Technical Review

CHROMATOGRAPHY, Vol.31 No.1 (2010)

Technical Review

Chiral separations − efficient, fast and productive
By Per Jageland*, Mattias Bengtsson and Kristina Hallman
AkzoNobel Eka Chemicals AB, Separation Products SE−445 80 Bohus Sweden

optimal thickness − both important to generate a high-performing,

Introduction

yet stable, product.

Chromatographic analysis and purification of optically active
compounds are still areas with large potential for improvement. In

Highly efficient 3 µm particles

the analytical field, chiral stationary phases (CSP) with better performance giving enhanced resolution and shorter analysis time areKromasil AmyCoat and CelluCoat are available in 3, 5, 10 anddesirable. Within recent years the Kromasil group has launched

25 µm particle sizes. The smallest particle size, 3 µm, gives hightwo new polysaccharide CSP’s: Kromasil AmyCoatTM which isefficiency, and consequently a high resolution. Table 1 illustrates

based on a tris-(3,5-dimethylphenyl) carbamoyl amylose selector,

the similarity in chiral recognition capability between Kromasil CelluCoat , which is based on a tris-(3,5-AmyCoat 3 µm and 5 µm. The higher resolution obtained using

TMand Kromasil

Kromasil AmyCoat 3 µm is a result of the higher plate countdimethylphenyl) carbamoyl cellulose selector.

achieved with a smaller particle size.
For difficult separations reducing the particle size could makeThe stationary phasethe crucial difference between achieving baseline separation or not,The in-house developed wide pore silica is specially designed toas illustrated in figure 2.

minimize the amount of achiral interactions with the silica surface,
while maintaining the mechanical strength of Kromasil silica. ThisHigh speed chromatographymechanical strength allows operating the columns without pressure

The mechanical strength of Kromasil AmyCoat and CelluCoat

restriction within the standard HPLC pressure range (<400 bar).
The amylose and cellulose selectors are well known for their

allow the columns to be operated at high flow rates. High flow rateability to resolve a broad range of racemates. This unique coating

combined with short column length provides very short analysis

technology ensures homogenous distribution of the selector and antime. Figure 3 a shows high speed chromatography with baseline

Figure 1.

FE-SEM pictures. a and b: pictures of uncoated Kromasil wide pore silica used for AmyCoat and CelluCoat; c:picture of CelluCoat

Correspondence: AkzoNobel Eka Chemicals AB, Separation Products SE−445 80 Bohus Sweden
Phone: +46 709 57 78 34
E-mail: per.jageland@akzonobel.com
Website: www.kromasil.com―５１―CHROMATOGRAPHY, Vol.31 No.1 (2010)

Table 1. Selectivity and resolution comparison of Kromasil AmyCoat 3 µm and 5 µm. Column size: 4.6×150 mm
AmyCoat 3 µmαRsAmyCoat 5 µm
αRsBenzoin

1.3

6.5

1.3

4.4

heptane/2−propanol (90/10)

1Bucetin

1.8

8.2

1.7

5.8

heptane/2−propanol (90/10)

2Racemate

Mobile Phase

Flow rate[mL/min]

Trifluoroanthrylethanol

1.4

6.4

1.4

4.2

heptane/2−propanol (90/10)

1

Hexobarbital

1.4

4.7

1.4

3.2

heptane/2−propanol (90/10)

1Oxamniquine

1.2

3.2

1.2

2.3

heptane/2−propanol/DEA (90/10/0.1)

0.8

Alprenolol

1.6

5.3

1.7

4.4

heptane/2−propanol/DEA (90/10/0.1)

1Metoprolol

1.53.21.4

2.0

methanol/DEA (100/0.1)

0.5

Figure 2.

Figure 3a.

Separation of 2-phenyl-1-cyclohexanol in heptane/2-propanol (95/5), detection UV @ 220 nm, temp. 25℃, flow rate 1 ml/min, column size: 4.6×150 mm

Separation of Troger’s base in heptane/2-propanol/DEA (90/10/0.1), detection UV @220 nm, temp. 22℃, column: Kromasil AmyCoat, 3 µm, 4.6×150 mm.

separation in less than 1 minute. Since the van Deemter plot, figure
3 b, is more flat for the smaller particle size, Kromasil AmyCoat 3µm should be the first choice when running at elevated flow rates.
Mechanical stability
Mechanical strength is an important product lifetime parameter,and makes it possible to pack columns at higher pressure, to obtainhigher efficiency. Kromasil AmyCoat and Kromasil CelluCoat are
based on a mechanically strong spherical silica matrix, withstanding repeated cycles of packing in columns at high pressure.
Stable performance
Figure 3b. van Deemter plots for Kromasil AmyCoat 3 and 5 µm.

It is most desirable to be able to run the compatible normal, po-―５２―CHROMATOGRAPHY, Vol.31 No.1 (2010)

Figure 4.

Figure 5.

Technical Review

The test shown above was designed to exert greater than normal mechanical stress on
the chiral stationary phases, and is performed at a packing pressure above the maximum 50 bar recommended by the manufacturer of competitor CA and competitor CO.

Maintained efficiency after extended use. Separation of trans-stilbene oxide in heptane/2-propanol(90/10), detection UV @ 229 nm, temp. 25℃, column Kromasil AmyCoat, 3 µm, 4.6×150 mm

lar organic, and reversed mobile phases without irreversibly damaging the stationary phase. Switching between compatible normal
to polar organic mobile phases will not lead to any degradation inperformance. By using Kromasil AmyCoat and CelluCoat there are
no need for solvent dedicated columns. In order to test the stabilityof Kromasil AmyCoat, in this sense, the chromatographic performance was evaluated before and after high flow rate conditions. As
shown in figure 5, the column efficiency was maintained even after
the harsh conditions of the test sequence.
Figure 6.
Scalability
Depending on the purpose of the chiral separation method theoptimum particle size of the CSP varies. Small particles (3 µm and5 µm) should be applied in analytical scale work, and larger parti-

Consistent retention and selectivity independent of particle size. Separation of trans-stilbene oxide in heptane/2-propanol (90/10), detection UV @ 220 nm, flow rate
0.5 ml/min, column Kromasil CelluCoat 3, 5, and 10µm, 4.6×150 mm

cles should be used when going to preparative scale.
separation of trans-Stilbene oxide on Kromasil CelluCoat 3 µm, 5Since particle sizes from 3 µm to 10 µm gives identical selectiv-µm, and 10 µm.

ity, it will be easy to scale up both Kromasil AmyCoat and CelluCoat, while maintaining excellent performance. Figure 6 shows the―５３―

CHROMATOGRAPHY, Vol.31 No.1 (2010)

Figure 7a.

Analytical injection. Solute: Ethyl mandelate 9 mg/ml
in mobile phase. Injection: 5 µl. Column: Kromasil
CelluCoat 3 µm, 4.6×150 mm. Flow rate: 1.0 ml/min.
Mobile phase: heptane/2-propanol (90/10). Detection:
UV @230 nm.

Figure 8 a.

Figure 7b.

Overloaded injections. Solute: Ethyl mandelate 140
mg/ml in mobile phase. Column: Kromasil CelluCoat
10 µm, 4.6×250 mm. Flow rate: 0.7 ml/min. Mobile
phase:heptane/2-propanol (90/10). Detection: UV @254 nm.

Separation with additive, 0.1% DEA

Figure 8 b.

Separation without additive

larly during evaporation. Screening without additives with analyti-

Semi-preparative chiral separations
Important aspects in preparative chiral separations are productiv-cal injections should however be made with careful considerations.

ity, loadability, selectivity and, solubility. Figure 7 a and 7 b illus-

A study performed on the separation of Metoprolol clearly indi-

trate a semi-preparative application of Ethyl mandelate on Kro-cated, using analytical injections, that 0.1% DEA was needed in themasil 10-CelluCoat. The analytical chromatogram was run on a 3

mobile phase. However, at overloaded conditions no additive wasµm CelluCoat column and since an identical manufacturing tech-needed in the mobile phase or in the injection solvent, as can benology is used for all particle sizes the results could easily be trans-

seen very clearly from the fraction analysis presented in Figure 8 alated to a column packed with 10 µm particles.

and 8 b. This behaviour could be explained by the fact that the injected substance itself will quickly saturate any acidic sites available on the surface, and thus “self-buffering” the phase at overload

Additives in the mobile phaseconditions.

In preparative chromatography additives are undesirable since
they complicate the solvent recovery process and they could also
reduce the stability of the enantiomers in the mobile phase, particu-―５４―

CHROMATOGRAPHY, Vol.31 No.1 (2010)

Technical Review

operated at pressures up to 400 bar.

Conclusion

The amylose and cellulose selectors are well-known for their

Kromasil AmyCoat and CelluCoat are two fully back-integrated

ability to resolve a broad range of racemates.

chiral stationary phases from Kromasil. The specially designed silica offers a high mechanical stability, which allows columns to be―５５―