CHIRAL ART

Chirall method
Chith d optimization
ti i ti off iionic
i compounds
dutilizing immobilized type column in reversedphase 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 arecommonly used for HPLC analysis.
 They can be used in both normal phase mode and reversed phase mode as they arecompatible 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 chiralseparation on reversed phase mode.

(*) We recommend that the column is dedicated to either phase. Frequent change of separation mode may resultin short column lifetime.
2

Retention Behavior of Ionic Compound on Reversed Phase Mode
Acidic compound● Influence of pH on retention behaviorColumn

CH 3pH 2.1OOH

EluentO

Flow rate
Temperature
Detection
Injection*140618_0001001.D

OpH 3.1
pWarfarin (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
5140704_1000002.D

pH 44.6
6
Peak shape may
deteriorated ataround pKa valueof the compound140619_0000003.D

pHH66.0025

Retention f actor (k')

Retention of Warfarin
4.0
3.0
202.0k' (1)1.0

k' (2)

0.0
2.0

3.0

4.0

5.0

6.0

7.0

pHH

140620_0000002.D

For acidic compound, retention is extended andgood separation is achieved when decreasing thepH of a mobile phase (ionization was suppressed).

pH 7.00 140620_0000004.D

02

468

101214 min

3Retention Behavior of Ionic Compound on Reversed Phase Mode
Basic compound● Influence of pH on retention behavior
ColumnN*

pH 4.6
Donepezil¥_002_140701_0000005.DO(pKa 8.62)

EluentOO

Flow rate
Temperature
Detection
Injection

CH 3CH3: 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 aactor (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 andgood separation is achieved when increasing thepH of a mobile phase (ionization was suppressed).

pH 88.990¥_005_140627_0000006.D024

681012min4

Tips for optimizing chiral separation method of ionic compounds onreversed 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))
ON

Donepezilp(pKa(p8.62))

CH 3*OHO*OOOO

CH 3CH3mAU
14Column
Eluent12: CHIRAL ART Cellulose-SB: 20 mM phosphoric acid (pH 2.1)/acetonitrile(50/50)121010886644220Column
EluentmAU
14: CHIRAL ART Cellulose-SB: 20 mM NH4HCO3-DEA (pH 9.0)/acetonitrile(40/60)0F140707A0F140618A

2468101214min

02468101214 min

5