% HAC TRYPTOPHAN 1G/L
10 NO MEOH NO IL 1% HAC TRYPTOPHAN 10G/L
1.0
8
0.8

0.6 6
C (g/L)
0.4 4
C (g/L)
0.2 2
0.0 0
0 100 200 300 400 500 600 700
Abs (mau) 0 200 400 600 800 1000 1200
BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 1G/L Abs (mau) (B)
1.0 BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 10G/L
10
0.8
8
0.6
6
0.4 C (g/L)
C (g/L)
0.2 4

0.0 2
0 100 200 300 400 500 600 700 0
Abs (mau) (C)

BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 1G/L 0200 400 600 800 1000 1200
1.0 BMIM 10MM NO MEOH Abs (mau)
1% HAC (D)
TRYPTOPHAN 10G/L
10
0.8
8
0.6
C (g/L)
6
C (g/L)
0.4 4
0.2 2

0.0 0

0 100 200 300 400 500 600 700 0 200 400 600 800 1000 1200
Abs (mau) (E) Abs (mau) (F)
Figure 7: Calibration curves of tryptophan determined by FA with different concentrations of BMIM on C18 X-terra column. tryptophanA-B:
No MeOH No IL; C-D: No MeOH, 5mM BMIM; E-F: No MeOH, 10 mM BMIM; left 1g/L Tryptophan and right 10g/L Tryptophan. Flow
rate 1.0 mL/ min; Wavelength 305 nm and room temperature. Mobile phases: aqueous mixture containing 1% HAC and NO methanol and
BMIM
 NO MEOH NO IL 1% HAC TRYPTOPHAN BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN
80

80
60

q(g/L)
40 40
q(g/L)

20
0
0 0 2 4 6 8 10
(B)
0 2 4 6 8 10 c(g/L)
C(g/L) (A)

BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN Tryptophan, No MeOH 1%HAC

100 black sqaures:10 mM BMIMBF4
red triangles: 5 mM BMIMBF4
80 80

60

q(g/L)
40 40
q (g/L)
20

0 0
0 2 4 6 8 10
0 2 4 6 8 10 c(g/L)
C (g/L) (C)

Figure 8: Experimental isotherm data with different concentrations of BMIM on X-Terra Column. Flow rate 1.0 mL/ min; Wavelength 305
nm and Room temperature. Mobile phases: -100% (v/v) mixtures of No methanol, acetic acid and HPLC water
The parameters of isothermal curve for tryptophan with different
concentrations IL’s in mobile phase on C18 Xterra column.
[BMIM] Model
5 mM Langmuir
IM
TRYPTOPHAN 195.7 0.09252
10g/L
220 0.0817
1 g/L
207.8 0.17422
AVG
220.95 0.0817
FA

10 mM Langmuir
IM
162.3 0.1111
TRYPTOPHAN 10g/L
158.5 0.1074
1g/L
160.4 0.10975
AVG

FA 218.02096 0.08614
 PREVAIL COLUMN
PREVAIL NO IL 1% HAC TRYPTOPHAN 1G/L
400 PREVAIL NO IL 1% HAC TRYPTOPHAN 0.1G/L 600

300
400
Abs (mau)
200
Abs (mau)
200
100

0 0
0 5 10 15 20 (A) 0 5 10 15 20 (B) 25
t (time)
t (time)

PREVAIL NO IL 1% HAC TRYPTOPHAN 10G/L

200

Abs (mau)

100

0
0 5 10 15 20
t (min) (C)

Figure 9: Effect of concentration of BMIM in mobile phase on breakthrough curves of Tryptophan A-C: No OMIM; D-F: 10% MeOH, 5 mM
BMIM; Column: C18Prevail, Flow rate: 1.0 ml/min, Wavelength: 305 and 310 for tryptophan 10 g and 1 g/L respectively.
 400 BMIM 5MM 10% MEOH 1% HAC TRYPTOPHAN 0.1G/L BMIM 5MM 10% MEOH 1% HAC TRYPTOPHAN 1G/L 1000 BMIM 5MM 10% MEOH 1% HAC TRYPTOPHAN 10G/L
BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 0.1G/L

120
300 400
100

200 500 80
200 Abs (mau) 60
Abs (mau) Abs (mau)
100 40
Abs (mau) 20
0 0 0
0 5 10 15 20 25 15 20 (C)
0
20 25 30 t (time)
0 5 10 15 20 25 30 35
t (min) (A)
t (time) (B) t (min) (D)

150
800 BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 1G/L 1200 BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 10G/L
BMIM 10MM 10% MEOH 1% HAC TRYPTOPHAN 0.1G/L
600
BMIM 10MM 10% MEOH 1% HAC TRYPTOPHAN 1G/L

1000 500
600
100
800 400
400 300
600 Abs (mau)
Abs (mau) 200
Abs (mau) 50
200 400 100

0 200 Abs (mau) 0

0 0 5 10 15 20
0 0 5 10 15 20 t (min) (H)
25 30 35 0 10 20 30 40 t (min) (G)
t (min)
(E)
t (min) (F)
BMIM 10MM 10% MEOH 1% HAC TRYPTOPHAN 10G/L
BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 1G/L
BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 10G/L
120 BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 0.1G/L 600
900
100 1000
600 80 400
60 Abs (mau)

40 500
300 200
20
Abs (mau) Abs (mau)
0 0
Abs (mau)0
0 5 10 15 20 25 30 35 40 45 50 0
0 5 10 15 20 0 20 40
(I)
0 5 10 15 20 25 30 35 40 t (min) (O)
t (min) t (time) (J) t (min) (K)

Figure 10: Effect of concentration of BMIM in mobile phase on breakthrough curves of Tryptophan A-C: No BMIM; D-F: 10% MeOH, 5
mM BMIM; G-I: No MeOH, 5Mm BMIM;. Column: C18Prevail, J-L: 10% MeOH, 10Mm BMIM; M-0: NO MEOH, 10 mM BMIM. Flow
rate: 1.0 ml/min, Wavelength: 305 and 310 for tryptophan 10 g and 1 g/L respectively.
 NO MEOH NO IL 1% HAC TRYPTOPHAN 0.1G/L NO MEOH NO IL 1% HAC TRYTOPHAN 1G/L
180 6
160
140
5
120 4
100
3
80 Abs (mau)
60 2
40 1
Abs (mau)
20
0
0 0 200 400 600 800 1000 1200 1400 1600
0 5 10 15 20 25 30
t (time) (A) t (time) (B)
NO IL NO MEOH 1% HAC TRYPTOPHAN 10G/L
600
500
400
300
200
100
Abs (mau)
0
0 5 10 15 20 25 30
t (time) (C)

Figure 11: The effect of concentration of BMIM in mobile phase on the overloaded band profilesof tryptophanA-C: No BMIM; Column:
C18 Prevail, Flow rate: 1.0ml/min, Wavelength: 305 and 310 for tryptophan 1 gand 10 g/L respectively
 BMIM 5MM 10% MEOH 1% HAC TRYPTOPHAN 0.1G/L
300 BMIM 5MM 10% MEOH 1% HAC TRYPTOPHAN 1G/L 70 BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 0.1G/L
400 300
250 60 BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 1G/L

200 300 50 250

40
150 200
200
30
Abs (mau)
100 150
20
100
50 10
Abs (mau) Abs (mau) 100
0 0 0 Abs (mau)
0 5 10 15 20 25 30 0 5 10 15 20 25 30 35 40 0 10 20 30 40 50 50
t (time) (C)
t (time) (A) (B) 0
t (time) 0 10 20 30 40
t (time) (D)
3500
BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 10G/L BMIM 10MM 1O% MEOH 1% HAC TRYPTOPHAN 0.1G/L
500 BMIM 10MM 10% MEOH 1% HAC TRYPTOPHAN 1G/L
3000 100
400 600 BMIM 10MM 10% MEOH 1% HAC TRYPTOPHAN 10G/L

2500 80
500
2000 300
60
400
1500 200
40 300
1000
20 100 200
500 Abs (mau) Abs (mau)
Abs (mau) 0 0 100
0 0 10 20 30 40 50 0 5 10 15 20 25 30 Abs (mau)
0 1 2 3 4 5 t (time) (F)
t (time) (G) 0
0 5 10 15 20 25
t (time) (E) t (time) (H)

250 BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 1G/L 300

BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 0.1G/L BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 10G/L
60
200 250
50
150 200
40
100 150
30
50
20 100
Abs (mau) Abs (mau)
0
10 50
Abs (mau) 0 10 20 30 40 50
0 t (time) (J)
0
0 10 20 30 40 50 0 10 20 30 40
t (time) (I)

t (time) (K)

Figure 12: The effect of concentration of BMIM in mobile phase on the overloaded band profiles of tryptophan A-B: BMIM 5mM 10%
MEOH, C-E:BMIM 5mM No MEOH;F-H:BMIM 10mM 10% MEOH;I-K:BMIM 10mM No MEOH ; Column: C18 Prevail, Flow
rate: 1.0ml/min, Wavelength: 305 and 310 for tryptophan 1 gand 10 g/L respectively
Parameters of the polynomial fit for the different
concentrations of BMIM in the mobile phase

Mobile phase C(g/L) Parameters of polynomifit : y=a+bx+cx2+dx3

a b c d

No IL No MeOH 0.1g/L -5.046* 2.0351* 2.386* -1.618*

1%HAC
10 g/L -0.0452 0.00661 -6.865* 1.143

1 g/L -1.521* 1.52101* 1.521 1.22701

BMIM 5 mM 10% 0.1 g/L -1.656 4.497 -1.418 2.438

MeOH 1%HAC 1 g/L

10 g/L
 NO IL NO MEOH 1% HAC TRYPTOPHAN 0.1G/L
0.10 1.0
NO IL NO MEOH 1% HAC TRYPTOPHAN 1G/L

0.08
0.8
0.06
0.6
0.04
0.4
0.02
C (g/L) 0.2
0.00 Abs (mau)

0 50 100 150 200 250 300 350 400 0.0

Abs (mau) (A)
0 50 100 150 200
t (time) (B)

10 NO IL NO MEOH 1% HAC TRYPTOPHAN 10G/L

4
C (g/L)
2

0
0 200 400 600 800 1000
t (time) (C)

.
Figure 13: Calibration curves of tryptophan determined by FA with different concentrations of
BMIM on C18 X-terra column. tryptophanA-C: No MeOH No IL; Flow rate 1.0 mL/ min;
Wavelength 305 nm and room temperature.Mobile phases: aqueous mixture containing 1% HAC
 BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 0.1G/L
BMIM 5MM 10% MEOH 1% HAC TRYPTOPHAN 0.1G/L 0.09
0.10 1.0 BMIM 5MM 10% MEOH 1% HAC TRYPTOPHAN 1G/L
0.8 BMIM 5MM 10% MEOH 1% HAC TRYPTOPHAN 10G/L
0.08
0.08 0.8
0.6 0.07
0.06 0.6
0.06
0.04 0.4 0.4
0.05
0.02 0.2 C (g/L)
C (g/L)
0.2
C (g/L) 0.04
0.00 C (g/L)
0.0
0.0 0.03
0 100 200 300 400 0.0 0.1 0.2 0.3 0.4 0.5
Abs (mau) (A) t (time) 40 60 80 100 120 140
(B) 0 200 400 600 800 1000
Abs (mau) (D)
1.1 11 Abs (mau) (C)
BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 1g/L
BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN 10G/L
1.0 10
0.9 9 BMIM 10MM 10% MEOH 1% HAC TRYPTOPHAN 1G/L
0.10 BMIM 10MM 10% MEOH 1% HAC TRYPTOPHAN 0.1G/L 1.0
0.8 8
0.7 0.08 0.8
7
0.6 0.06
6 0.6
0.5 C (g/L)
C (g/L) 5 0.04
0.4 0.4
4 C (g/L)
0.3 0.02
3 C (g/L) 0.2
0.2
200 300 400 500 600 700 800 2 0.00
Abs (mau) (E) 400 500 600 700 800 900 100011001200 0.0
Abs( mau)
0 20 40 60 80 100 120 140 0 100 200 300 400 500 600 700
(F) Abs (mau) (G) Abs (mau) (H)

10
BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 10G/L
1.0 BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 1G/L
8 0.10 BMIM 10MM NO MEOH 1% HAC TRYPTOPHAN 0.1G/L

0.8
6 0.08
0.6
4 0.06
C (g/L) 0.4
2 C (g/L) 0.04
0.2 C (g/L)
0 0.02
0.0
0 200 400 600 800 1000 1200 0.00
Abs (mau) (L) -100 0 100 200 300 400 500 600 700 0 20 40 60 80 100 120
Abs (mau) (K) Abs (mau) (J)

Figure 14: Calibration curves of tryptophan determined by FA with different concentrations of

BMIM on C18 Prevail column. TryptophanA-C: BMIM 5 mM 10% MeOH: D-F: BMIM 5 mM No MeOH; G-I: BMIM 10 mM 10%
MeOH; J-L: BMIM 10 mM No MeOH; Flow rate 1.0 mL/ min; Wavelength 305 nm and room temperature. Mobile phases: 10-100% (v/v)
mixtures of methanol, acetic acid and HPLC water.
 120 120 BMIM 5MM NO MEOH 1% HAC TRYPTOPHAN
BMIM 10MM 10% MEOH 1% HAC TRYPTOPHAN
BMIM 5MM 10% MEOH 1% HAC TRYPTOPHAN
60
100 100
50
80 80
60
40
60
q(g/l)
40 q (g/L) 30
40
20 20
20
q (g/L)
0 10
0
0 2 4 6 8 10
0 2 4 6 8 10 0
(A)
c(g/l) (B)
C (g/L) 0 2 4 6 8 10
C (g/L) (C)

100 NO IL NO MEOH 1% HAC TRYPTOPHAN 120 tryptophan, 5mMBMIM NO MeOH 1%HAC

tryptophan, 10mMBMIM NO MeOH 1%HAC
80 100

60 80
without IL 10%MeOH

40 60

20 40
q (g/L)
q (g/L) 20 5mMBMIM 10% MeOH 1%HAC
0
0 2 4 6 8 10 0
C (g/L) (E)
0 2 4 6 8 10
C (g/L) (F)

Figure 15: Experimental isotherm data with different concentrations of BMIM on C18 PREVAIL
column. Flow rate 1.0 mL/ min; Wavelength 305 nm and Room temperature. Mobile phases: 10-
100% (v/v) mixtures of methanol, acetic acid and HPLC water
Estimation of the parameters of the adsorption isotherm from the
inverse method using Prevail C18 column.
[BMIM] MODEL
5 mmol 10% MEOH SS
shaped IM
140.2 0.1057 0.000000004936
TRYPTOPHAN 0.1g/L
132.142 0.0096 0.00545
FA

5 mmol NO MEOH IM
Langmuir 620.4 0.0537
TRYPTOPHAN 0.1 g/L
184.99 0.1452
FA

10 mmol 10% MEOH IM

Langmuir 146.18 0.6357
TRYPTOPHAN 0.1 g/L

FA 146.8 0.06357

10 mmol NO MEOH IM
Langmuir
TRYPTOPHAN 0.1 g/L 220 0.19159

FA 157.0 0.19
 BMIM 5MM 10% MEOH 0.5 min 0.1g/L
BMIM 5MM TRYP 0.4 MIN 10g/L
0.08
BMIM 5MM TRYP 0.4 MIN 1g/L 4 0.07
0.8
0.7 0.06
0.6 3
0.05
0.5
2 0.04
0.4 C (g/L)
0.3 C (g/L) 0.03
1
0.2 0.02
0.1 0.01
C (g/L) 0
0.0
-200 0 200 400 600 800
0.00
-0.1 0 200 400 600 80010001200140016001800
0 200 400 600 800 1000 (B)
(A)
t (time) t (time)
t (time) (C)

BMIM 10MM NO MEOH 0.1g/L 0.5 min BMIM 10MM 10% MEOH 0.1g/L 0.5 min BMIM 5MM NO MEOH 0.5 min 0.1g/L
0.05 0.08 0.030
0.04 0.025
0.06
0.03 0.020
0.02 0.04 0.015
C (g/L) C (g/L)
0.01
0.02
0.010
0.00 0.005
C (g/L)
0.00
-0.01 0.000
600 800 1000
-500 0 500 1000 1500 2000 2500 3000 t (time) (F) -500 0 500 1000 1500 2000 2500
t (time) (D) t (time)
BMIM 10MM NO MEOH 1g/L 0.5 min (G)

0.30
0.25
0.20
0.15
0.10
C (g/L)
0.05
0.00
-500 0 500 10001500200025003000
t (time)
(H)
Figure 17 :Experimental (dotted) and the calculated of profiles (solid lines) with different concentrations of Tryptophan and
Phenylalanine on C18 X-terra column. Flow rate 1.0 mL/ min; Wavelength 305 nm and Room temperature. Mobile phases: 10-100%
(v/v) mixtures of methanol, acetic acid without methanol .
 CONCLUSION
The adsorption isotherm behavior of tryptophan depends on the composition of
mobile phase and concentration of Ionic liquid used.

Inverse method was used to calculate adsorption parameters. Here it is important

to choose a good isotherm model.

The model can be guessed from the shape of the overloaded band profiles.

 This method is useful for purification process and in the industrial field because
the parameters for adsorption isotherm is known in a very short time when
compared to that of Frontal analysis.

 Our results indicate that the shape of the profiles, the isotherms, and the retention
of tryptophan are affected by the amount of IL added to the mobile phase.

The amount of analyte adsorbed on the column and the retention factor can be
manipulated by changing the amount of BMIM in the mobile phase.

Mobile phase containing no methanol as modifier and containing only BMIM can
be used as mobile phase to elute tryptophan
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