LM1105EC

ULTRON ES-OVM
ULTRON ES-OVM-CULTRON ES-OVM-E
Chiral Chromatography Columns

Instruction Manual
1.

Introduction
Thank you for purchasing an ULTRON ES-OVM series column for High-performance liquid chromatography. The packing

material used in these columns consists of a stable; highly denaturation resistant acidic glycoprotein (ovomucoid) covalently bonded
to aminopropyl silica gel particles (5 μm, 3 μm). These columns are mainly designed for separations of the optical isomers ofcompounds consisting of aromatic rings possessing basic and acidic substituents under reversed phase analytical conditions.

2.

Specifications
Inner diameter2.0φ (5 µm)

3.0φ

4.6φ, 6.0φ(Particle size)

2.1φ (3 µm)(3 µm)(5 µm)

Fittings
pH
Organic solvent concentration
Temperature
Pressure

3.

Waters compatible
Recommended range: pH 3.0~7.5
Recommended range: 1~10% (Maximum: 50%)
Recommended range: 5 ~ 3 0 ℃ ( M a x i m u m : 4 0 ℃ )5 µm: Recommended range: Up to 15 MPa (Maximum: 20 MPa)
3 µm: Recommended range: Up to 40 MPa (Maximum: 50 MPa)

Precautions when using these columns

・The same packing material is used for both the ULTRON ES-OVM, the ULTRON ES-OVM-C and the ULTRON ES-OVM-E withthe result that the performance for these columns is the same. The main difference between these columns is the fact that
confirmation of the separation of benzoin is used for shipping checks for ES-OVM columns, while that for clopidogrel bisulfate
and its related compounds are used for ES-OVM-C columns, and for Escitalopram oxalate and R-citalopram oxalate and used for
ES-OVM-E columns.
・ES-OVM (5 μm)columns are shipped containing 33% aqueous solutions of 2-propanol. ES-OVM (3 μm) columns,
ES-OVM-C columns and ES-OVM-E columns are shipped containing 30% aqueous solutions of acetonitrile.
When replacing these solutions with mobile phases with high salt concentrations or high viscosity levels, in order to avoid thedanger of sudden pressure increases, when using 150 x 4.6 mm I.D. columns, flow the mobile phase at an initial flow rate of 0.2
ml/min, determine that the pressure is stable, and, determining that the pressure is stable at each step, increase the flow rate to thedesired value in steps of 0.2 mL/min.
・Ensure to filter samples and mobile phases using a membrane filter with a mesh size of 0.45 μm or smaller before using these columns.
Failure to filter mobile phases, etc. can lead to blockages of column filters and increases in analytical pressures.
・Ensure to thoroughly degas mobile phases prior to use. Insufficient degassing of mobile phases can lead to the formation of bubbles
inside analytical instruments and columns resulting in problems with analyses.
・Although the packing material used in these columns consists of a highly denaturation resistant protein bound to silica gel particles,
the maximum allowable organic solvent concentration under reversed phase conditions is 50%. However, the recommended organic
solvent concentration range of 1~10% is most effective in terms of separation performance. Excessively high concentrations oforganic solvents can lead to the denaturation of the protein resulting in adverse effects with respect to analyses.
・The recommended analytical temperature range for these columns is 5~30℃. In addition, although the maximum operating
temperature for these columns is 40℃, excessively high analytical temperatures can lead to the denaturation of the protein used inthese columns resulting in adverse effects with respect to analyses.
・In order to extend column operating lifetimes as much as possible, we recommend using columns at analytical pressures up to 15
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LM1105EC
MPa. (ES-OVM (3 μm): u p t o 2 0 MPa) In addition, although the maximum allowable analytical pressure for these columns is 20
MPa (ES-OVM (3 μm): u p t o 5 0 MPa), excessively high analytical pressures can lead to the formation of voids inside columns
resulting in degradations in peak shapes.
・The pH range of mobile phases should be kept between 3.0 and 7.5. Excessively high levels of acidity can lead to detachment anddenaturation of the protein used in these columns. In addition, excessively high levels of basicity can lead to dissolution of the silica
gel used in these columns and denaturation of the protein.
・In order to avoid the build up of contamination such as scale and extend column lifetimes, we recommend the use of guard columns.
The use of guard columns does not have any adverse effects on separation performance, etc.
・With regard to the equilibration of mobile phases, one of the separation modes of these columns is ionic interactions, with the resultthat a certain amount of time is required for mobile phase equilibration. In particular, it should be noted that equilibrations aroundthe isoelectric point of ovomucoid (pI 4.1) used in the packing material for these columns require extended amounts of time.
When changing the pH of a mobile phase from 3.0 to 4.6 for example, an extended amount of time will be required to thoroughly
equilibrate the mobile phase.

4.

Optimizing analytical conditions

・Sample loading amounts
Theoretical plate numbers decrease with increases in sample loading amounts. In order to achieve sharp peaks, we recommend
keeping sample loading amounts as small as possible.
(1) Effects of sample loading weights

Column：ULTRON ES-OVM ( 4.6mmφ×150mm )Mobile phase：20mM KH2PO4 (pH=5.5)/C2H5OH= 100/4
Flow rate：1.0 mL/min.
Column temp.：25 ℃Detector：UV-254nm

Injection Volume：0.5 µL
Sample：Tolperison( 2 ) E ff e c t s o f s a m p l e l o a d i n g v o l u m e s

Analytical conditions same as those shownin “Effects of sample loading weights” above.
Injection volumes: 0.5, 1.0, 3.3, 6.7, 10 μL

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LM1105EC

5. Flow diagram for optimization of mobile phase conditions
Prepare mobile phase (20 mM KH2PO4 (pH 4.6)/CH3CN = 90/10)

Flow mobile phase for 60~80 minutes
Inject sample

PeakNot eluteddetection
Change proportion of CH3CN to 5, 20%
Elutedto elute sample*Proportion of organic solvent can beincreased up to a maximum of 50%

Acidic sample

Basic sample

Maintain pH of mobile phase at 3~4, andMaintain pH of mobile phase at 5~7, andcontrol elution by changing type orcontrol elution by changing type oramount of organic solvent usedamount of organic solvent used

Decrease mobile phase flow rate to improve peak separation

6.

Effects of analytical temperatureWith some exceptions, relatively good peak separation can be achieved by using analytical temperatures around 20℃. Shown
below is a reference table showing the effects of analytical temperature on separations of propranolol.

Column Temperature
5℃

11 ℃

19℃

28℃

40℃

k’1

18.67

11 . 4 9

7.79

4.97

2.59

k’2

23.94

14.59

9.68

6.05

3.06

α

1.28

1.27

1.25

1.22

1.18

N1446

722

1059

1224

1899

N2380

563

796

890

1282

Rs

1.12

1.35

1.44

1.21

1.08

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Column：ULTRON ES-OVM ( 4.6mmφ×150mm )Mobile phase：20mM KH2PO4 (pH=4.6)/C2H5OH= 100/10
Flow rate：1.0 mL/min.
Detector：UV-220nm(0.08AUFS)

Sample：PropranololLM1105EC

7. Cleaning and storage of columns
・When cleaning 150 x 4.6 mm I.D. columns, replace the buffer solution of the mobile phase used (e.g. 20 mM KH2PO4/acetonitrile= 90/10) with purified water, and then clean the column overnight using a solution with an organic solvent concentration the sameas (water/acetonitrile = 90/10) or slightly higher than that of the mobile phase at a flow rate of 0.2 mL/min. When columns arecontaminated, clean for at least 2 hours using an 80/20 solution of pH 3.5 phosphoric acid aqueous solution and 2-propanol at a flowrate of 0.2 mL/min.
・When not performing analyses for 2 or 3 days, replace the mobile phase inside the column with a 30% aqueous solution of acetonitrile,and store the column in the fridge, around 4℃ is recommended. (allowable storage temperature is between 4℃ to 25℃.
・Please note that deviating from the storage temperature may cause deterioration of the column during storage.
ULTRON series packed columns are shipped under highly controlled conditions. However, if you should find any defect, pleasecontact your dealer or Shinwa.
Note that Shinwa does not warrant the product against column life or deterioration caused by the failure to follow the above
instructions.

50-2 Kagekatsu-cho, Fushimi-ku, Kyoto 612-8307 JAPANTEL +81-75-621-2360

URL https://shinwa-cpc.co.jp
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