Column Category. Inertsil 3 Series. GL Sciences Inc.

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1 Column Category Inertsil 3 Series New Inertsil ODS Sprint New Inertsil ODS-3 4um Inertsil ODS-3 Inertsil ODS-3V Inertsil ODS-EP Inertsil ODS-P New Inertsil Sulpha C18 Inertsil C8-3 Inertsil Ph-3 New Inertsil Hilic Inertsil Diol Inertsil CN-3 Inertsil NH2 Inertsil AX Inertsil CX Inertsil SIL-100A

2 Column Category Inertsil 2 Series Inertsil ODS-2 Inertsil C8 Inertsil C4 Inertsil Ph Inertsil SIL-150A

3 Column Category Inertsil for Proteins and Peptides Inertsil WP300-C18 Inertsil WP300-C8 Inertsil WP300-C4 Inertsil WP300-Diol Inertsil Peptides-C18 Inertsil SIL-150A

4 Controlling Factors: Base Silica-Gel Chemical Bonding Selectivity of Packings Inertness of Packings Durability of Packings Column Performance

5 QC Tests for Lots: Category Method Description Base Silica-Gel SEM Sphericality and Surface Smoothness by Scanning Electron Microscopy. Ph Particle Size Uniformity by Nitrogen Adsorption Laser Ray Particle Analyzer. M Metals by Atomic Emission and ICP Ph Particle Size, Surface Area, Pore Diameter and Pore Volume. by Nitrogen Adsorption, Laser Ray Particle Analyzer Chemical Bonding C Carbon Loading by Elementary Analysis Si NMR Residual Silanol by Si-NMR Schro Residual Silanol by Chromatographic test Selectivity of Packings T Hydrophobicity Tanaka Test Mixture T Stereoselectivity Tanaka Test Mixture T Hydrogen-bonding capability Tanaka Test Mixture Inertness of Packings Py Inertness to Basic compounds Pyridine Am Inertness to Basic compounds Ethylaniline, Benzylamine, Aminopyridine Ac Inertness to Acidic compounds Formic Acid, Acetic Acid Cu Inertness to Complexes Oxine-Copper Durability of Packing D Durability to Low ph mobile phase at ph 2.0 D Durability to High ph mobile phase at ph 9.0

6 Sphericality and Surface Smoothness by Scanning Electron Microscopy

7 Particle Size, Surface Area, Pore Diameter and Pore Volume are Controled by Using Nitrogen Adsorption and Laser Ray Particle Analyzer. Pore Volume & Surface Area FSQ5-138 FSQ5-139 FSQ5-140 FSQ5-141 FSQ5-142 FSQ5-143 FSQ5-144 FSQ5-145 FSQ5-146 FSQ Pore Size Particle Size Surface Area (m2/g) Pore Volume (ul/g) Pore Size (A ) Particle Size (μm)

8 Packings with an Uniform Size and Smooth Surface Show Very Low Column Back Pressure Column back pressure PRODIGY 5 ODS-2 (5um) S/NO TSKgel ODS-80Ts QA (5um) C-No.W0004 YMC-Pack Pro C18 (5um) No (W) J'sphere ODS-M80 (4um) No (W) PRODIGY 5u ODS(3) 100A (5um) S/NO COSMOSIL 5C18-AR (5um) Manf.No.H11279 L-column ODS (5um) No ZORBAX SB-C18 (5um) Serial No.CL3106 Mightysil RP-18 (5um) Lot.No Inertsil ODS-3 (5um) TQ Pressure(kgf/c m2 ) Methanol concentration in H2O (%)

9 High Base Silica-Gel Purity Amount of Metals Are Inpected by Atomic Emission and ICP

10 High Base Silica-Gel Purity ( Amount of Metals Are Inpected by Atomic Emission and ICP)

11 Chemicaly Bonded Carbon Loading Are Inspected by Elementary Analysis

12 Chemical Bonding - Residual Silanol Groups are inspected by 29 Si-NMR Silanol Si-OH Slica gel structure Si-(O-Si) 4 Silanol Si-(OH) 2

13 Chemical Bonding - Residual Silanol Groups are inspected by 29 Si-NMR ODS groups C18H37- Si-O- remaining silanol

14 Chemical Bonding - Residual Silanol Groups are inspected by 29 Si-NMR End-capped groups (CH 3 ) 3 - Si-O-

15 29 Si-NMR spectra of Inertsil ODS-3 and commercially available ODS packing materials E nd-capping group (CH 3 ) 3 - Si-O- endcappi ng residual silanol Inertsil ODS-3 Commercially available ODS column A residual silanol residual silanol endcappi ng endcappi ng Commercially available ODS column B Commercially available ODS column C

16 -Residual Silanol Groupsby Chromatographic Tests (Pyridine/Phenol) Column : 250x4.6mmI.D. Eluent:CH 3 OH/H 2 O=30/70 Detection : UV at 254 nm Column Temperature : 40 Peak identification : 1) Pyridine 2) Phenol Time (min) Time (min) Time (min) Time (min)

17 GL Sciences is using Prof. Tanaka s selectivity test for QC k (n-amylbenzene) /k ( n-butylbenzene) : Hydrophobicity, α (CH2) k (triphenylene) /k ( o-terphenyl) : Stereo selectivity, α (t/o) k (caffeine ) / k (phenol) : Hydrogen-bonding capacity, α (c/p) According to Prof. Tanaka s group: The α(ch2) value is a better measure of the surface coverage than the carbon content. The α(t/o) values are determined by the functionality of silylating reagents and the surface coverage. The α(c/p) divided by α(ch2) illustrates the effect of hydrogen bonding by canceling the difference in the hydrophobic property.

18 Separation selectivity test for ODS columns k (n-amylbenzene) / k (n-butylbenzene) : the hydrophobicity of the ODS k (Triphenylene) / k (o-terphenyl) : the stereo-selectivity of the ODS k (Caffeine) / k (Phenol) : the quantity of unreacted silanol groups in the ODS 1)Uracil 2) Caffeine O CH 3 H3C N N N O N 7) Triphenylene H N O O NH CH3 3) Phenol4) Butylbenzne 5)o-Terphenyl 6) Amylbenzene OH ( CH 2 ) 3 CH 3 ( CH 2 ) 4 C H Time (min)

19 -Inertness to Basic Compounds- Pyridine/Phenol

20 -Inertness to Acidic Compounds- Formic acid/ Acetic acid

21 -Inertness to Chelating Compounds- Oxine Copper

22 -Durability to Acidic Condition - 0.1% Trifluoroacetic acid in H2O ph2.0

23 -Durability to Alkaline Condition CH3OH/0.01M Na2HPO4(pH9)=40/60

24 -Column Performance Measurement- Calucalation N : Number of theoretical plates Rt : Retention time W/2 : Half-height width 150x4.6mmI.D. 13,000 more 250x4.6mmI.D. 22,000 more S : peak asymmetry 5%peak height 1<leading,fronting 1>Tailing Peak asymmetry 0.95 ~ 1.10 a w/2 b

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