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Technical support – Care & Use
- Packing instruction for Kromasil in DAC columns
- Care and use of Kromasil HPLC columns
- Care & Use of Kromasil Coated Chiral HPLC Columns
- Guidelines for chiral chromatography using DMB and TBB
- Optimizing your large-scale application with the software KromaGuide
- Unpacking and care of used packings
- Frequently Asked Questions
Care and Use of Kromasil Coated Chiral HPLC Columns
Please read this information carefully before using the column. All Kromasil columns are individually manufactured and tested to meet strict specification criteria. The following measures will maintain their performance and lifetime.
Products intended
Those instructions are applicable for the following Kromasil coated chiral phases:
for normal phase chromatography (NP):
- Kromasil AmyCoat
- Kromasil CelluCoat
for reversed phase chromatography (RP):
- Kromasil AmyCoat RP
- Kromasil CelluCoat RP
Important
The entire HPLC system, including solvent lines, injector, loops or
autosampler has to be purged with a solvent compatible with the Kromasil coated chiral column. A
thorough instrument purge with 2-propanol is recommended. Solvents commonly used for other normal
phase separations, such as e.g. TFA, dichloromethane,
DMSO, DMF,
ethyl acetate, acetone orchloroform could severely damage the Kromasil coated chiral column even at residual quantities.
Changing mode (from RP to NP or vice-versa) on a coated chiral phase column may permanently
impair its performance.
Column installation
Shipping solvent:
The normal and reversed phase columns are shipped in heptane /2propanol (90/10)¹ and acetonitrile/water (40/60)¹, respectively
System dead volume:
Reduce dead volume in the system to a minimum by using small internal diameter connection tubing, for analytical columns 0.010”. Keep the tubing length between injector, column and detector as short as possible.
Column connection:
The column should be mounted according to the flow direction indicated on the column. For optimum performance, it is important that the tubing used to connect the column to the injector or detector is swaged into position such that it abuts the internal shoulder of the fitting. Excessive tightening of the column end and fittings will result in damage to the column tubing and/or fitting.
Performance Testing:
It is recommended that the performance of columns is tested upon arrival and at periodic intervals during use. The test conditions are described on the test chromatogram, the calculations of plates and symmetry as described below.
Plates: N = 5.54·(tr/W0.5)2
Asymmetry factor: As0.1 = B/A
Mechanical damage:
Protect the column from mechanical chock. Dropping or banging a column can impair its performance.
Storage:
Wash out all additives from normal phase columns with a neutral mobile phase such as heptane/2propanol (90/10)¹ and buffer from reversed phase columns with a salt-free mobile phase such as acetonitrile/water (40/60)¹. Close the column openings with the end-caps in order to prevent the packing from drying out and keep the column at ambient temperature (15-25°C).
Operational guidelines
Flow rate and pressure limitation:
Maximum operating pressure = 400 bar ( ≈ 6000 psi)
Temperature limits:
Generally, Kromasil coated chiral columns can be operated between 0 and 40°C, except for buffered basic mobile phases. See compatibility table below for actual limits.
Compatible mobile phases
If you wish to use solvents, buffers or additives others than mentioned on this page, please consult the Kromasil technical support team first (kromasil@akzonobel.com).
Changing mode (from RP to NP or vice-versa) on a coated chiral phase column may
permanently impair its performance.
Switching between acetonitrile and methanol in the mobile phase on coated chiral phases
may irreversibly damage the coating. When switching between mobile phases containing these
solvents, run an intermediate wash with 100% ethanol for Kromasil AmyCoat / AmyCoat RP or
2-propanol for Kromasil CelluCoat / CelluCoat RP.
Compatible normal phase mobile phases:
Possible combinations and compositions for Kromasil AmyCoat and Kromasil CelluCoat are described in the tables below:
| alkane/2-propanol | 100/0 to 0/100 |
| alkane/ethanol | 100/0 to 0/100 |
| alkane/methanol² | 100/0 to 0/100 |
| alkane/MTBE | 100/0 to 50/50 |
| ethanol/methanol | 100/0 to 0/100 |
| (SFC) CO2/alcohol | 100/0 to 50/50 |
| acetonitrile/methanol | 0/100 to 15/85
85/15 to 100/0 |
| acetonitrile/2-propanol | 100/0 to 0/100 |
| ethanol/MTBE | 100/0 to 70/30 |
| acetonitrile/methanol | 85/15 to 100/0 |
| ethanol/MTBE | 100/0 to 50/50 |
Mobile phase additives for normal phase:
For basic samples we recommend the addition of 0.1% (< 0.5%) DEA and for acidic samples the addition of 0.1% (< 0.5%) TFA.
Switching between mobile phases on normal phase columns.
When switching between non-miscible solvents, use 100% 2-propanol as a transition mobile phase. Kromasil AmyCoat will also need a subsequent wash with 100% ethanol.
When switching from 100% polar mode to alkane/alcohol run a transition wash with 100% ethanol for Kromasil AmyCoat or 2-propanol for Kromasil CelluCoat.
Otherwise, no intermediate column wash is necessary. An adequate equilibration time is depending on the column dimension. By increasing the flow rate the equilibration time can be reduced. However, stable base line should always be reached before a separation is started.
Compatible reversed phase mobile phases:
Possible combinations and compositions for Kromasil AmyCoat RP and Kromasil CelluCoat RP are described in the table below.
To avoid salt precipitation, a wash with acetonitrile/water (40/60)¹ is recommended when switching from buffer to 100% organic modifier.
| Aqueous solution | Organic modifiers | Organic part | Temperature |
|---|---|---|---|
| water | acetonitrile³,
methanol³, ethanol, 2-propanol |
10-100 % | 5-40°C |
| potassium phosphate buffer
0-0.5 M, pH 2.0-8.0 Suggestions: 50 mM at pH 2.0 20 mM at pH 8.0 |
10-85% | pH < 7: 5-40°C
pH > 7: 5-25°C |
|
| phosphoric acid, aqueous solution at pH 2.0 | |||
| sodium hexafluorophosphate aqueous solution
Suggestions: 100 mM at pH 2.0 50 mM at pH 5.0 |
|||
| sodium borate buffer
0-0.2 M, pH 7.5-9.0 Suggestion: 20 mM at pH 9.0 |
5-25°C | ||
| acetic acid, 0.1%¹ | 5-40°C |
¹: volume by volume.
²: due to limited miscibility of methanol in alkanes, ethanol should be added as a mediator when exceeding 5% methanol.
³: Read the red-boxed note about these solvents
