Company Profile of GL Sciences Inc.

H PLC Sem ina r Introducing Inertsil HPLC Columns How to select HPLC best eluent How to select best column packing UHPLC and FastLC using u particles How to maintain best performance

Company Profile of GL Sciences Inc. M ain office 0 branch offices factories R& D center GL U SA Total 470 Employees M ain Office (Tokyo), 70 Em ployees Technical Headquarters (R& D, Customer Support) 00 Employees

M ain Products INERT FAMILY IN ERTSIL Colum ns f o r H PLC IN ERTCAP Colum ns for GC IN ERTSEP SPE Cartridge for Sample Preparation

Introduction of Inertsil Columns Features and Benefits Inertsil Quality Control Quality comparison among leading companies

Inertsil Series H PLC Colum n High Purity, Spherical Silica-gel High Theoretical Plates Highly Uniform ed Particle Size Distribution Lo w Back Pressu re Highly Stable Chem ical Bonding High Durability Extreme End capping Treatment Lower Adsorption Rigorous Inspection Tests (M ore than 0 Tests) Batch- to-batch Reproducibility The biggest feature is W e synthesize the material of the silica- gel and conduct chem ical bonding, inspection and the packing of the column by ourselves. H ighly Stable Excellent Support Continue to Evolve

H ighly Pure Base Silica- gel Completely spherical Smooth surface Synthesized from pure ingredients Focused only for chromatography W hole manufacturing process within GLS Inertsil Silica- gel One com petitor's Silica- gel

H ighly Pure Base Silica- gel Amount of M etals are inspected by Atom ic Em ission and ICP. M anufacturer's Validation C ertificate Inertsil OD S-3V 5u Lot.N o. VQ5 Atomic Emission ppm Specification R esults Fe < 0 5 Na < 0 4 Al < 0 Ti < < 0.5

Influences of M etals

Durability of Inertsil Durability Test: Introducing 500 volumes (times) of 0.% TFA against the column volume. Upper Chromatogram: Before Lower Chromatogram: After Phenol Pyridine 0 4 6 8 0 4 6 Time (min) Retention Tim e Volatility (%) 00 90 80 70 60 0 Comparison of Durability Test with other manufacturer s column Inertsil ODS- 3 00 00 300 400 500 600 Immersed time of TFA (hrs) Column A Column B Column C Column D Column E Column F Column G 0 4 6 8 0 4 6 Time (min)

Adsorption of analyte Basic Compounds: Acidic Compounds: Caused by residual hydroxyl group (silanol) on silica surface. Caused by reaction reagent. These reagent includes amine group for the purpose of resistance to basic compounds adsorption. Chelating Compounds: Caused by metal- ions on silica surface. Adsorption mechanism of basic compounds C8 C8 C8 C8 C8 C8 OH OH OH OH OH OH OH OH OH Si Si O Si Si O O O O Silica ODS Si O OH OH Si Si O O O Silica OH OH Si O C Si O End - Capping C OH Si Si O O O Silica O C Si C -Si-O Acidic H hydroxyl group

Adsorption comparison Column : 50 4.6 mm I.D. Eluent : Acetonitrile / Water=30 / 70 Detection : UV54nm Column Temp. : 40 ) Pyridine ) Phenol of basic compound Inertsil ODS- 3 Company A Company B Company C

Adsorption comparison Column : 50 4.6 mm I.D. Eluent : M ethanol / Water=30 / 70 Detection : UV54nm Column Temp. : 40 ) Pyridine ) Phenol of basic compound Inertsil ODS- 3 Company A Company B Company C

Inertsil Quality Control Scanning Electron M icroscopy Sphericity and surface smoothness of Silica- gel Atom ic Em ission and Inductivity Coupled Plasm a Trace metals N itrogen Adsorption & Laser Ray Particle Analyzer Particle size and its uniform ity Surface area Pore diameter and pore volume 9 Si- NMR Residual Silanol Chromatographic Tests Inertness (Basic, A cid ic, Co m p lexes) Durability (Low ph, H igh ph ) Selectivity (Hydrophobicity, Stereoselectivity, Hydrogen- bonding capacity)

Inertsil Quality Control Evaluation Test of Inertness to Basic compounds 0 4 6 8 0 4 6 Time (min) 0 4 6 8 0 4 6 Time (min) 0 4 6 8 0 4 6 Time (min) LOT# VQ 5-43 LOT# VQ5-44 LOT# VQ5-45

Inertsil Quality Control Evaluation Test of Inertness to Acidic compounds Column : Inertsil ODS- 3V 50 x 4.6 m m I.D. Eluent : 0.% H 3 PO 4 Detector : UV 0nm Col. Tem p.: 40 Sam ple : ) Form ic acid ) Acetic acid 0 4 6 0 4 6 0 4 6 Tim e (m in ) LOT# VQ 5-43 LOT# VQ5-44 LOT# VQ5-45

Inertsil Quality Control Evaluation Test of Inertness to Complex compounds Column : Inertsil ODS- 3V 50 x 4.6 m m I.D. Eluent : CH 3 CN / 0.% H 3 PO 4 = 0 / 90 Detector : UV 40nm Col. Tem p.: 40 Sam ple : ) Oxine-Copper ) Caffeine 0 4 6 8 0 4 6 0 4 6 8 0 4 6 0 4 6 8 0 4 6 LOT# VQ 5-43 LOT# VQ5-44 LOT# VQ5-45

Inertsil Quality Control Evaluation Test of Selectivity. 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 hydrogen-bonding capacity of the ODS 7 ) Uracil ) Caffeine 3) Phenol 3 4 5 6 4) Butylbenzne 5) o- Terphenyl 6) Amylbenzene 7) Triphenylene 0 0 0 Time (min)

Inertsil Quality Control Evaluation Test of Selectivity. Column : Inertsil ODS- 3V 50 x 4.6 mm I.D. Eluent : CH 3 OH / HO = 80 / 0 Detector : UV 54nm Col. Tem p.: 40 A:Hydrophobicity of the ODS B:Stereoselectivity of the ODS C:Hydrogen- bonding capacity of the ODS A=.47 B=.3 C= 0.49 A=.47 B=.30 C= 0.5 A=.47 B=.30 C= 0.5 0 0 Ti m e (m i n ) 0 0 Ti m e (m i n ) 0 0 Ti m e (m i n ) LOT# VQ 5-68 LOT# VQ5-03 LOT# VQ 5-06

Inertsil Quality Control Evaluation Test of residual silanol by 9 Si- NMR. Isolated silanol Si- OH Gem inal silanol Si- (OH) Silica gel Si- (OSi) 4 - O - Si- (CH 3 ) 3 - O - Si- C 8 H 37 Residual silanol Octadecyl Bonding End- Capping

Inertsil Quality Control Evaluation Test of residual silanol by 9 Si- NMR. LOT# V Q 5-68 LOT# V Q 5-03 LOT# V Q 5-06

Si- N M R spectrum of commercially ODS Residual silanol Residual silanol OD S A OD S B Residual silanol Residual silanol OD S C OD S D

Quality Comparison Among Leading Companies High Quality at Reasonable Prices by 30 Year s Experience Execution of whole manufacturing process with reliable and consistent high technologies is essential for the reproducibility of high colum n efficiency resulting in best custom er satisfaction. This is also the only way to reduce the cost which enable us to supply our customer with HPLC colum ns at a reasonable price. Execution of whole manufacturing process Synthesis of base silica- gel from pure ingredients Bonding functional groups on silica- gel m edia Packing silica gel m edia into HPLC column hardware

Quality Com parison ~Selectivity~ Colum n Size:4.6 50 mm Eluent :CH3OH / HO = 80 / 0 Flow Rate :.0 m L/ m in Col. Tem p. :40 Detector :UV54 nm Sample Size :5 ul Samples :)Uracil 0.005 mg/ml )Caffeine 0.04 mg/ml 3)Phenol 0.08 mg/ml 4)n-Butylbenzene. mg/ml 5)o-Terphenyl 0.04 mg/ml 6)n-Amylbenzene.37 mg/ml 7)Triphenylene 0.04 mg/ml 3 4 0 0 0 30 Inertsil ODS- 3 5 6 7 0 4 6 8 0 4 6 8 Column A 0 0 0 30 Column B 0 0 0 Column C

Quality Com parison ~Selectivity~ 0 0 0 30 Inertsil ODS- 3 0 0 0 Column D 0 0 0 30 Column E 0 0 0 30 0 4 6 8 0 0 0 0 Column F Column G Column H

Quality Com parison ~Selectivity~ 0 0 0 30 0 0 0 30 0 0 0 30 Inertsil ODS- 3 Column I Column J 0 0 0 30 0 0 0 30 0 0 0 30 Column K Column L Column M

Quality Com parison ~Selectivity~ 0 0 0 30 0 0 0 30 0 0 0 Inertsil ODS- 3 Column N Column O 0 0 0 0 0 0 0 0 0 Column P Column Q Column R

Quality Com parison ~Selectivity~ 0 0 0 30 0 0 0 30 0 0 0 Time (min) Inertsil ODS- 3 Column S Column T 0 0 0 0 0 0 0 0 0 Column U Column V Column W

Quality Com parison ~Selectivity~ 0 0 0 30 0 0 0 0 0 0 30 Inertsil ODS- 3 Column X Column Y 0 0 0 30 Column Z

Quality Com parison ~Pyridine~ Colum n Size:4.6 50 mm Eluent :CH3OH / HO = 30 / 70 Flow Rate :.0 m L/ m in Col. Tem p. :40 Detector :UV54 nm Sam ple Size :4 ul Samples :)Pyridine 0.09 mg/ml )Phenol 0.4 mg/ml 0 4 6 8 0 4 6 8 Inertsil ODS- 3 0 4 6 8 0 0 0 0 0 4 6 8 0 4 Column A Column B Column C

Quality Com parison ~Pyridine~ 0 0 0 0 4 6 8 0 0 0 0 Inertsil ODS- 3 Column D Column E 0 0 0 0 0 0 0 0 0 Column F Column G Column H

Quality Com parison ~Pyridine~ 0 0 0 0 4 6 8 0 0 4 6 8 0 4 6 Inertsil ODS- 3 Column I Column J 0 4 6 8 0 4 0 4 6 8 0 4 6 0 4 6 8 0 4 6 Column K Column L Column M

Quality Com parison ~Pyridine~ 0 0 0 0 4 6 8 0 4 0 4 6 8 0 4 6 Inertsil ODS- 3 Column N Column O 0 4 6 8 0 4 6 0 4 6 8 0 4 6 0 4 6 8 0 4 6 Column P Column Q Column R

Quality Com parison ~Pyridine~ 0 0 0 0 4 6 8 0 4 6 0 4 6 8 0 4 6 Inertsil ODS- 3 Column S Column T 0 4 6 8 0 4 6 0 4 6 8 0 4 6 0 4 6 8 0 Column U Column V Column W

Quality Com parison ~Pyridine~ 0 0 0 0 4 6 8 0 4 6 0 4 6 8 0 4 Inertsil ODS- 3 Column X Column Y 0 4 6 8 0 4 6 Column Z

Quality Com parison ~Am inopyridine~ Colum n Size :4.6 50 mm Eluent :CH3OH / 0mM Phosphate Buffer (ph7.6) = 0 / 90 Flow Rate :.0 m L/ m in Col. Tem p. :40 Detector :UV54 nm Sample Size : ul Samples :) 4- Am inopyridine 0. m g/ m L ) 3- Am inopyridine.0 m g/ m L 3) - Am inopyridine.0 m g/ m L 3 0 4 6 8 0 Inertsil ODS- 3 0 4 6 8 0 0 0 0 4 6 8 0 4 Column A Column B Column C

Quality Com parison ~Am inopyridine~ 0 0 0 0 4 6 8 0 0 0 0 Inertsil ODS- 3 Column D Column E 0 0 0 0 0 0 0 0 Column F Column G Column H

Quality Com parison ~Am inopyridine~ 0 0 0 0 0 0 0 4 6 8 0 Inertsil ODS- 3 Column I Column J 0 4 6 8 0 0 4 6 8 0 4 0 4 6 8 0 4 Column K Column L Column M

Quality Com parison ~Am inopyridine~ 0 0 0 0 4 6 8 0 4 6 0 4 6 8 0 4 Inertsil ODS- 3 Column N Column O 0 4 6 8 0 4 6 8 0 4 6 8 0 4 6 0 0 0 Column P Column Q Column R

Quality Com parison ~Am inopyridine~ 0 0 0 0 4 6 8 0 4 6 0 4 6 8 0 4 6 Inertsil ODS- 3 Column S Column T 0 4 6 8 0 4 6 8 0 4 6 8 0 0 4 6 8 0 Column U Column V Column W

Quality Com parison ~Am inopyridine~ 0 0 0 0 4 6 8 0 0 4 6 8 0 4 6 Time (min) Inertsil ODS- 3 Column X Column Y 0 4 6 8 0 4 6 8 Column Z

Quality Com parison ~Acids~ Colum n Size :4.6 50 mm Eluent :0.% H3PO4(v/v) Flow Rate :.0 m L/ m in Col. Tem p. :40 Detector :UV0 nm Sample Size :4 ul Samples :) Form ic Acid 0. %(v/ v) ) Acetic Acid 0. %(v/ v) 0 4 6 8 Inertsil ODS- 3 0 4 6 0 4 6 0 4 6 Column A Column B Column C

Quality Com parison ~Acids~ 0 4 6 8 0 4 6 0 4 6 8 Inertsil ODS- 3 Column D Column E 0 4 6 8 0 4 6 8 0 4 6 8 Column F Column G Column H

Quality Com parison ~Acids~ 0 4 6 8 0 4 6 8 0 4 6 8 Inertsil ODS- 3 Column I Column J 0 4 6 8 0 4 6 8 0 4 6 8 Column K Column L Column M

Quality Com parison ~Acids~ 0 4 6 8 0 4 6 8 0 4 6 8 Inertsil ODS- 3 Column N Column O 0 4 6 8 0 4 6 8 0 4 6 8 Column P Column Q Column R

Quality Com parison ~Acids~ 0 4 6 8 0 4 6 8 0 0 4 6 8 Inertsil ODS- 3 Column S Column T 0 4 6 8 0 4 6 8 0 4 6 8 Column U Column V Column W

Quality Com parison ~Acids~ 0 4 6 8 0 4 6 8 0 4 6 8 Inertsil ODS- 3 Column X Column Y 0 4 6 8 Column Z

Quality Com parison ~Oxine- copper~ Colum n Size :4.6 50 mm Eluent :CH3CN / 0mM H3PO4 = 0 / 90 Flow Rate :.0 m L/ m in Col. Tem p. :40 Detector :UV54 nm Sam ple Size :.5 ul Samples :) O xine- Copper 0.0 mg/ml ) Caffeine 0.4 mg/ml 0 4 6 8 0 4 6 Inertsil ODS- 3 0 4 6 8 0 0 4 6 8 0 0 4 6 8 0 Column A Column B Column C

Quality Com parison ~Oxine- copper~ 0 4 6 8 0 4 6 0 4 6 8 0 0 4 6 8 0 Inertsil ODS- 3 Column D Column E 0 4 6 8 0 0 4 6 8 0 4 6 0 0 0 Column F Column G Column H

Quality Com parison ~Oxine- copper~ 0 4 6 8 0 4 6 0 4 6 8 0 4 0 4 6 8 0 Inertsil ODS- 3 Column I Column J 0 4 6 8 0 0 4 6 8 0 0 4 6 8 0 Column K Column L Column M

Quality Com parison ~Oxine- copper~ 0 4 6 8 0 4 6 0 4 6 8 0 0 4 6 8 0 Inertsil ODS- 3 Column N Column O 0 4 6 8 0 4 6 0 4 6 8 0 4 0 4 6 8 0 4 Column P Column Q Column R

Quality Com parison ~Oxine- copper~ 0 4 6 8 0 4 6 0 4 6 8 0 0 4 6 8 0 4 6 Inertsil ODS- 3 Column S Column T 0 4 6 8 0 0 4 6 8 0 0 4 6 8 0 Column U Column V Column W

Quality Com parison ~Oxine- copper~ 0 4 6 8 0 4 6 0 4 6 8 0 0 4 6 8 0 4 6 Inertsil ODS- 3 Column X Column Y 0 4 6 8 0 Column Z

How to select HPLC eluent Select organic solvent for RP- H PLC Reduce running costs using M ethanol Improving the results of chromatogram

Organic solvent for RP- H PLC Almost all customers often use acetonitrile. If they can not get good separation... Change column Use buffer or ion- pair Change ph Isocratic Gradient to obtain satisfactory result s... Takes a long time N eed skills and knowledge Difficult to validate W hy are people using acetonitrile?

Organic solvent for RP- H PLC Same instrument, column and sample were used. Only changed the type of an organic solvent. 7% CH3CN 80% CH3OH 56% THF Phenol Caffeine o-terphenyl Butylbenzene Triphenylene Am ylbenzene Phenol Caffeine Butylbenzene Am ylbenzene o-terphenyl Triphenylene Phenol Caffeine Triphenylene o-terphenyl Butylbenzene Am ylbenzene 0 0 0 0 0 0 0 0

Obtaining better results by changing the organic solvent Column : Inertsil ODS- 3 5μm 50 4.6 mm I.D. Eluent : Organic / 0.% H 3 PO 4 Flow Rate :.0 m L/ m in Detector : UV 84nm Col. Tem p. : 40 4,5 Sample : ) Sulfamerazine ) Furazolidone 3) Oxolinic acid 4) Sulfadim ethoxine 5) Sulfaquinoxaline 6) N alidixic acid 3 4 3 4 5 5 6 6 3 6 0 0 0 30 40 Time (min) 0% CH 3 CN 0 0 0 Time (min) 30% CH 3 CN 0 0 0 Time (min) 40% CH 3 OH

UV spectrum of organic solvents M ethanol Acetonitrile Tetrahydrofuran absorption.0 0.5 A B A :H PLC- Grade B:Special- Grade First- Grade absorption.0 0.5 A B C A :H PLC- Grade B:Special- Grade C:First- Grade absorption 4.0.0 A B A:H PLC- Grade B:Special- Grade First- Grade 0 0 0 00 50 300 00 50 300 00 50 300 wavelength(nm) wavelength(nm) wavelength(nm) UV spectrum of HPLC- grade and special- grade are almost in the same level. W hen you use UV- detector, you can obtain satisfactory results even if you use special- grade methanol. UV spectrum of each grade were different. You must be careful when you use low wavelength or highsensitive analysis or gradient analysis. Tetrahydrofuran oxidizes very easily. Special- grade or first- grade THF contains oxidation inhibitor ( 3,5- Di- tert- butyl p- hydroxytoluene). If you use HPLC- grade THF, you must use it as soon as possible.

Reduce Running Costs Cost (in Japanese Yen) Solvent S-Grade HPLC-Grade Pressure Toxicity Acetonitrile, / L 3,833 / L Low High Methanol 36 / L 933 / L High Low M ethanol.5 pressure ratio.0 0.5 Acetonitrile as.0 when W ater 00% 0 0 40 60 80 00 solvents ratio in W ater %

Acetonitrile vs.. M ethanol Ability of Separation Eq ual (depends on the character of analyte) Peak Shape Almost equal (depends on the column performance) Running cost M ethanol is better Toxicity M ethanol is better W hy are people not using M ethanol? Because the Back Pressure is too high!!

Reduce Running Costs W hen selecting a low- p ressure co lum n Easy to reduce running costs. Can keep yourself healthy. Pressure(kgf/cm) 00 80 60 40 0 00 80 60 40 Back pressure of com m ercially available colum ns Inertsil ODS- 3 0 0 40 60 80 00 Methanol ratio(%) Conditions Column Size : 50 4.6 mm I.D. Flow Rate :.0 m L/ m in Col. Tem p. : 40

W hat makes the column back pressure different? It depends on the size of the silica gel uniform ity of the particle size particle shape 5μm Uniform ed Spherical Back pressure = Low 3μm Uniform ed Spherical Back pressure = H igh 5μm Not uniformed Spherical Back pressure= H igh 5μm Uniform ed Irregular Back pressure= H igh

W hat makes the column back pressure different? Com petitor Y s silica gel Reasons why Inertsil can offer the lowest back pressure column in the world! Packing - Low ba ck pressure - High theoretical plates - Lot- to- Lot reproducibility Inertsil Packing - H igh ba ck pressure - Low theoretical plates - Poor Lot- to- Lot reproducibility

W hy is the particle size distribution different? Every commercial columns have a particle size distribution. But it s distribution width are quite different. Com m ercial silica- gel Inertsil silica- gel 35 60 30 50 Percentage(%) 5 0 5 0 Percentage(%) 40 30 0 5 0 0 3 4 5 6 7 8 0 3 4 5 6 7 8 Particle Size (μm) Particle Size (μm) Almost all column manufactures do not synthesize their silica- gel. They purchase commercial silica- gel from silica- gel specialty manufacture. GL Sciences synthesizes the original silica- gel focused for HPLC columns. As a result, the particle size distribution width is highly controlled.

Changing another factor of eluent to improve Separation Peak shape Throughput Buffer concentration Ion- pair reagent concentration Isocratic Gradient Column temperature

Improving Peak Shape Effect of Buffer concentration Eluent : CH 3 CN / Buffer = / 88 Sample : ) Thiam in ) Riboflavin mm N ah PO 4 0mM NaH PO 4 00mM NaH PO 4 It is efficient to use high concentration buffer to improve the peak shape. H owever, it will cause the colum n to deteriorate as well. W hen analyzing a strong adsorptive compound, it is important to select a column that has high inertness but durability too.

Improving Separation Effect of ion- pair reagent concentration 3 4 5 Ion-pair reagent: Heptafluoro butyric acid 3 45 3 45 0.30 % 0.5 % Sam ple.tolazoline.prazo sin 3.Yohim bin 4.Phentolamine 5.Labetalol 0.0 %

Improving Throughput Effect of Gradient elution CH 3CN / H O = 50 / 50 CH 3 CN / H O = 40 / 60 Gradient

Improving Throughput Effect of Column Temperature Inertsil ODS- 3 3μm ; 00 4.0 m m I.D. ; 70% CH 3 CN ;.5 ml/min 0 40 60 80 0 4 6 0 4 6 0 4 6 0 4 6 Press:7M pa N(No.5):7985 Press:M pa N(No.5):3643 Press:0M pa N(No.5):349 Press:8M pa N(No.5):50

How to select Column packing Difference and features of ODS Colum n line-up except ODS Reduce running costs by Scale- down Im prove Rs using sm all particle size colum n

Differences and features of ODS The first choice column should be an Octadecyl groups bonded (ODS) column. There are various ODS columns that offers different characteristics? Base Silica Physical Properties and Chem ical M odification of Inertsil ODS Series Silica-gel Chemical Treatment Column Surface Carbon Size Pore Size Purity Area Loading ODS Type End-Cap Inertsil ODS-3 3,4,5μm 450 m /g 00A 99.999% 5% Monomeric Well Inertsil ODS-P 5μm 450 m /g 00A 99.999% 9% Polymeric None Inertsil ODS-EP 5μm 450 m /g 00A 99.999% 9% Embedded None Inertsil ODS-SP 5μm 450 m /g 00A 99.999% 8.5% Monomeric Well Inertsil ODS- 5μm 30 m /g 50A 99.999% 8.5% Intermediate Well Inertsil ODS-80A 5μm 450 m /g 80A 99.99% 7.5% Monomeric Well

M onomeric ODS vs. Polym eric ODS Planar compounds M onomeric ODS Silica- gel surface C8 group Planar compounds Non- planar compounds Polym eric ODS

M olecular Recognition of ODS ) Benzo[a]pyrene (BaP) ) Tetrabenzonaphthalene (TBN ) Analytical conditions Column : 50 x 4.6 mm I.D. Eluent : CH 3 CN / H O = 85 / 5 Flow Rate :.0 m L/ m in Detector : UV 54nm Col. Tem p.: 30 Selectivity(α ) α =k TBN / k Bap 3) Phenanthro[3,4- c]phenanthrene (PhPh) α.7 monomeric.7>α>.0 intermediate α.0 polym eric

M olecular Recognition of ODS Inertsil ODS- P Inertsil ODS- Inertsil ODS- 3 α = 0.7 α =.65 α =.5 TBN PhPh BaP + PhPh TBN BaP PhPh TBN BaP 0 0 0 Time (min) 0 4 6 8 0 4 6 8 Time (min) 0 0 0 Time (min) Polym eric Intermediate M onomeric

Application of PAH s. Naphthalene. Acenaphthylene 3. Biphenyl 4. - M ethylnaphthalene 5. - M ethylnaphthalene 6. Acenaphthene 7. Fluorene 8. Phenanthrene 9. Anthracene 0. Fluoranthene. Pyren e. Ben zo - [a]- anthracene 3. Chrycene 4. Ben zo - [ j]- fluoranthene 5. Ben zo - [e]- pyrene 6. Ben zo - [b]- fluoranthene 7. Ben zo - [k]- fluoranthene 8. Ben zo - [a]- pyrene 9. D ib en zo - [a,h ]- anthracene 0. Ben zo - [g,h,i]- perylene. Indeno- [,,3- cd]pyrene Analytical Conditions Colum n Size : 50 4.6 m m I.D. 5μm Eluent : A = H O ; B= CH 3O H A/B= 0/80(7min Hold) ---0min --- 0/00 Flo w rat e : ml/min Detector : UV 54nm Col. Temp. : 40 Sam ple size : 5 μl (each 00ng / ml) 3 7 8 9 0 3 5 4 6 7 8 9 0 45 6 0 0 0 30 Time (min) 0 0 0 30 Time (min) Inertsil ODS- 3 (monomeric) Inertsil ODS- P (polym eric)

Application of Vitam in D & D3 Analytical Conditions Colum n size : 50mm x 4.6mm I.D Eluent : Acetonitrile / M ethanol (75:5) Flo w rat e :.0m L/ m in Te m p. : 30 Detection : UV 80nm Sam p les :. Vitamin D (450ng). Vitamin D3 (50ng) H O H C C H 3 H 3 C Vitam in D C H 3 C H 3 C H 3 H O H C H 3 C H 3 C Vitam in D3 C H 3 C H 3 0 4 6 8 0 4 Inertsil ODS- 3 (monomeric) 0 4 6 8 0 4 Inertsil ODS- P (polym eric)

Application of Glycyrrhizin Analytical methods written in the Japanese Pharmacopoeia Column I.D.: Column length: Column packing: Flow Rate: 4.0~6.0mm 5~5cm ODS (The elution pattern shall be from Glycyrrhizin to p - Hydroxybenzoate propyl, which is an internal standard. Each peak shall be completely separated.) Adjust it until the retention tim e of Glycyrrhizin is about 0m in. Column size : 50 x 4.6 mm I.D Eluent : H O / Acetic acid / CH 3 CN =560 / 40 / 400 Flow rate : ODS-3 0.8mL/min HO ODS-P 0.4mL/min HO Col. Tem p. : 5 Detector : UV 54nm Sample :. Glycyrrhizin (750ng). p- hydroxy benzoic acid propyl (50ng) CO O H O H O O HO H O CH 3 O O H O H CH 3 CO O H H CH 3 CH 3 CH 3 CH 3 CH 3 CO O H Structural formula of Glycyrrhizin

Application of Glycyrrhizin Column size : 50 x 4.6 mm I.D Eluent : H O / Acetic acid / CH 3 CN = 560 / 40 / 400 Col. Tem p. : 5 Detector : UV 54nm Sample : ) Glycyrrhizin (750ng) ) p- Hydroxy benzoic acid propyl (50ng) Flow rate = 0.4 ml/min Flow rate = 0.8 ml/min 0 4 6 8 0 4 6 Time (min) Inertsil ODS- P (Polym eric) 0 4 6 8 0 4 6 Time (min) Inertsil ODS- 3 (M onomeric)

Effect of Silanol Good End capping No End capping Treatment Polym eric ODS 0 4 6 8 0 4 6 8 Time (min) 0 4 6 8 0 4 6 8 Time (min) Good End capping M onomeric ODS Poor End capping No End capping Treatment 0 4 6 8 0 Time (min) 0 4 6 8 0 Time (min) 0 4 6 8 0 Time (min)

Selectivity of Embedded ODS Analytical Conditions Column : 50 4.6 mm I.D. Eluent : CH 3 CN / H O = 70 / 30 Flow Rate :.0 ml/min Detector : UV 54nm Sam ple : ) Ethylbenzene ) N aphthalene 3 ) n- Propylbenzene 4 ) n- Butylbenzene 5 ) Anthracene Inertsil ODS- EP =Embedded 0 4 6 8 0 3 4 5 Inertsil ODS- 3 3 4 3 4 Inertsil C8-3 5 5 0 4 6 8 0 0 4 6 8 0

79 GL Sciences Application of Antibacterial Column : 50 4.6 mm I.D. Eluent : Organic / 0.% H 3 PO 4 Flow Rate :.0 m L/ m in Detector : UV 84nm Col. Tem p. : 40 4,5 Sample : ) Sulfamerazine ) Furazolidone 3) Oxolinic acid 4) Sulfadim ethoxine 5) Sulfaquinoxaline 6) N alidixic acid 0% CH 3 CN 3 30% CH 3 CN 0% CH 3 CN 3 4 3 5 6 6 4 6 5 0 0 0 30 40 Time (min) 0 0 0 Time (min) 0 0 0 Time (min) Inertsil ODS- 3 Inertsil ODS- EP

80 GL Sciences W eakness of Embedded ODS Ordinary embedded ODS are bonded with amine polar groups. Therefore, acidic com pounds cannot be analyzed since it will be adsorbed to the embedded amine groups. However, our Inertsil ODS- EP does not use an amine polar group. As a result, it can also be used for analyzing acidic compounds. Form ic acid Acetic acid Form ic acid + Acetic acid Acetic acid Form ic acid Acetic acid Form ic acid 0 4 Time (min) Inertsil ODS- EP 0 4 Time (min) Column A 0 4 Time (min) Column B 0 4 Time (min) Column C

Low Carb on Loading O D S Features of Low Carbon Loading ODS Retaining hydrophilic compounds without excessive retention of hydrophobic compounds. Elut es sharp p eaks. Compatible with 00% aqueous eluents. 3 Inertsil ODS- SP =Low Carbon Loading 4 Analytical Conditions Column Size : 50x4.6 mm I.D. Eluent : A) Acetonitrile B) 0mM NaHPO4 (ph3.0) (A/B) = (70/30) Flow rate :.0 m L/ m in Col. Tem p. : 40 Detector : UV 80 nm Injection Vol. : 5μL ) Phenol (50ppm) ) Bisphenol- A (30ppm) 3) 4- Octylphenol (00ppm) 4) 4- Nonylphenol (00ppm) (in CH 3 OH) 0 0 0 30 Time (min) Inertsil ODS- 3 =General ODS 0 0 0 30 Time (min) 3 4

Features of Low Carbon Loading ODS Retention for hydrophilic compounds are about the same as an ordinary ODS! Analytical Conditions Column Size : 50x4.6 mm I.D. Eluent : A) M ethanol B) 0mM NaHPO4 (A/B) = (0/90)-30min-(50/50) Flow Rate :.0 m L/ m in Col. Tem p. : 40 Detector : UV at 80 nm Injection Vol. : 0μL (each mg/ L) ) Gallocatechin (GC) ) Epigallocatechin (EGC) 3) Catechin (C) 4) Epigallocatechin Gallate (EGCg) 5) Epicatechin (EC) 6) Gallocatechin Gallate (GCg) 7) Epicatechin Gallate (ECg) 8) Catechin Gallate (Cg) Inertsil ODS- SP =Low Carbon Loading Inertsil ODS- 3 =General ODS 3 4 5 6 7 8 0 0 0 Time (min)

Advantages of Low Carbon Loading ODS W hen increasing the concentration of an organic solvent to reduce the analysis time... Possibility in deteriorating the separation. Im purity Im purity Target compound Target compound Target compound 3 Im purity Enlarged view Target compound 0 0 0 Time (min) 0 0 0 Time (min) 30% Organic 40% Organic

Advantages of Low Carbon Loading ODS Possible to reduce the analysis time when using a Low Carbon Loading ODS without changing the eluent conditions. Of course, the separation will not be deteriorated. Im purity Target compound Target compound Target compound 3 Im purity Target compound 0 0 0 Time (min) Inertsil ODS- 3 =General ODS 0 0 0 Time (min) Inertsil ODS- SP =Low Carbon Loading

Advantages of Low Carbon Loading ODS Analytical Conditions Column: 5um, 50 x 4.6mmI.D. M obile Phase: CH 3CN / H O = 5/95 (5min hold) (7min) 00/0 (5min hold), v/v Flow Rate:.0mL/min Tem p.: 40C Detection: UV5nm ) M ethamidophos ) Acephate A highly uniformed low carbon loading ODS enables shorter equilibration time for gradient analyses and can be used for high- throughputs. Of course, the time for purging and cleaning the column can be reduced as well in an isocratic. Fluctuations in retention time are shown on Column C and Column D. On the other hand, Inertsil ODS- Sprint shows a complete equilibration after 0mins with stable rententivity.

Application of Food Colorants 3 4 5 6 7 Analytical Condition 0 0 0 30 40 Time (min) 8 9 Column : Inertsil ODS- SP 5um 50X4.6 mm I.D. M obile Phase: A) Acetonitrile B) 0mM Phosphate buffer (ph6.9) (A/B) = (0/90)-50min-(35/65) Flow Rate :.0 m L/ m in Column Temp.: 40C Detector : UV at 70nm Sam ple Vol.: 0uL ) Acid Yellow N o.4 7.6mg/L ) Acid Red No. 3.8mg/L 3) Acid Blue N o. 7.6mg/L 4) Acid Red No.0 3.8mg/L 5) Acid Yellow N o.5 5.3mg/L 6) Uranine 3.8mg/L 0 3 4 5 7) Acid Red No.40 5.3mg/L 8) Ponceau R 7.6mg/L 9) Ponceau SX 5.3mg/L 0) Orange 5.3mg/L ) Acid Green N o.3 3.0mg/L ) Acid Blue N o. 3.0mg/L 3) Acid Red No.3 5.3mg/L 4) Azul Blue VX 3.0mg/L 5) Orange 7.6mg/L 6) Acid Red No.06 3.0mg/L

Comparison among Leading Companies Inertsil ODS- Sprint This m ethod is em ployed 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 m easured. Iner t sil columns show very good peak shapes for such significant basic compound. Comparison of Tailing Factor Inertsil ODS-3 Inertsil ODS-Sprint Sunfire Inertsil ODS-3 4um Symmetry Xterra Ec lips e 0 0.5.5 Colum n Tailing Factor Inerts il O D S -3.04 Inertsil ODS-Sprint.06 Sunfire.06 Inertsil ODS-3 4um.07 Symmetry. Xterra.3 E c lips e.8 Tailing Factor

Comparison among Leading Companies 0 0 0 mau 0 0 40 60 80 m Volt Inertsil ODS-Sprint 3 4 5 6 Analytical Condition Eluent: 0mM Phosphoric Acid Buffer (ph7.0) / M eoh = 0/80 (v/v) Flow:.0m L/ m in Detect: UV54nm Oven: 40C Sample: 0uL- inject m Volt mau 0 0 40 60 80 0 0 0 3 Zorbax Eclipse XDB- C8 4 5 6.Uracil ug/ml.propranolol 99ug/mL 3.n-Butyl paraben ug/ml 4.N aphthalene 37ug/mL 5.Acenaphthene 50ug/mL 6.Am itryptyline 30ug/mL

Quality Comparison Among Leading Companies Pyridine Test for Base Deactivation m Volt 0 0 40 0 0 0 Inertsil ODS- SP m Volt mau 0 0 40 0 0 0 Inertsil ODS- 3 40 m Volt 0.00 0.0 0.04 0 0 0 0 Sym m etry C8 Pyridine tailing due to poor end- capping. 0 0 0 SunFire C8 Pyridine tailing due to poor end-capping. m Volt 0 0 40 Pyridine tailing due to poor end- capping. Column Length: 4.6X50m m 5um Eluent CH3OH / HO = 30 / 70 Flo w Rat e:.0 m L/ m in Co l. Tem p.: 40C Detector:: UV54nm Sample Volume: 4uL Sam p les: ) Pyridine 0.09 mg/ml ) Phenol 0.4 m g/ m L 0 0 0 XTerra M S C8 0 0 0 Atlantis dc8 0 0 0 Zorbax Eclipse XDB- C8

Quality Comparison Among Leading Companies Aminopyridine Test for Base Deactivation m Volt 0 00 0 0 Inertsil ODS- SP 3 3 m mau Volt 0 00 m Volt 0 00 00 0 0 Inertsil ODS- 3 3 3 m Volt 0.00 00 0.0 00 0.0 All peaks are adsorbed. 0 0 0 3 Sym m etry C8 All peaks are adsorbed. 3 0 0 SunFire C8 3 Column Length: 4.6X50m m 5um Eluent CH3OH / 0m M Phosphate Buffer (ph7.6) 0:90 Flo w Rat e:.0 m L/ m in Co l. Tem p.: 40C Detector:: UV54nm Sample Volume: 4uL Sam p les: ) 4- Am inopyridine 0.m g/ m L ) 3- Aminopyridine.0mg/mL 3) - Aminopyridine.0mg/mL 0 0 0 XTerra M S C8 0 0 Atlantis dc8 0 0 Zorbax Eclipse XDB- C8

Quality Comparison Among Leading Companies Oxine- copper Test for Silica Purity m Volt 0 0 40 60 80 mau 0 0 40 60 80 m Volt 0.00 0.0 0.04 0.06 0.08 0.0 Oxine- copper shows tailing. 0 0 0 0 0 0 0 Sym m etry C8 0 0 SunFire C8 Inertsil ODS- SP Inertsil ODS- 3 m Volt 0 0 40 60 80 Oxine- copper shows tailing. Oxine- copper shows tailing. Column Length: Eluent Flo w Rat e: Co l. Tem p.: Detector:: Sample Volume: Sam p les: 4.6X50m m 5um CH3OH / 0m M H3PO4 (0/ 90, w:w).0 m L/ m in 40C UV54nm.5uL ) Oxine- copper 0.0 mg/ml ) Caffeine 0.4 m g/ m L 0 0 0 XTerra M S C8 0 0 Atlantis dc8 0 0 Zorbax Eclipse XDB- C8

Quality Comparison Among Leading Companies Carboxylic Acid Test m Volt 0 0 40 60 m mau Volt 0 0 40 m Volt 0 40 60 0.00 0.0 0.04 0.06 All peaks are adsorbed. All peaks are adsorbed. 0 4 6 8 0 Inertsil ODS- SP 0 4 6 8 0 Inertsil ODS- 3 0 4 6 8 Sym m etry C8 0 4 6 8 0 SunFire C8 All peaks are adsorbed. m Volt 0 0 40 60 Form ic acid is adsorbed. Column Length: 4.6X50m m 5um Mobile Phase: 0.% H3PO4(v/ v) Flo w Rat e:.0 m L/ m in Co l. Tem p.: 40C Detector: UV0nm Sample Volume: 4uL Sam p les: ) Form ic Acid 0. %(v/ v) ) Acetic Acid 0. %(v/ v) 0 4 6 8 0 XTerra M S C8 0 4 6 8 0 Atlantis dc8 0 4 6 8 0 Zorbax Eclipse XDB- C8

Other Column Lineups For low molecular compounds Inertsil C8-3 Inertsil Ph- 3 Inertsil CN - 3 Inertsil N H Inertsil AX Inertsil H ilic Inertsil SIL- 00A Inertsil CX Inertsil Diol For high molecular compounds Inertsil W P300- C8 Inertsil W P300- C8 Inertsil W P300- C4 Inertsil W P300- SIL Inertsil W P300- Diol Fo r sp ecial use Inertsil Sulfa C8 Inertsil Peptides C8 (Designed for analysis of Sulfa Drugs) (Designed for analysis of Peptides)

Analysis tim e com parison of ODS- 3 vs. C8-3 3 4 5 Column : 50 x 4.6 mm I.D. Eluent : CH 3 CN / H O = 70 / 30 Flow rate :.0 ml/min Detector : UV 54nm Co l. Tem p. : 40 Sam ple : ) N aphthalene ) Ethylbenzene 3) Propylbenzene 4) Anthracene 5) Butylbenzene 3 4 5 0 4 6 8 0 Inertsil ODS- 3 0 4 6 8 0 Inertsil C8-3

Application of Fat- soluble Vitam ins Column : Inertsil C8-3 50 4.6 mm I.D. Eluent : CH 3 OH / H O = 95 / 5 Flow Rate :.0 m L/ m in Detector : UV 80nm Col. Tem p. : 40 ) Vitam in K3 ) Vitam in D3 3) Vitam in E 3 4 5 4) Vitamin E acetate 5) Vitam in K 0 4 6 8 0 Time (min)

A nalysis co m p ariso n of ODS- 3 vs. Ph- 3 + Column Eluent : 50 x 4.6 mm I.D. : O D S 90% CH 3 OH Ph 70% CH 3 OH Flow rate :.0 ml/min Detector : UV 54nm Co l. Tem p : 30 Sam ple : ) Fluorene ) Phenanthrene Fluorene 0 4 6 8 0 Time (min) 0 4 6 8 0 Time (min) Inertsil ODS- 3 Inertsil Ph- 3 Phenanthrene

Feature of Inertsil Ph- 3 Ordinary phenyl columns are bonded with alkylphenyl groups. For our Inertsil Ph- 3, a phenyl group is directly bonded to the silica- gel to maximize the effect of an electron affinity. Inertsil Ph- 3 Si 3 4 5 3 4+5 5 0 0 0 Time (min) Inertsil ODS- 3 3 0 0 0 Time (min) Si ( CH ) n 4+5 5 0 0 0 Time (min) Analytical Conditions Column Size:4.6m m I.D. 50mm Eluent :60% Acetonitrile (Ph Column) 75% Acetonitrile (ODS column) Flow Rate:.0m L/ m in Col. Tem p. :40 Detector :UV 0nm Samples ) Flucythrinate ) Cyhalothrin 3) Deltamethrin 4) Fluvalinate 5) Tralomethrin (two isom ers)

A nalysis co m p ariso n of ODS- 3 vs. CN - 3 Inertsil CN - 3 Inertsil ODS- 3 0 4 6 8 0 + 3 3 4 4 Analysis of Organic acids Column : 50 4.6 m m I.D. 5μm Eluent : 0mM Phosphate buffer (ph4.0) Flow Rate :.0 m L/ m in Detector : UV 0nm Col. Tem p. : 40 Sample : asparagine aspartic acid H N COOH C H CH CONH H N COOH C H CH COOH 3 fum aric acid 4 m aleic acid 0 4 6 8 0 H HOOC C C COOH H H HOOC C C H COOH

A nalysis co m p ariso n of ODS- 3 vs. CN - 3 Column : 50 4.6 m m I.D. 5μm Flow Rate :.0 m L/ m in Detector : UV 360nm Col. Tem p. : 40 A nalysis of D N PH - Aldehydes Sample : ) Formaldehyde ) Acetaldehyde 3) Propionaldehyde 4) n- Butylaldehyde 5 ) iso - Butyraldehyde 4 3 3 5 4+5 0 4 6 8 0 Time (min) Inertsil ODS- 3 Eluent : CH 3 CN / H O = 60 / 40 0 0 0 Time (min) Inertsil CN 3 Eluent : CH Cl / Hexane = 0 / 90

W hat is HILIC? HILIC = ( H ydrophilic Interaction Chromatography ) HILIC is a variation of normal phase mode where a high concentration of nonpolar solvent is used in a mobile phase and an ideal method for separation and retaining highly polar analytes unretained by traditional reversed- phase mode. Applications for HILIC mode Am ino acids Water-soluble vitam ins Saccharides Drug m etabolites 3 4 0% CH 3 CN 60% CH 3 CN 90% CH 3 CN Column : Inertsil HILIC 5μm 50 x 3.0 mm I.D. Eluent : 0.%TFA in % CH 3 CN Flow rate : 0.4 m L/ m in Detector : UV 54nm Col. Tem p.: 40 Sam ple : ) Thiam ine 8μg/ ml ) N icotinam ide 8μg/ ml 3) L- Ascorbic Acid 80μg/ ml 4) Riboflavin 8μg/ ml 0 4 6 8 0

Application of Oligosaccharides 3 4 Analytical Conditions Column : Inertsil HILIC 5um 50 4.6 mm I.D. Eluent : Acetonitrile / HO = 85/5 (w/w) Detection : RI Col. Tem p.: 40 Flow Rate:.0 m l/ m in Samples : ) M alt ose ) M altotriose 3) M altotetraose 4) M altopentaose 5) M altohexaose 6) M altoheptaose 5 6 0 4 6 8 0 Time (min)

Scale- Down of column I.D. Advantages Amount of Eluent and waste solution can be reduced by setting a low flow rate. Since the sensitivity will increase the usage amount of sample can be reduced or trace amount of compounds can be analyzed. Can reduce the labor hours of making the eluent composition and sample preparation procedures. Improving the working environment and reduce the harmful effect to nature. ISO400: Save Energy and Resources!!! Reduce Amount of Eluent with Smaller ID Inertsil Columns!!! Weakness The influence of dead volume will deteriorate the peak shape and separation.

Example of Scale- Down Column :Inertsil ODS- 3 5μm 50mm I.D. 4.6 m m Flow.0 m L/ m in I.D. 3.0 m m Flow 0.4 m L/ m in I.D.. m m Flow 0. m L/ m in 5mV 0 0 0 0 0 0 0 0 0 Sensitivity Consumption of eluent Liquid waste volum e Separation twofold 60% cut 60% cut Com plete fourfold 80% cut 80% cut Incom plete

Optim izing the colum n length M obile Phase: Column Temp.: 65% Acetonitrile 40C To improve separation 5μm;50 mm Theoretical Plate 3,000; 6M Pa 3μm;50 mm Theoretical Plate 0,000: M Pa 3μm;50 mm Theoretical Plate 30,000; 8M Pa 4μm;50 mm Theoretical Plate 7,000; 8.5M Pa Inertsil ODS- 3 5μm;50 mm Theoretical Plate 3,000;3.5M Pa

Performance comparison between various Particle Sizes Theoretical Plates of N umbers Back Pressure 35,000 30,000 5,000 3,500 7,00 30,500 6 8 4 4.7 M Pa 6.8 M Pa 3.8 M Pa 0,000 5μm 4μm 3μm 0 5μm 4μm 3μm The theoretical plates are inversely proportional to the particle size The Pressure is inversely proportional to the sq uare of the particle size

Features of 4um column Low Back Pressure!(/ the back pressure of 3um column) Particle size 5μm 4μm 3μm Column length 50 mm 50 mm 50 mm 50 mm Pressure 9.3 M Pa 3.5 M Pa 9.0 M Pa 7.5 M Pa Higher Theoretical Plates of Numbers! 50% M ethanol ; 4.6mm ;.0mL/min (Increase in column efficiency by over 5% over 5um column) Particle size 5μm 4μm 3μm Column length 50 mm 50 mm 50 mm 50 mm Theoretical plate 3,5 7,06 30,484 0,998 65% Acetonitrile ; 4.6mm ;.0mL/min ; Naphthalene Extended Column Life Time! (Using the same filter of a 5um column) The column durability is the same as a 5um column.

Optim izing the colum n length Inertsil ODS- 3 5μm;50 mm Theoretical Plate 3,000;3.5M Pa M obile Phase: Column Temp.: Analysis Tim e: 65% Acetonitrile 40C 5 minutes To shorten the analysis tim e 3μm;00 mm Theoretical Plate,500; 7.5M Pa 3μm;75 mm Theoretical Plate 9,000: 5.5M Pa 0mins Increased flow rate folds 7.5mins 3μm;00 mm Theoretical Plate,000; 5M Pa 3μm;75 mm Theoretical Plate 8,500; M Pa 5m ins 3.8mins 4μm;00 mm Theoretical Plate,000: 5.5M Pa 0mins 4μm;00 mm Theoretical Plate 9,000; M Pa 5m ins To reduce more analysis time

Advantages of small particle size column Inertsil ODS- 3 5μm 50 4.6 mm I.D. CH 3 CN / H O = 70 / 30.0 ml/min 40 Pressure =.9 M Pa Particle Size Column Size Eluent Flow Rate Co l. Tem p. Sam ple Ethylbenzene Propylbenzene Butylbenzene iso - Amylbenzene n- Amylbenzene Inertsil ODS- 3 3μm 00 4.0 mm I.D. CH 3 CN / H O = 70 / 30.5 ml/min 40 Pressure =0.5 M Pa 0 4 6 8 0 4 6 Time (min) Analysis Tim e Consumption of eluent Separation Pressure 0 4 6 Time (min) /3 45% less Com plete Very H igh

Van Deemter Plot of HETP vs. Flow Rate Column: 50 X.mmI.D.

Comparison of um vs. 5um

Pressu re vs. Flow Rate

Exam ple of H igh- Speed Separation Analytical Condition Sam ple Column: Inertsil ODS- 3. Gallic acid (0.7mg/mL) Eluent: A: Acetonitrile. Vanillic acid (0.7mg/mL) B: 0.% Phosphoric Acid `. Vanillin (Im purity of Vanillic acid) A/B=5/85 3. p - Coum aric acid (0.7mg/mL) Flow Rate: 0.5m L/ m in.,.0m L/ m in. 4. Ferulic acid (0.7mg/mL) Column Temperature: 40C 5. m - Coum aric acid (0.7mg/mL) Detector: PDA 75nm Injection Volum e: 0uL (4.6m m I.D.), 5uL (3.0m m I.D.) Analysis Time: 9 mins Result s: All peaks were well separated.

Exam ple of H igh- Speed Separation - Shorten the column length - Changing the packing material to a 3um Analysis Time: 4 mins Result s: Obtained the sam e resolution with shorter analysis tim e.

Exam ple of H igh- Speed Separation - Shorten the column length - Using the same flow rate Analysis Time: 3.5 mins Result s: The analysis time was shortened to 3.5 minutes. However, peak ` and 3 were not separated.

Exam ple of H igh- Speed Separation - Changing the packing material to a um Analysis Time: 3.5 mins Result s: The analysis time was shortened to 3.5 minutes. In addition, complete separation between peak ` and 3 is obtained.

H igh- Speed Separation Inertsil ODS- 3 um Base Silica Physical Properties and Chem ical M odification Base Silica: High Purity Silica Gel 99.999% Surface Area: 450 m /g Pore Size: 00A Pore Volume:.05 ml/g Bonded Phase: Octadecyl Groups Carbon Loading: 5% End capped: Yes Column Sizes: 50. mm I.D. 50 3.0 mm I.D. M aximum Operating Pressure: 50 M Pa Guaranteed Theoretical Plates: 60,000/m (GLS Standard Column Performance Test)

Features of Inertsil ODS- 3 um Advanced classification technologies for sizing silica gels realized an ideal balance between theoretical plate number and column back pressure enabling high throughput analysis in the existing standard HPLC system. Fine particles are thoroughly eliminated and proper mesh size is employed, which result in less column clogging problems. Customers who are currently using Inertsil ODS- 3 5um, 4um or 3um in their analyses can now simply readily achieve High- Speed Separation without changing the analytical conditions. A newly- developed column joint is used to minimize the dead- volum e, which is an ideal design for high throughput analyses, resulting in high/sharp peak shapes for those fast eluted samples. * M aximal performance can be obtained at the following flow rate:.m m I.D.: From 0.4 to 0.6 ml/min 3.0m m I.D.: From 0.8 to. ml/min

Durability Test -Colum n Clogging- Confirmed no column clogging or no increase of column back pressure after a,000 times injection of pretreated 5uL serum. Before Test Pretreated serum: Using an human serum supernatant, which was centrifuged mixed with commercially available human serum adding equal volumes of methanol. The pressure fluctuation of the column 00 0 4 Time (min) 80 60 40 0 00 After Test 80 60 40 0 0 0 00 400 600 800 000 0 4 Time (min)

Durability Test -Pressure Durability- - The analytical condition was purposely set to have the maximum pressure reach at 50M Pa. - The test was repeatedly conducted and observed no variation in retention and peak shape. - Instead of an isocratic, a gradient condition was employed since it generates more pressure fluctuation resulting in more rigorous durability test. Analytical Conditions M obile Phase: Water00% 0min M ethanol00% Flow Rate: 0.5 m L/ m in M aximum Pressure: 50M Pa (At W ater/ M ethanol = 50/ 50) Sample: Alkylphenol 6 8 Time (min) 6 8 Time (min) 6 8 Time (min) st Injection After 00 Injections After 500 Injections

Inertsil ODS- 3 um Instruction for Use W hen using an ultra high pressure specification instrument, please be careful with the fitting type. To m axim ize colum n life, operate at pressures within 50M Pa (approx. 7,00psi). To maximize the performance of the column, extreme care should be taken on the internal diameter of piping and detector cell. The below mentioned Seal Tight Fitting (Upchurch) can also be tightened by hand, however, leakage may be observed due to tolerance of an outer diameter of the piping or worn piping. Use stainless steel male nut and ferrule when analyzing precious samples. Please be careful with the setting of response speed of detector and retrieving interval on the data processor in a high throughput analysis. Description Cat.No. Seal Tight Fitting, Short Nut, 0PCS/PACK, P/N F-95X (Upchurch) *600-7304 Seal Tight Fitting, Long Nut, 0PCS/PACK, P/N F-96X (Upchurch) *600-7305

H igh-speed Separation Columns Inertsil C8-3 μm The same base silica synthesizing and bonding technologies used for Inertsil ODS- 3 are also employed for Inertsil C8-3 um. The difference between these two phases is just the length of the hydrocarbon ligands. This enables faster analyses to be performed by converting from an Inertsil ODS- 3 column to an Inertsil C8-3 column without changing the separation pattern. Base Silica Physical Properties and Chem ical M odification Inertsil ODS- 3 um, 50 3.0 mm I.D. Inertsil C8-3 um, 50 3.0 mm I.D. 3 4 Base Silica: High Purity Silica Gel 99.999% Particle Size: um Surface Area: 450 m/g Pore Size: 00A Pore Volum e:.05 ml/g Bonded Phase: Octadecyl Groups Carbon Loading: 9% End capped: Yes 3 4 0 4 6 8 5 5 Conditions Syst em : GL- 7400 system Columns: Inertsil ODS- 3, C8-3 Eluent: A : CH 3 CN B: H O A/B = 50/50, v/v Flow rate:.0 ml/min Detector: PDA 60 nm In j. Vo l.: 5 ml Sam ple:. Naphthalene. Ethylbenzene 3. n - Propylbenzene 4. Anthracene 5. n - butylbenzene 0 4 6 8 Ordering Information Length / I.D..mmI.D. 3.0mmI.D. 30mm 500-84930 500-84935 50mm 500-8493 500-84936 List Price US$ 490.00 490.00 The shorter alkyl group a functional group has, the less hydrophobic interaction it has. Therefore, the retentivity of C8-3 is weaker than ODS- 3.

H igh-speed Separation Columns Inertsil Ph- 3 μm Ordinary phenyl columns are bonded with alkylphenyl groups. For our Inertsil Ph- 3, phenyl groups are directly bonded the silica- gel to m axim ize the effect of an electron affinity. Inertsil Ph- 3 shows inherent inertness and unique selectivity. Base Silica Physical Properties and Chem ical M odification Inertsil C8-3 um, 50 3.0 mm I.D. Inertsil Ph- 3 um, 50 3.0 mm I.D. 3 4 0 4 6 3 4 5 Base Silica: High Purity Silica Gel 99.999% Particle Size: um Surface Area: 450 m/g Pore Size: 00A Pore Volum e:.05 ml/g Bonded Phase: Phenyl Groups Carbon Loading: 9.5% End capped: No 5 Conditions Syst em : GL- 7400 system Columns: Inertsil C8-3, Ph - 3 Eluent: A : CH 3 CN B: H O A/B = 50/50, v/v Flow rate:.0 ml/min Detector: PDA 60 nm In j. Vo l.: 5 ml Sam ple:. Naphthalene. Ethylbenzene 3. n - Propylbenzene 4. Anthracene 5. n - butylbenzene 0 4 6 Inertsil Ph- 3 not only m akes the analysis tim e shorter than C8-3, but also provides an unique retention for No. and No.4, which offers a different separation pattern. Ordering Information Length / I.D..mmI.D. 3.0mmI.D. 30mm 500-8530 500-8535 50mm 500-853 500-8536 List Price US$ 490.00 490.00

How to Maintain Column perform a nce N ecessity of cleaning the water for eluent Using guard column How to wash and store columns

Cleaning Water for eluent Im purities (organic compounds or bacteria ) in water ODS column 00% Water 60min hold - 0min 00% Acetonitrile UV 0nm (Retain the impurity) ----- (Eluted) <Deionnization+Ultraviolet oxidation+ M em brane filtration> <Deionization+ Activated carbon filtration+ M em brane filtration > <Deionization+ Distillation> <Deionization>

Retained im purities on the colum n Flow Accumulation of stains (im purities in water) Silica- gels became hard Resulting in increase in back pressure, deterioration of separation, poor peak shape and unknown peaks.

Colum n for rem oving Im purities Pre- clean ORG rem oves organic im purities from water. This is placed in the line between pump and injector. Seal scum of pumping system also can be retained on Pre-clean ORG before flowing into analysis column.

Another way of cleaning eluent By using the Solid Phase Extraction procedure, the phenomena of base line drifts or ghost peaks can be prevented by filtering the mobile phase. Reference Title: M obile-phase Cleanup Using Solid-Phase Extraction Disks Journal Title: LCGC N O RTH AM ERICA (http://www.chromatographyonline.com/lcgc/) Volume, N umber, Year: V olume, Number, 003 Page: P68~78 Author: M.C.Ringo, J.R. Allen, D.M. M attocks, (GlaxoSm ithkline)

Recommendation of Guard Column The purpose of using a Guard Column Removing high lipophilic impurities from the sample (for reversed phase mode) Removing high polarity impurities from the sample (for normal phase mode) Rem oving im purities from the sam ple The head of the column view when using a guard column The head of the column view without a guard column

Recommendation of Guard Column Concerns when using a Guard Column The separation efficiency was poor The retention time was late Possibility that a different packing material is used for the guard column All of our Guard Columns are using the absolutely same packing m aterials that are packed in the analytical colum ns. Therefore, please feel safe on using our guard columns. W ithout guard column W ith Cartridge guard column E W ith GL cart Cartridge Guard Column E GL- Cart Rt. = 6.5 min T.P. = 3,693 As. =.03 Rt. = 6.48 min T.P. = 3,464 As. =.05 Rt. = 6.49 min T.P. =,75 As. =.07

Before using a N ew column Inertsil series are shipped with the solvents as below for the final test of the column. Before using the column, please confirm miscibility to the below solvents that you will be using as a mobile phase. W hen using a mobile phase that is non-miscible to the below solvents, please purge the column with Isopropanol by 5 to 0 volumes of the column volume. After purging the column with Isopropanol, substitute with the mobile phase. Colum n ODS, C8, C4, Ph, HILIC CN, N H, Diol, SIL Shipping solvents Acetonitrile / W ater Hexane / Ethanol Solvents that cannot be mixed with the shipping solvent Hexane, Heptane, Isooctane, Chloroform, Dichloromethane etc Water, M ethanol, Acetonitrile etc AX, CX M ethanol Hexane, Heptane, Cyclohexane, Isooctane, etc

Daily maintenance of the column Eluent with acids, buffer, ion- pair At first you must wash the column using same organic solvent and ratio(%) that was used as an eluent without any additives. Eluent without additives Example: Eluent = 0.% H3PO4 in 35% M ethanol W ash so lvent = 35% M ethanol Wash column using 00% organic. = same as an eluent Store column with eluent. (without additives) W hen using a gradient elution, use 30 ~ 50% of organic to store column.

How to wash column W hen is the best timing to wash the column? W hen you observe fluctuation in retention time, increase in pressure, deterioration of separation, and poor peak shape. In case of using reverse phase mode including HILIC mode In case of using normal phase mode To remove polar compounds ) Water ) M ethanol 3) THF * 4) M ethanol 5) Water 6) Eluent ) Isopropanol ) M ethanol 3) Water * 4) Isopropanol 5) Eluent To remove non- polar compounds ) Isopropanol ) THF * 3) Dichloromethane 4) Hexane 5) Isopropanol 6) Eluent ) Isopropanol ) M ethanol 3) Dichloromethane 4) Hexane 5) Isopropanol 6) Eluent * Skip this process when you use none chemical bonded silica- gel. * Do not introduce it to fittings that are made out of PEEK!

How to wash column W hen there are no problem in separation, peak shape and retention tim e, but still observe an increase in pressure, the cause m ay be from buffer decomposition or filter clogging on the column inlet. Reverse the column and connect it by introducing the following solvents In case of using reverse phase mode (including HILIC mode) Water Isopropanol Water In case of using normal phase mode Isopropanol Caution!! Do not connect the column outlet to the detector! Reduce the flow rate when the pressure is above 0M Pa! Contact each manufacturer before contacting a reversed column washing method. It is difficult to fully regenerate the colum n once it experiences a colum n clogging. The above procedure will tem porary regenerate the colum n only.

How to Store column Long term storage differs from overnight or short term storage Colum n ~0 days Few weeks ~ ODS,C8,C4,Ph Eluent without additives 00% M ethanol CN,N H,Diol,SIL Eluent without additives 00% Hexane AX,CX Eluent without additives 00% M ethanol * When the column was not used for a long period of time, please wash the colum n before use.