The Evolving HPLC Column Packings

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2 The Evolving HPLC Column Packings GL Sciences has been steadily supplying with columns from Inertsil ODS, first-generation to InertSustain series, integration of state-ofthe-art technologies, and has established a very good reputation around the world. They are available with the same quality and the same performance anywhere in the world. Europe Asia North America Africa Oceania South America Inertsil Series Inertsil Series 00 Å 0 Å 0 m /g 0 m /g Inertsil ODS Inertsil ODS- Inertsil Ph etc. 80 Å 0 m /g Inertsil ODS-80A Inertsil Series InertSustain Series 00 Å 00 Å 0 m /g 0 m /g For Low Molecular For High Molecular 00 Å Inertsil WP Series : Inertsil WP00 C8 Inertsil WP00 C8 Inertsil WP00 C Inertsil WP00 DIOL Inertsil WP00 SIL Inertsil ODS- Inertsil ODS- Inertsil ODS-SP Inertsil ODS-P Inertsil ODS-EP Inertsil C8- Inertsil Ph- Inertsil CN- Inertsil HILIC Inertsil Amide Inertsil SIL-00A etc. 00 Å 0 m /g InertSustain InertSustain InertSustain InertSustain 00 m /g C8 C8 Phenyl NH InertSustainSwift C8 Base on Evolutionally Surfaced Silica (ES Silica) evolved from Inertsil, InertSustain has developed InertSustain employs a radically new type of silica, in which the surface of the silica is uniquely modified, the amount of silanols are controlled. Owing to this state-of-the-art technology ES silica allows easy surface modification including endcapping, which grants the following three favors.. Exceptionally improved inertness. Robust bonded phase. High reproducibility batch to batch Owing to above benefits InertSustain is recommended as the first choice column for almost all compounds analyzed. Batch to batch reproducibility Keeping on stable supplies with high quality and performance, GL Sciences continues to evolve. Year 0 TQ-68 Year 0 TQ-9 Year 0 TQ-6 Year 00 TQ-08 Year 009 TQ-86 Year 008 TQ-688 Year 007 TQ-6 Year 006 TQ-8 Year 00 TQ-9 Year 00 TQ-0 Year 00 TQ-97 Year 00 TQ-86 Year 00 TQ-688 Year 000 TQ- Year 999 TQ-6 Column : Inertsil ODS- ( μm, 0.6 mm I.D.) CHOH/HO=80/0,v/v.0 ml/min 0 C UV nm : : : : : ) Uracil ) Caffeine ) Phenol ) Butylbenzene ) o-terphenyl 6) Amylbenzene 7) Triphenylene

3 InertSustain C8 Physical Properties Silica : Newly Developed ES Silica Gel : μm, μm, μm Surface Area : 0 m /g Pore Size : 00 Å (0 nm) Pore Volume : 0.8 ml/g Bonded Phase : Octadecyl Groups End-capping : Complete Carbon Loading : % USP Code : L ph Range : ~0 Generally, silica based columns are mechanically stable and provide high effi ciencies, however, they cannot be used under alkaline conditions and their residual silanol groups tend to adsorb organic bases. InertSustain C8 employs a radically new type of silica, in which the surface of the silica is uniquely modifi ed, enabling precise control of the silica properties. InertSustain C8 inherits the advantages of all the current Inertsil HPLC columns (e.g., extremely low operating back pressure, superior inertness to typically any analytes, high effi ciency and compatibility with a wide range of solvents), but now can be used for wide ph analysis with consistent performance from column to column and lot to lot. Figure : Basic Compounds InertSustain C8 X SELECT CSH C8 ZORBAX Eclipse Plus C8 Figure : Acidic Compounds No peak InertSustain C8 X SELECT CSH C8 ZORBAX Eclipse Plus C : μm, 0. mm I.D. : A) CHCN B) mm KHPO (ph 7.0, KHPO) A/B = 0/70, v/v : 0. ml/min : 0 C : UV 0 nm :. Uracil. Pyridine. Phenol. Berberine chloride. Dextromethorphan : μm, 0. mm I.D. : A) CHCN B) 0. % HPO A/B = /7, v/v : 0. ml/min : 0 C : UV nm :. Brilliant Blue FCF. Phenol. Salicylic acid Figure : Chelating Compound InertSustain C8 st Injection X SELECT CSH st Injection : No Peak : μm, 0.6 mm I. D. : A) CHCN B) 0. % HPO A / B = 0 / 60, v / v :.0 ml / min : 0 C : UV nm :. Hinokitiol Time (min) Time (min) nd Injection nd Injection Time (min) Time (min)

4 InertSustain C8 Comparison of Performance to Core-Shell Columns As shown below, core-shell columns show peak tailing due to the presence of trace metals or silanol groups in their silica gel. Quantitative and qualitative analysis will be a source of concern since the adsorption of compounds can be expected. InertSustain C8 µm Kinetex C8 (.7 μm) Intensity, cps.e.0e.0e Intensity, cps.e.0e.0e 0.0 Adsorption of Peak Column Injection Vol. : ODS Column(00. mm I.D.) : A) mm CHCOONH in 9 % CHCN B) mm CHCOONH in 0 % CHCN A / B = 0 / 80 - min 00 / 0 -. min 00 / min - 0 / 80, v / v (Mixed by a gradient mixer) : 0. ml / min : 0 C : LC / MS / MS (000 QTRAP : ESI, Positive, MRM) : 0 μl :. Nitrofurazone (00 μg / L). Lasalocid A (00 μg / L) Wide ph compatibility with Long Column Lifetime As shown in the experiment below, due to the introduction of Evolved Surface Silica, InertSustain C8 maintained high effi ciency and peak shape for 00 hours while other wide ph column brands failed InertSustain C8 Xterra MS C8 Gemini-NX 60 % Initial N Hours of Purging(h) Purging : μm, 0.6 mm I.D. : A)CHOH B)0 mm Triethylamine (ph 0.0) A/B = 0/70, v/v :.0 ml/min : 0 C Analytical : A) CHCN B) HO A/B = 6/, v/v :.0 ml/min : 0 C : UV nm : Naphthalene Experience the InertSustain! (Inertness and Sustainability) Highly end-capped ODS column such as InertSustain C8 offers an opportunity to fl ush out contaminants from the column surface easily using an organic solvent. Coffee melanoidins are brown heterogeneous polymers contained in coffee. Its components are not clarifi ed yet, but it is considered to contain several ionic compounds, which a poorly end-capped column will adsorb those ionic compounds leading to short column lifetime. As for ODS column, which is commonly used for HPLC and LC/MS/MS, its inertness has an infl uence not only on peak shape but also detection sensitivity and durability. It is highly recommended to use highly end-capped column which provides good peak shape for both basic and acidic compounds such as InertSustain C8. The packing material was visually confirmed by removing the column Before Experiment Brand A InertSustain C8 Flow Injection of Coffee After loading Coffee Still Contaminated After washing the column with CH CN 00 % 0 minute washing Ionic contaminants were hard to be washed out from the Column Clean!

5 InertSustain C8 Reliable Reproducibility, Performance and Quality Rigorous quality control of physical properties and strict chromatographic tests for inertness and selectivity, contribute to the production of InertSustain C8 with an outstanding reproducibility and long column lifetime. Strong Basic Compound Test Superior peak shape with highly reproducible chromatograms can be obtained even for those strong basic compounds. Lot. Same Retention Behavior between Various s µm 0.0 MPa Reliable reproducibility and same retention behavior between various particle size columns µm.8 MPa Lot. Lot. : µm, 0.6 mm I.D. : A) CHCN B) mm phosphate buffer (ph 7.0) A / B = 0 / 60, v / v :.0 ml / min : 0 C : UV 0 nm :. Dextromethorphan µm. MPa : 0. mm I.D. : A) CHOH B) mm phosphate buffer (ph 7.0) A / B = 0 / 70, v / v : 0. ml / min : 0 C : UV 0 nm :. Uracil. Pyridine. Phenol. Berberine chloride. Dextromethorphan Strong Acidic Compound Test Strong Chelating Compound Test Lot. Lot. Lot. Lot. Lot. Lot : µm, 0.6 mm I.D. : A) CHCN B) 0. % HPO A / B = / 7, v / v :.0 ml / min : 0 C : UV nm :. Brilliant Blue FCF. Phenol. Salicylic acid : µm, 0.6 mm I.D. : A) CHCN B)0. % HPO A / B = 0 / 60, v / v :.0 ml / min : 0 C : UV nm :. Hinokitiol

6 InertSustain C8 Analytical Columns : μm HPSeries : μm 0 MPa (00 Bar) Length \ I.D Length \ I.D * End-fi ttings are /6 Waters-compatible. * UHPLC compatible end-fi ttings are also available upon request for UHPLC systems (Ex: UPLC) to avoid dead volume. * Indicate UP Type end-fi ttings when ordering. (Please note that UP type is not available for a.6 mm I.D. column) * For maximum operating pressure information, please refer to page 6. Length \ I.D : μm Length \ I.D Length \ I.D : μm Length \ I.D * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6. Cartridge Guard Column E I.D. of the Analytical Column Applicable Length I.D. Replacement Cartridge E Guard Column ( EA.) Cartridge E Holder / Cartridge Set ( Cartridge E Guard Columns & Holder) μm μm μm μm , , , , , For 0 mm Length Holder for Cartridge Guard Column E For 0 mm Length * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6.

7 InertSustainSwift TM C8 Physical Properties Silica : Newly Developed ES Silica Gel :.9 μm, μm, μm Surface Area : 00 m /g Pore Size : 00 Å (0 nm) Pore Volume :.00 ml/g Bonded Phase : Octadecyl Groups End-capping : Complete Carbon Loading : 9.0 % USP Code : L ph Range :.0~0.0 As shown in figure, InertSustainSwift C8 maintains the same extreme inertness, wide ph range and provide rapid separations with symmetric peaks. The optimization of surface area, pore size and chemical bonding delivers superior peak shapes (Figure ). Figure proves InertSustainSwift C8 is also ideal for LC/MS/MS applications which offer highly sensitive results and enables MS compatible buffers to be used due to the extremely inert silica gel. Figure : Comparison of Retentivity. min InertSustainSwift C8 (.9 μm) InertSustain C8 ( μm). min : 0. mm I.D. : A) CHOH B ) HO A/B = 80/0, v/v : 0. ml/min : 0 C : UV nm :. Uracil. Toluene. Ethylbenzene. Propylbenzene. n-butylbenzene 6. n-amylbenzene Figure : Comparison of Efficiency 7. MPa N : 600 N : MPa N : 00 N : 900 : 0. mm I.D. : A) CHOH B) HO A/B = 0/70, v/v : 0. ml/min : 0 C : UV nm :. Pyridine. Phenol.0.0 InertSustainSwift C8 (.9 μm) Figure : Rapid LC/MS/MS Analysis of Basic Drugs Complete Separation in 60 Seconds.0.0 Acguity BEH C8 (.7 μm) Column : InertSustainSwift C8(.9 μm, 0. mm I.D.) : A) 0 mm Ammonium Formate in HO B) 0 mm Ammonium Formate in CHOH A/B = 70/0 0. min - 0/60 0. min - 0/00 0. min 0/ min -70/0-0. min - 70/0,v/v : 0.6 ml/min : 0 C : LC/MS/MS (ESI, Positive, MRM) :. Acebutolol. Atenolol. Labetalol. Nadolol. Pindolol

8 InertSustainSwift C8 Analytical Columns :.9 μm HP Series : μm Max. Operating Pressure : 0 MPa (00 Bar) * End-fi ttings are /6 Parker style. * For maximum operating pressure information, please refer to page 6. Length / I.D Length / I.D Length / I.D : μm Length / I.D Length / I.D : μm Length / I.D * End-fi ttings are /6 Parker style. * For maximum operating pressure information, please refer to page 6. Cartridge Guard Column E I.D. of the Analytical Column Applicable Length I.D. Replacement Cartridge E Guard Column ( EA.) Cartridge E Holder / Cartridge Set ( Cartridge E Guard Columns & Holder) μm μm μm μm , , , , , For 0 mm Length Holder for Cartridge Guard Column E For 0 mm Length * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6.

9 InertSustain C8 Physical Properties Silica : Newly Developed ES Silica Gel : μm, μm Surface Area : 0 m /g Pore Size : 00 Å (0 nm) Pore Volume : 0.8 ml/g Bonded Phase : Octyl Groups End-capping : Complete Carbon Loading : 8 % USP Code : L7 ph Range : ~ 0 IInertSustain C8 is an octyl group (C8) bonded column delivering the same extreme inertness to any type of compounds just like InertSustain C8, which enables rapid analysis of highly hydrophobic compounds delivering symmetric peaks at a wide range of ph. Figure : Comparison of Retentivity between InertSustain C8 and InertSustain C8 9.9 min 6. min 6 : μm, 0.6 mm I.D. : A)CHOH B)HO A/B = 80/0, v/v :.0 ml/min : 0 C : UV nm :. Uracil. Toluene. Ethylbenzene. Propylbenzene. n-butylbenzene 6. n-amylbenzene InertSustain C InertSustain C8 Figure : Analysis of Losartan Potassium and Hydrochlorothiazide Tablets, Assay Test (Based on the Condition of United States Pharmacopeia 6-NF) Column Col.Temp. InjectionVol. : InertSustainC8( μm,0.0 mm I.D.) : A)CHCN/Buffer* = 7/9,v/v(Premix) B)CHCN A/B=00/0 min 9/8 6min 8/6 min 00/0 min 00/0,v/v :.0mL/min : C : UV80 nm(gl-70 UV Detector) : 0 μl :. Hydrochlorothiazide 0. mg/ml. Losartan Potassium 0. mg/ml Tailing factor of :.79 ( <.) * Buffer :. g/l of monobasic potassium phosphate and. g/l of dibasic sodium phosphate n water

10 InertSustain C8 Analytical Columns : μm HPSeries : μm 0 MPa (00 Bar) Length \ I.D Length \ I.D * End-fi ttings are /6 Waters-compatible. * UHPLC compatible end-fi ttings are also available upon request for UHPLC systems (Ex: UPLC) to avoid dead volume. * Indicate UP Type end-fi ttings when ordering. (Please note that UP type is not available for a.6 mm I.D. column) * For maximum operating pressure information, please refer to page 6. : μm : μm Length \ I.D Length \ I.D Length \ I.D Length \ I.D * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6. Cartridge Guard Column E I.D. of the Analytical Column Applicable Length I.D. Replacement Cartridge E Guard Column ( EA.) Cartridge E Holder / Cartridge Set ( Cartridge E Guard Columns & Holder) μm μm μm μm , , , , , Holder for Cartridge Guard Column E * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6. For 0 mm Length For 0 mm Length

11 InertSustain Phenyl Physical Properties Silica : Newly Developed ES Silica Gel : μm, μm, μm Surface Area : 0 m /g Pore Size : 00 Å (0 nm) Pore Volume : 0.8 ml/g Bonded Phase : Phenyl Groups End-capping : None Carbon Loading : 0 % USP Code : L ph Range : ~ 7. InertSustain Phenyl delivers an extremely unique reverse phase characteristics that are critical to resolving compounds that could not be separated on a C8 or C8 phase. InertSustain Phenyl provides not only pi-pi interactions, but also hydrogen bonding secondary interactions, which results in retaining polar compounds at the same time. As the phenyl groups are directly bonded to the silica gel, InertSustain Phenyl is compatible with the analysis of structural isomers due to it s high stereo-selectivity (fi gure ) while other alkyl phenyl type columns fails to separate. Figure : Comparison of Selectivity between InertSustain Phenyl and InertSustain C8, InertSustain Phenyl InertSustain C8 : μm, 0.6 mm I.D. : A) CHOH B) HO A/B = 70/0, v/v : 0.8 ml/min :0 C : UV nm. Butylbenzene. o -Terphenyl. Amylbenzene. Triphenylene Non - Planar Structure Planar Structure Figure : Analysis of Structural Isomers (Positional Isomers of Cresol),, Not Separated Not Separated InertSustain Phenyl Ascentis Phenyl XBridge Phenyl : μm, 0.6 mm I.D. : A)CHOH B)HO A/B = 0/80, v/v : 0 C : 0.8 ml/min : UV nm OH OH OH CH CH CH. o-cresol. m-cresol. p-cresol

12 InertSustain Phenyl Analytical Columns : μm HPSeries : μm 0 MPa (00 Bar) Length \ I.D Length \ I.D * End-fi ttings are /6 Waters-compatible. * UHPLC compatible end-fi ttings are also available upon request for UHPLC systems (Ex: UPLC) to avoid dead volume. * Indicate UP Type end-fi ttings when ordering. (Please note that UP type is not available for a.6 mm I.D. column) * For maximum operating pressure information, please refer to page 6. : μm : μm Length \ I.D Length \ I.D Length \ I.D Length \ I.D * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6. Cartridge Guard Column E I.D. of the Analytical Column Applicable Length I.D. Replacement Cartridge E Guard Column ( EA.) Cartridge E Holder / Cartridge Set ( Cartridge E Guard Columns & Holder) μm μm μm μm , , , , , Holder for Cartridge Guard Column E * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6. For 0 mm Length For 0 mm Length

13 InertSustain Phenylhexyl Physical Properties Silica : Newly Developed ES Silica Gel : μm, μm Surface Area : 0 m /g Pore Size : 00 Å (0 nm) Pore Volume : 0.8 ml/g Bonded Phase : Phenylhexyl Groups End-capping : Yes Carbon Loading : 9.0 % USP Code : L ph Range :.0 ~ 0.0 InertSustain Phenylhexyl columns are bonded with phenylhexyl groups, which employs a phenyl ring with a hexyl (6- carbon) linker and is densely bonded to our newly developed ES silica gel delivering complementary selectivity to straight alkyl-chain columns, but with industry leading inertness, lot-to-lot reproducibility and low back pressure. Figure : Comparison of Selectivity with other brands, 7, , InertSustain Phenylhexyl : μm, 0.6 mm I.D. : A) CHOH B) HO A/B = 70/0, v/v :.0 ml / min : 0 C : UV nm :. Uracil. Caffeine. Phenol. Butylbenzene. o-terphenyl 6. Amylbenzene 7. Triphenylene XBridge Phenyl Luna Phenyl-Hexyl Figure : Analysis of Acidic Compounds InertSustain Phenylhexyl XBridge Phenyl Luna Phenyl-Hexyl : μm, 0.6 mm I.D. : A) CHCN B) 0.% HPO A/B = /7, v/v :.0 ml/min : 0 C : UV nm :. Brilliant Blue FCF. Phenol. Salicylic acid 0 0 0

14 InertSustain Phenylhexyl Analytical Columns HP Series : μm 0 Mpa(00 Bar) Length \ I.D * End-fi ttings are /6 Waters-compatible. * UHPLC compatible end-fi ttings are also available upon request for UHPLC systems (Ex: UPLC) to avoid dead volume. * Indicate UP Type end-fi ttings when ordering. (Please note that UP type is not available for a.6 mm I.D. column) * For maximum operating pressure information, please refer to page 6. : μm : μm Length \ I.D Length \ I.D Length \ I.D Length \ I.D * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6. Cartridge Guard Column E I.D. of the Analytical Column Applicable Length I.D. Replacement Cartridge E Guard Column ( EA.) Cartridge E Holder / Cartridge Set ( Cartridge E Guard Columns & Holder) μm μm μm μm , , , , , Holder for Cartridge Guard Column E * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6. For 0 mm Length For 0 mm Length

15 InertSustain NH Physical Properties Silica : Series Newly Developed ES Silica Gel : μm, μm Surface Area : 0 m /g Pore Size : 00 Å (0 nm) Pore Volume : 0.8 ml/g Bonded Phase : Aminopropyl Groups End-capping : None Carbon Loading : 7 % USP Code : L8 ph Range : ~ 7. InertSustain NH is a newly developed ES silica column, compare to the other normal silica columns, InertSustain NH performs a sharp peaks, and high reproducible results with exceptional stability and durability that will maintain performance over the lifetime of the method. Also, InertSustain NH is an easily equilibrate column. Figure is an analysis sample to show the equilibrate time of InertSustain NH column. Using an column was replaced with isopropanol, phthalic acid ester as the sample and measured the time until stabilized the analysis conditions. From the chromatogram we can know at the total fl ow time of minutes of th injection, the column was stabled to be analyze. Figure : Analysis of Vitamin E with InertSustain NH & Inertsil NH column. α-tocopherol. β-tocopherol. γ-tocopherol. δ-tocopherol FLU InertSustain NH FLU Inertsil NH R= Column Injection Vol. : μm, 0.6 mm I.D. : A) n-hexane B) Ethyl acetate A/B = 8/, v/v :. ml/min : 0 C : FL Ex. 98 nm Em. nm : 0 μl : α-tocopherol ( μg/ml in Hexane) β-tocopherol ( μg/ml in Hexane) γ-tocopherol ( μg/ml in Hexane) δ-tocopherol ( μg/ml in Hexane) (C6H) Figure : Evaluation of equilibration time st injection (0 min) 0 6 Di-n-propylphthlate nd injection (7 min) 0 6 rd injection ( min) 0 6 Diethy phthalate th injection ( min) 0 6 Column : InertSustain NH ( μm, 0.6 mm I.D.) : A) n-hexane/isopropanol = 00/, v/v B) Isopropanol A/B = 9/, v/v (gradient mixer) :.0 ml/min : 0 C : UV 00 nm th injection (8 min) 0 6

16 InertSustain NH Physical Properties Silica : Newly Developed ES Silica Gel : μm, μm Surface Area : 0 m /g Pore Size : 00 Å (0 nm) Pore Volume : 0.8 ml/g Bonded Phase : Aminopropyl Groups End-capping : None Carbon Loading : 7 % USP Code : L8 ph Range : ~ 7. InertSustain NH shows far superior stability compared to other brand available aminopropyl columns, as our newly developed Evolved Surface Silica is chemically bonded with aminopropyl group. Generally, aminopropyl columns are used for applications that are hard to be separated in a reversed phase mode, such as simultaneous analysis of sugars or vitamin E. However, the shift in retention time has been an issue for a long time. InertSustain NH delivers highly reliable reproducibility and stability with accurate qualitative and quantitative results. Further more, aminopropyl columns generally can not be washed by weakly acidic eluent, however InertSustain NH was improved and it can be washed by weakly acidic eluent. Figure : Comparison of preventing anomer resolution of sugar analysis : A) CHCN B) HO A/B = 7/ :.0 ml/min, v/v : 0 C : RI :. Fructose ( mg/ml). Glucose ( mg/ml). Sucrose ( mg/ml). Maltose (0 mg/ml) InertSustain NH other brand Amide Column Figure : Comparison of stability between various Amino column Figure : Reliable reproducibility 0 InertSustain NH 00 Other Brands NH Column A Other Brands NH Column B Initial retention time (%) Lot. 80 Lot : μm, 0.6 mm I.D. :.0 ml/min : 0 C : RI Volume of eluent passing through the column (ml) :. Maltose : μm, 0.6 mm I.D. : A)CHCN B)HO A/B = 8/, v/v :.0 ml/min : 0 C : RI :. Fructose. Glucose. Sucrose. Moltose

17 InertSustain NH Analytical Columns : μm : μm Length \ I.D Length \ I.D Length \ I.D Length \ I.D * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6. Cartridge Guard Column E I.D. of the Analytical Column Applicable Length I.D. Replacement Cartridge E Guard Column ( EA.) Cartridge E Holder / Cartridge Set ( Cartridge E Guard Columns & Holder) μm μm μm μm , , , , , Holder for Cartridge Guard Column E * End-fi ttings are /6 Waters-compatible. * For maximum operating pressure information, please refer to page 6. For 0 mm Length For 0 mm Length