INSTRUCTION MANUAL FOR

CHIRALPAK® IE COLUMNS
Please read this instruction sheet completely before using this column
IE columns can also be used in reversed phase mode.
Please refer to the corresponding instruction sheet for details.

Column description

CHIRALPAK® IE

Amylose tris(3,5-dichlorophenylcarbamate) immobilised on 5µm silica gel.

OR
Cl
O
O
RO
R:
OR O N Cl
n H

silica gel

Shipping solvent:n-Hexane / 2-Propanol solvent mixture (90:10 v/v)

All columns have been pre-tested before packaging. Test parameters and results, as
well as the Column Lot Number, are included on a separate (enclosed) page.

THIS INSTRUCTION SHEET IS NOT APPLICABLE TO ANY OTHER DAICEL COLUMNS

Operating Instructions

2.1 x 150 mm 4.6 x 150 mm

10 x 250 mm 20 x 250 mm
2.1 x 250 mm 4.6 x 250 mm
Semi-prep. columns Semi-prep. columns
Analytical columns Analytical columns

Flow rate direction As indicated on the column label

Typical Flow rate
~ 0.1 - 0.2 ml/min ~ 1 ml/min ~ 5 ml/min ~ 18 ml/min

Should be maintained < 300 Bar (4350 psi) for maximum column life
Pressure limitation
Adapt flow rates to column size.
Temperature 0 to 40°C

The maximum flow rate depends on the mobile phase viscosity (mobile phase composition), and
should be adjusted in accordance with the pressure upper’s limit (i.e. 300 Bar).
Method Development / Normal Phase

A - Mobile phases
CHIRALPAK® IE can be used with all ranges of organic miscible solvents, progressing from the traditional
mobile phases used with other DAICEL columns (mixtures of alkanes/alcohol, pure alcohol or acetonitrile
(CH3CN)) to mobile phases containing methyl tert-butyl ether (MtBE), tetrahydrofuran (THF),
dichloromethane (DCM), chloroform (CHCl3), ethyl acetate (EtOAc) among others.

B - Method Development - Screening

When developing methods we would recommend a screening approach.
1. The conditions described in Table 1 should be used as a Primary Screening.
2. If the compound or compound series are not soluble in any of these mobile phases, we recommend
progressing directly to the Secondary Screening (Table 2).

Table 1. Immobilised Primary Screening Solvents

Primary solvent
Alkane/2-PrOH Alkane/EtOH Alkane/MtBE/EtOH Alkane/THF Alkane/DCM/EtOH
mixtures

Typical starting
80:20 80:20 0:98:2 70:30 50:50:2
conditions

99:1 99:1 80:20:0 95:5 85:15:0

Advised
to to to to to
optimisation range
50:50 50:50 0:40:60 0:100 0:80:20

 Alkane = n-Hexane, iso-Hexane or n-Heptane. Some small selectivity differences may sometimes be found.
 In absence of alkane, methanol is more efficient than ethanol when combined with MtBE.
 In the case of no environmental restrictions, use of DCM is preferred to THF in terms of better enantioselectivity that the former may
induce.
 For excessively retained samples, addition of ethanol up to 20% in pure DCM would be helpful.

If a suitable chiral separation is not found using the Immobilised Primary Screening strategy, we recommend
an Immobilised Secondary Screening to be applied using the following conditions:

Table 2. Immobilised Secondary Screening Solvents

Secondary solvent
EtOAc/Alkane CH3CN/Alcohol
mixtures
Typical starting
50:50 100:0
conditions
20:80 100:0
Advised optimisation
to to
range
100:0 0:100

 Alcohols () or THF can be added into EtOAc to enhance the eluting strength for strongly retained compounds.
 Alkane: n-Hexane, iso-Hexane or n-Heptane. Some small selectivity differences may sometimes be found.
 Transfers between alkane mixtures and CH3CN are preferably made with a transition in alcohol in order to avoid miscibility issues.
 Alcohol: MeOH, EtOH and 2-PrOH.

Note: All solvent proportions indicated in this manual are by volume.

C – General Comments
 Additional solvent combinations such as CHCl3/Alkane, 1,4-Dioxane/Alkane, Toluene/Alkane or
Acetone/Alkane can also be investigated with CHIRALPAK® IE column.
 The typical starting conditions represent the mobile phases of upper middle eluting strength. Under such
conditions, most of the analytes can be eluted within a reasonable time range with a good probability of
full resolution of the enantiomers.
 Toluene, MtBE and chlorinated solvents can be used in their pure form as the mobile phase.
 For fast eluting solvents, such as THF, we recommend to add alkane in order to modulate the retention.
 Detection with a regular UV detector may become difficult depending on a combination of sample and
mobile phase (e.g. EtOAc, high percentages of DCM). In these cases an alternative detector, such as RI
detector or ELSD (Evaporative Light Scattering Detector), may be more effective than the UV detector.

D – Additives Basic Samples Acidic Samples

require require
Basic additives Acidic additives
For basic or acidic samples, it is necessary to
incorporate an additive into the mobile phase in Diethylamine (DEA) Trifluoroacetic acid (TFA)
order to optimise the chiral separation. 2-Aminoethanol (AE) Acetic acid
Ethylenediamine (EDA) Formic acid
 It has been found that certain amines, such as EDA and Butyl amine (BA)
AE induce much better behaviour for certain basic
compounds than the most commonly used DEA. < 0.5% < 0.5%
Typically 0.1% Typically 0.1%
 The addition of a low percentage of an alcohol (e.g. 2%
 STRONGLY BASIC solvent additives or sample
EtOH or MeOH) in the mobile phase may be helpful to ensure the
solutions MUST BE AVOIDED, because they are likely to
miscibility of EDA and AE with the low polarity mobile phases.
damage the silica gel used in this column.

Column care / Maintenance

 The use of a guard cartridge is highly recommended for maximum column life.

 Samples should be dissolved in the mobile phase. The mobile phase and the sample solution should be
filtered through a membrane filter of approximately 0.5µm porosity to ensure that there is no
precipitate before using.

 Column cleaning and regeneration procedures

Following extensive use of the column in multiple solvents there may be a change in separation
reproducibility. In order to ensure consistent performance, a regeneration method may be implemented
to eliminate any change in chiral recognition due to the history of the column (mobile phases,
additives…).

• Flush with ethanol at 0.5 ml/min(*) for 30 min, followed by 100% N,N-dimethylformamide (DMF)
at 0.3 ml/min(*) for 3 hours.
• Flush with ethanol at 0.3 ml/min(*) for 50 min and then equilibrate with n-Hexane/IPA = 90/10
(v/v) prior to retesting the column.

(*)
Recommended flow rate for analytical columns (4.6mm ID).

Column storage
 For column storage, remove the acidic or basic additives by flushing the column with the same mobile
phase without the additive. Columns can be stored end capped with additive-free mobile phases.

 If you have any questions about the use of these columns, or encounter a problem, please contact
DAICEL CORPORATION for assistance ([email protected]).

Operating this column in accordance with the guidelines outlined here will result in a long
column life.

CHIRALCEL®, CHIRALPAK® and CROWNPAK® are registered trademarks of

DAICEL CORPORATION.
 Internal Column Length
Column Name Ref. Product Type
Diameter (mm) (mm)
85311 Guard cartridge (x3) 4.0 10
85324 Analytical 4.6 150
85325 Analytical 4.6 250
CHIRALPAK® IE 85394 Analytical 2.1 150
5µm 85395 Analytical 2.1 250
85335 Semi prep 10 250
85345 Semi prep 20 250
85337 Guard column for Semi prep 10 20

CPI Company
2-18-1, Konan, Minato-ku, Tokyo 108-8230, Japan
TEL: +81-(0)3-6711-8222, FAX: +81-(0)3-6711-8228
http://www.daicelchiral.com/
E-mail: [email protected]

DAICEL CHIRAL TECHNOLOGIES(CHINA) CO., LTD. DAICEL CHIRAL TECHNOLOGIES(INDIA) PVT., LTD.
Part C, FL5 No.16, Xiya Road No.69, Waigaoqiao Free Laboratory No. 4A, Innovation Corridor 1,
Trade Zone, Shanghai, 200131, China Phase III, ICICI Knowledge Park, Turkapally Village,
TEL: +86-21-5046-0086, FAX: +86-21-5046-2321 Shameerpet Mandal, Ranga Reddy District,
http://www.daicelchina.com/ Hyderabad -500 078, INDIA
E-mail: [email protected] TEL: +91-40-2348-0103, FAX: +91-40-2348-0104
E-mail: dia.com" [email protected]
 INSTRUCTION MANUAL FOR
CHIRALPAK® IE COLUMNS

Please read this instruction sheet completely before using this column

<REVERSED PHASE>
IE columns can also be used in normal phase mode.
Please refer to the corresponding instruction sheet for details.

Switching between RP and NP mode

To switch from reversed phase mode to normal phase mode, and vice versa, column should be carefully
flushed with miscible solvent.
It is highly recommended to apply the regeneration procedure described in the instruction sheet for
normal phase mode. Before applying this protocol, any traces of salts should be removed by flushing with a
mobile phase that does not contain any salts / buffers.

Method Development / Reversed Phase

A - Mobile phases / For both UV and Mass detections

ACIDIC
NEUTRAL BASIC
(AMPHOTERIC)
Compounds Compounds

Compounds
20mM NH4HCO3 aq.
Aqueous HCOOH aq. pH 9.0
Water
solution  pH 2.0 adjusted with a basic
additive
CHIRALPAK® IE Organic
CH3CN or MeOH or EtOH or IPA or THF
modifier 
Typical starting Aqueous solutions 60%
conditions  CH3CN 40% 
 NOTE 1: If you cannot achieve sufficient resolution, try the complementary aqueous solutions

B – Complementary aqueous and buffer solutions / For UV detection

ACIDIC
NEUTRAL BASIC
(AMPHOTERIC)
Compounds Compounds

Compounds
50mM Phosphate Buffer
pH 2.0 20mM Borate Buffer
pH 9.0
OR
OR
H3PO4 aq. pH 2.0
Aqueous
CHIRALPAK® IE Water 20mM Phosphate Buffer
solution  OR pH 8.0 
100mM KPF6 (or NaPF6) OR
aq.
pH 2.0 100mM KPF6 (or NaPF6) aq.
adjusted with H3PO4

 NOTE 2: The concentration of all the buffering salt should be less than 500mM.

 Refer to section C for preparation of aqueous solution and choice of basic additives.
  It is recommended to use CH3CN to start the investigation
 The elution power of organic modifiers for these columns is in the descending order of
CH3CN > EtOH > MeOH: 50%CH3CN ≈ 65-70%EtOH ≈ 75-80%MeOH.
  The use of other organic solvents –except THF- has not been investigated and could be harmful
to the columns.
 The use of alcohols causes the back pressure to be significantly higher compared to CH3CN due
to their high viscosity in mixtures with water.

  Retention can be adjusted by changing the proportion of CH3CN. Retention may be very sensitive
to the amount of CH3CN present into the mobile phase.
 Lowering the column temperature may increase the retention time and the selectivity.
 Increasing the column temperature and decreasing the flow rate may increase the
resolution.

 To maximize column life the use of a guard cartridge is essential when basic
conditions are employed.
 The use of strong basic conditions (> pH 9) must be avoided, as they are known to damage
the silica gel matrix.
 When these columns are used at pH > 7, the temperature should be maintained between
5°C and 25ºC for maximum column life.

 High percentages of organic modifier in the mobile phase may precipitate the buffering salt from
the solution, and lead to consequent clogging of the column (refer to the table below).

Water / Organic Modifier Buffer solution / Organic Modifier

90 / 10 to 0 / 100 90 / 10 to 15 / 85

 Do not use the phosphate buffer for pH > 8. When pH 9 is necessary, use the ammonium
bicarbonate solution or borate buffer for maximum column life.

C – Buffer preparation – Examples

Preparation of pH 2 Phosphate buffer:
Solution A: 50mM potassium dihydrogenphosphate
3.40g KH2PO4 / FW 136.09, make up the volume to 500ml with HPLC grade water
Solution B: phosphoric acid (H3PO4 85% by weight)
Adjust the pH of solution A to a value of 2.0 using solution B.

Preparation of pH 2 KPF6 (NaPF6) solution:

Solution A: 100mM potassium (sodium) hexafluorophosphate
9.20g KPF6 / FW 184.06 or 8.40g NaPF6 / FW 167.95, make up the volume to 500ml with HPLC
grade water
Solution B: phosphoric acid (H3PO4 85% by weight)
Adjust the pH of solution A to a value of 2.0 using solution B.

Preparation of pH 9 Ammonium bicarbonate solution:

Solution A: 20mM ammonium bicarbonate
0.78g NH4HCO3 / FW 78.05, make up the volume to 500ml with HPLC grade water
Solution B Basic additive such as diethylamine (DEA), triethylamine (TEA), ammonia (NH3) and so on.
* DEA tends to give better peak shape than other bases.
Adjust the pH of solution A to a value of 9.0 using solution B.

Preparation of pH 8 Phosphate buffer:

Solution A: 20mM potassium hydrogenophosphate
1.74g of K2HPO4 / FW 174.18, make up the volume to 500ml with HPLC grade water
Solution B: 20mM potassium dihydrogenophosphate
1.36g KH2PO4 / FW 136.09, make up the volume to 500ml with HPLC grade water.
Adjust the pH of solution A to a value of 8.0 using solution B.

Preparation of pH 9 Borate buffer:

Solution A: 20mM sodium tetraborate decahydrate
3.81g of Na2B4O7.10H2O / FW 381.37, make up the volume to 500ml with HPLC grade water
Solution B: 20mM boric acid
0.62g H3BO3 / FW 61.83, make up the volume to 500ml with HPLC grade water
Adjust the pH of solution A to a value of 9.0 using solution B.

Column care / Maintenance

 Any traces of salts should be removed before column storage and /or before switching to 100%
organic solvent (use Water/CH3CN 60:40 (v/v) for instance)

Refer main instruction for normal phase and column care/maintenance.