CHIRAL ART
Chirall method
Chi
th d optimization
ti i ti off iionic
i compounds
d
utilizing immobilized type column in reversed
phase mode
~ Influence of p
pH on retention behavior ~
(F140718AE)
Features of immobilized type columns
 Immobilized type CHIRAL ART columns can be used with various solvents that are
commonly used for HPLC analysis.
 They can be used in both normal phase mode and reversed phase mode as they are
compatible with non-aqueous and aqueous solvents (*).
 Reversed phase mode will be effective in case where a sample is hydrophilic and has
limited solubility in organic solvent (e. g. Hexane)
 In this technical data sheet, we will introduce influence of pH of mobile phase on
retention behavior of ionic compounds. We also introduce several example of chiral
separation on reversed phase mode.
(*) We recommend that the column is dedicated to either phase. Frequent change of separation mode may result
in short column lifetime.
2
Retention Behavior of Ionic Compound on Reversed Phase Mode
Acidic compound
● Influence of pH on retention behavior
Column
CH 3
pH 2.1
O
OH
Eluent
O
Flow rate
Temperature
Detection
Injection
*
140618_0001001.D
O
pH 3.1
p
Warfarin (pKa 5.56)
: CHIRAL ART Cellulose-SB 5 µm
250 X 4.6 mmI.D.
: 20 mM phosphate buffer/acetonitrile
(50/50)
: 1.0 mL/min
: 25℃
: UV at 254 nm
: 2 µL (0.2 mg/mL)
140619_0000001.D
pH 3
3.5
5
140704_1000002.D
pH 4
4.6
6
Peak shape may
deteriorated at
around pKa value
of the compound
140619_0000003.D
pH
H6
6.0
0
25
Retention f actor (k')
Retention of Warfarin
4.0
3.0
20
2.0
k' (1)
1.0
k' (2)
0.0
2.0
3.0
4.0
5.0
6.0
7.0
pH
H
140620_0000002.D
For acidic compound, retention is extended and
good separation is achieved when decreasing the
pH of a mobile phase (ionization was suppressed).
pH 7.0
0 140620_0000004.D
0
2
4
6
8
10
12
14 min
3
Retention Behavior of Ionic Compound on Reversed Phase Mode
Basic compound
● Influence of pH on retention behavior
Column
N
*
pH 4.6
Donepezil
¥_002_140701_0000005.D
O
(pKa 8.62)
Eluent
O
O
Flow rate
Temperature
Detection
Injection
CH 3
CH3
: CHIRAL ART Cellulose-SB 5 µm
250 X 4.6 mmI.D.
: 20 mM phosphate buffer/acetonitrile
(40/60)
: 1.0 mL/min
: 25℃
: UV at 254 nm
: 2 µL (0.2 mg/mL)
pH 6.0
¥ 001 140701 0000002 D
¥_001_140701_0000002.D
Retention f a
actor (k')
Retention of Donepezil
pH 7.0
¥_004_140627_0000206.D
pH 8.0
4.0
3.0
20
2.0
k' (1)
1.0
k' (2)
0.0
4.0
5.0
6.0
7.0
8.0
9.0
pH
30
¥_003_140627_0000203.D
For basic compound, retention is extended and
good separation is achieved when increasing the
pH of a mobile phase (ionization was suppressed).
pH 8
8.9
9
0
¥_005_140627_0000006.D
0
2
4
6
8
10
12
min
4
Tips for optimizing chiral separation method of ionic compounds on
reversed phase mode
 Mobile phase
> Optimal pH that ionization of analyte is suppressed is recommended.
(Retention will be extended and possibility of greater resolution is expected.)
Column
> Immobilized type CHIRAL ART columns are the best option.
Reversed phase analyses with optimized separation methods
Warfarin (p
(pKa 5.56))
O
N
Donepezil
p
(pKa
(p
8.62))
CH 3
*
OH
O
*
O
O
O
O
CH 3
CH3
mAU
14
Column
Eluent
12
: CHIRAL ART Cellulose-SB
: 20 mM phosphoric acid (pH 2.1)/acetonitrile
(50/50)
12
10
10
8
8
6
6
4
4
2
2
0
Column
Eluent
mAU
14
: CHIRAL ART Cellulose-SB
: 20 mM NH4HCO3-DEA (pH 9.0)/acetonitrile
(40/60)
0
F140707A
0
F140618A
2
4
6
8
10
12
14
min
0
2
4
6
8
10
12
14 min
5