Comparison among Leading Companies High Quality at Reasonable Prices There are many companies which have a relation with supply of HPLC columns, for example base media manufacturers, chemical bonding companies, packers, companies which implement more than two of the above manufacturing process, OEM suppliers, dealers etc. GL Sciences Inc. synthesizes base silica-gel media from pure ingredients, bonds functional groups on the media and packs them into HPLC column hardware. We think the execution of whole manufacturing process with reliable and consistent high technologies is essential for the reproducibility of high column efficiency resulting in best customer satisfaction. We convince that this is the only way we can reduce the cost which enable to supply our customer with HPLC columns at a reasonable price

Execution of whole manufacturing process

Synthesis of base silica-gel from pure ingredients

Bonding functional groups on silica-gel media

Packing silica-gel media into HPLC column hardwares

High Quality with Excellent Column Reproducibility GL Science’s Inertsil HPLC columns ushered in the high-purity silica revolution in early 1990 and set a new standard for excellence. The purity (99.999%) and physical properties of silica-gel and figures of chemical bonding are strictly controlled during the manufacturing process. Chromatographic inertness, selectivity and durability are also investigated for every batch. All those Quality tests below guarantee “High Quality with Excellent Column Reproducibility”.

Quality Control of Base Silica-Gel The following instruments are used for the control of physical properties et al. and the figure shows very good batch-to-batch reproducibility.

Scanning Electron Microscopy: - Sphericity and surface smoothness of Silica-gel Atomic Emission and ICP: -Trace metals Nitrogen Adsorption & Laser Ray Particle Analyzer: - Particle size & its uniformity, Surface area, Pore diameter, Pore volume

Elementary Analysis: - Carbon load

Si NMR: - Residual silanol after bonding and endcaping

Quality Control by Chromatographic Tests The following chromatographic tests are used for column quality and property control and the figures show very good batch-to-batch reproducibility.

Tanaka Test Mixture for Selectivity: - Hydrophobicity, Stereoselectivity, Hydrogen-bonding capability Inertness of Packing to Basic compounds: - Pyridine Ethylaniline, Benzylamine, Aminopyridine Inertness of Packing to Acidic compounds: - Formic Acid, Acetic Acid Inertness of Packing to Chelating compounds: -Oxine-Copper Durability of Packing at High pH & Low pH: - At pH 2.0 & pH 9.0 Column Performance Test: - Theoretical number plates, Peak symmetry, Retention Column back pressure

Industry Leader’s Test Method for Peak Shape of Basic Compounds This method is employed by one of industry leaders to investigate peak shape of basic compounds. Amitriptyline, basic prove was analyzed under the most demanding mobile phase condition at pH 7.0 and USP tailing factor was measured. Inertsil columns show very good peak shapes for such significant basic compound.

Inertsil ODS-Sprint

0 10 20Time (min)

020

4060

80m

AUm

Volt

1

2

3 4

5 6

Zorbax Eclipse XDB-C18

0 10 20Time (min)

020

4060

80m

AUm

Volt

1

2

3

4

5

6

Analytical Condition Eluent: 20mM Phosphoric Acid Buffer (pH7.0) / MeOH = 20/80 (v/v) Flow: 1.0mL/min Detect: UV254nm Oven: 40C Sample: 10uL-inject 1.Uracil 12ug/mL 2.Propranolol 199ug/mL 3.n-Butyl parabene 11ug/mL 4.Naphthalene 37ug/mL 5.Acenaphthene 50ug/mL 6.Amitryptyline 30ug/mL

0 0.5 1 1.5 2

Eclipse

Xterra

Symmetry

Inertsil ODS-3 4um

Sunfire

Inertsil ODS-Sprint

Inertsil ODS-3

Comparison of Tailing Factor

Tailing Factor

Column Tailing Factor

Inertsil ODS-3 1.04

Inertsil ODS-Sprint 1.06

Sunfire 1.06

Inertsil ODS-3 4um 1.07

Symmetry 1.11

Xterra 1.13

Eclipse 1.8

Another Industry Leader’s Test Method for Peak Shape of Basic Compounds This method is employed by another industry leader to investigate peak shape of basic compounds. Procainamide and N-acetylprocainamide are difficult basic compounds to analyze with symmetry peak. Inertsil columns show very good peak shapes for such significant basic compound. Analytical Condition Eluent: 20mM Phosphoric Acid Buffer (pH7.0) / ACN = 90/10(v/v) Flow: 1.5mL/min Detect: UV254nm Oven: 40C Sample 10uL-inject 1. Procaine amide 50ug/mL 2. n-Acetylprocaine amide 50ug/mL

0 2 4 6 8 10Time (min)

020

40

60

80

10

0m

Vm

Vol

t 60

0 2 4 6 8 10Time (min)

020

40

60

80

10

0m

Vm

Vol

t 60

Inertsil ODS-Sprint

Zorbax Eclipse XDB-C18

1

2

0 2 4 6 8 10Time (min)

020

40

60

80

10

0m

Vm

Vol

t 60

XTterra

1

2

0 2 4 6 8 10Time (min)

020

40

60

80

10

0m

Vm

Vol

t 60

Symmetry

1

2

0 2 4 6 8 10Time (min)

020

40

60

80

10

0m

Vm

Vol

t 60

SunFire

1

2

1

2

0 2 4 6 8 10Time (min)

020

40

60

80

10

0m

V

1

2

Inertsil ODS-3

Pyridine Test for Base Deactivation

Inertsil ODS-SP

0 10 20Time (min)

020

40m

Vol

t1

2 Column Length: 4.6X250mm 5um Eluent CH3OH / H2O = 30 / 70 Flow Rate: 1.0 mL/min Col.Temp.: 40C Detector:: UV254nm Sample Volume: 4uL Samples: 1) Pyridine 0.09 mg/mL 2) Phenol 0.41 mg/mL

0 10 20Time (min)

1 2

SunFire C18

0 10 20Time (min)

020

40m

Volt

Atlantis dC18

1

2

Pyridine tailing due to poor

end-capping.

0 10 20Time (min)

XTerra MS C18

1 2 Pyridine tailing due to poor

end-capping.

0 10 20Time (min)

1

2

Zorbax Eclipse XDB-C18

0 10 20Time (min)

0.00

0.02

0.04

mV

olt

Symmetry C18

1

2

Pyridine tailing due to poor

end-capping.

20

40

0 10 20Time (min)

020

40m

AU

Inertsil ODS-3

1

2

mV

olt

Inertness to Basic Compounds The Pyridine-Phenol Test is widely known as a simple but informative method to evaluate the residual silanol Groups on the surface of reversed-phase packing materials. The residual silanol groups are acidic and Strongly retain basic compounds. The retained basic compounds elute late or elute as tailing peaks. GL Sciences inspects and controls the retention time and the tailing factor of the pyridine peak using an Eluent of pH 7.6, conditions under which the residual silanols are completely dissociated and most likely to cause tailing of bases. Besides the Pyridine-Phenol Test, if necessary, Ehtylaniline Test developed by Prof. Engelhardt, Benzylamine Test developed by Prof. Tanaka and Aminopyridine Test developed by Prof. Barret are also Adopted as part of the quality control to understand the total profile of each packing material produced.

Aminopyridine Test for Base Deactivation

Column Length: 4.6X250mm 5um Eluent CH3OH / 20mM Phosphate Buffer (pH7.6) 10:90 Flow Rate: 1.0 mL/min Col.Temp.: 40C Detector:: UV254nm Sample Volume: 4uL Samples: 1) 4-Aminopyridine 0.1mg/mL 2) 3-Aminopyridine 1.0mg/mL 3) 2-Aminopyridine 1.0mg/mL

Inertsil ODS-SP

0 20Time (min)

020

0m

Volt

1

2

3

0 20Time (min)

SunFire C18

1 2

3

XTerra MS C18

0 10 20Time (min)

1 2

3

Zorbax Eclipse XDB-C18

0 20Time (min)

All peaks are adsorbed.

1

2

3

0 20Time (min)

020

0m

Volt

Atlantis dC18

1

2

3

100

0 10 20Time (min)

0.00

0.10

0.20

mVolt

1

2

3

All peaks are adsorbed.

200

100

Symmetry C18

0 20Time (min)

020

0m

AU

Inertsil ODS-3

1

2

3

mV

olt

This method was published by D.A. Barret, etal., J. Chromatogr. Sci. 34 (1996) 146. Determination of Aminopyridines is a very demanding analysis. Only Inertsil columns show very good peak shapes.

All peaks show tailing.

Peak 2 shows tailing. Peak 1 and 3 are absorbed.

Carboxylic Acid Test for Silanol Activity

Column Length: 4.6X250mm 5um Mobile Phase: 0.1% H3PO4(v/v) Flow Rate: 1.0 mL/min Col.Temp.: 40C Detector: UV210nm Sample Volume: 4uL Samples: 1) Formic Acid 0.1 %(v/v) 2) Acetic Acid 0.1 %(v/v)

0 2 4 6 8 10Time (min)

020

4060

mVo

lt

1

2

Inertsil ODS-SP

0 2 4 6 8 10Time (min)SunFire C18

All peaks are adsorbed.

1

2

XTerra MS C18

0 2 4 6 8 10Time (min)

All peaks are adsorbed.

1

2

Atlantis dC18

0 2 4 6 8 10Time (min)

020

4060

mVolt

1

2

0 2 4 6 8 10Time (min)

Zorbax Eclipse XDB-C18

1

2 Formic acid is adsorbed.

0 2 4 6 8Time (min)

0.00

0.02

0.04

0.06

mVolt

Symmetry C18

1

2

All peaks are adsorbed.

20

40

60

0 2 4 6 8 10Time (min)

020

40m

AU

Inertsil ODS-3

1

2 mV

olt

Inertness to Acidic Compounds Acidic compounds of low molecular weight like acetic acid or formic acid may be absorbed into the Reversed-phase packing materials. We have sometimes experienced the phenomena that a packing material showing a good performance and a good peak shape for pyridine gives tailing peaks for low molecular weight carboxylic acids. This might be caused by the procedure and the reagents used in the process of the chemical modification of the reversed-phase packing materials. Inertness for acidic compounds as well as basic compounds is important for reliable and reproducible reversed-phase selectivity.

0 10 20Time (min)

0.00

0.02

0.04

0.06

0.08

0.10

mVo

lt 1

2

Oxine-copper shows tailing.

20

40

60

mV

olt

80

100

Oxine-copper Teest for Silica Purity Column Length: 4.6X250mm 5um Eluent CH3OH / 20mM H3PO4 (10/90, w:w) Flow Rate: 1.0 mL/min Col.Temp.: 40C Detector:: UV254nm Sample Volume: 2.5uL Samples: 1) Oxine-copper 0.01 mg/mL 2) Caffeine 0.4 mg/mL

0 20Time (min)

020

4060

80m

Vol

t1

2

Inertsil ODS-SP

XTerra MS C18

0 10 20Time (min)

1

2

Atlantis dC18

0 20Time (min)

020

4060

80m

Volt

1

2

Oxine-copper shows tailing.

Zorbax Eclipse XDB-C18

0 20Time (min)

1

2

Oxine-copper shows tailing.

0 20Time (min)

1

2

Oxine-copper shows tailing.

SunFire C18

0 20Time (min)

020

4060

80m

AU

Inertsil ODS-3

1

2

mV

olt

Symmetry C18

Inertness to Chelating Compounds Chelating compounds have been widely used as pesticides, disinfectants, etc. Strong chelating compounds sometimes elutes from HPLC columns as tailing peaks or, in the worst case, they never elute from the column due to the strong chelation with metal impurities in the packing materials. The inertness for chelating compounds is inspected by chromatographing oxine-copper in addition to determining the level of metal impurities by ICP or AA spectrum analyses.