HPLC Column Technical Guide - GL Sciences

1 hplc ColumnTechnical GuideIntroductionSpecificationsShipping SolventMobile PhaseSample Diluent/Injection SolventColumn Cleaning and StorageTips to Increase hplc Column LifetimeBenefits of Scaling Down Column Internal Diameter SizeTips on Maximizing Efficiency on UHPLC ColumnsBenefits of Metal-Free PEEK ColumnsTips on Maximizing Purification Efficiency on Preparative columns (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(1 1)P 1P 1 4P 5 8P 9 11P 12P 13 17P 18 21P 22 26P 27 28P 29 30P 31 3322-1 Nishishinjuku, 6-ChomeShinjuku-ku, Tokyo 163-1130, JapanTEL: +81-35323-6620 FAX: +81-35323-6621 Web: : IntroductionThank you for choosing GL Sciences hplc columns .

2 GL Sciences hplc columns are subjected to a rigorous array of QC test in the ISO 9001 compliant facility, with special emphasis on reagent purity, raw material traceability and consistency in raw materials, and final products. To maintain and maximize peak performance of GL Sciences hplc columns , and ensure long life and stability, please read the following before use.(2) Specifications(2-1) Recommended Operating Pressure (Maximum Operating Pressure)GL Sciences hplc columns can tolerate up to pressures shown below. Although the columns are packed by high pressure slurry method, it is recommended to keep the operating pressure under the pressure shown below to maintain peak performance and ensure long Column life and ColumnsParticle SizeRecommendedOperating Pressure(MPa)

3 Inertsil series, InertSustain series,InertSustainSwift m, 2 m80 Inertsil series, InertSustain series,InertSustainSwift series3 m HP50 Inertsil series, InertSustain series, InertSustainSwift series, Titansphere3 10 m20 InertCore m100 InertSphere Sugar-15 m15 Capillary EX columns3 m, 5 m20 Capillary EX Nano columns3 m, 5 m15 Guard ColumnsRecommendedOperating Pressure(MPa)Guard Column for UHPLC80 Cartridge Guard Column ECartridge Guard Column EiGL-Cart, Pre-clean ORGPREP Guard Cartridge20 Conventional Guard ColumnConventional Mini Guard ColumnCapillary Micro GuardPreparative Guard Column20 Recommended Operating Pressure for analytical columns Recommended Operating Pressure for Guard Columns1 ColumnsMaximumOperatingTemperature20 40 C40 50 C50 60 CInertsil series, TitansphereInertSustain C18, InertSustain AQ-C18 InertSustainBio C18, InertSustainSwift C181 - 7 InertSustain C8, InertSustain Phenylhexyl,InertSustainSwift C81 - 10 (*)1 - 9 (*)

4 2 - 7 InertSustain Amide2 - 7 InertSustain Phenyl, InertSustain NH2 InertCore C18 InertSphere Sugar-180 C2 - 14 (5 80 C)60 C2 - Range1 - 10 (*)2 - - - (*)(2-2) Temperature and pH RangeThe recommended operating pH range and temperatures are shown below. To maximize Column lifetime, set the pH of the mobile phase within the range shown below. Additionally, use mixed mobile phase such as organic solvent and buffer rather than 100 % buffer. When operating at high pH, lower operating temperatures are recommended for longer Column lifetime. Working at high temperatures may also result in shorter Column lifetimes.

5 Please note that the Column lifetime will vary depending upon the operating temperature, the type, and concentration of buffer used.* For method development at low pH (pH 1 2), the usage of either trifluoroacetic acid, formic acid, acetic acid or phosphate buffer is recommended. At high pH (pH 10), the buffer concentration should be adjusted at 5 mM using buffers such as trimethylamine. In case an organic solvent is not present in the buffer mobile phase, set the pH in the range within 2 operating temperatures are recommended for longer Column lifetime at pH 1 2 or pH 9 10. Additionally, it is recommended to have an organic solvent ( , methanol) present in yourmobile (2-3) Column End-fittingsThe specification of GL Sciences end-fitting styles are summarized below.

6 The chromatographic separation and result can be negatively impacted if the style of the Column end-fittings does not match the existing tubing ferrule ColumnsParticle SizeEnd-fitting Style InertSustain m UP TypeInertSustainSwift C18, InertSustainSwift m 3 m HPUP TypeInertSustain C18, InertSustain C8, InertSustain PhenylInertsil series2 mW TypeInertSustain AQ-C18, InertSustainC18, InertSustain C8,InertSustain Phenylhexyl, InertSustain Phenyl,InertSustain Amide, Inertsil series, Unisil Q series,Titansphere3 m HP 3 10 mW TypeUHPLC PEEK, PEEK columnsAll Particle SizesUP TypeInertCore mUP TypeInertSphere Sugar-15 mW TypeCapillary EX columns3 m 5 mUP TypeCapillary EX Nano columns3 m 5 mUP Type End-fitting Styles for analytical columns * Each end-fitting style differs in the required length of the tubing protruding from the ferrule.

7 The UP type requires a mm ferrule depth. The W type requires a mm ferrule more details, please see page ColumnsEnd-fitting StylesCartridge Guard Column ECartridge Guard Column EiGL-Cart, Pre-clean ORGPREP Guard CartridgeW TypeGuard Column for UHPLCUP TypeConventional Guard ColumnConventional Mini Guard ColumnCapillary Micro GuardPreparative Guard ColumnW Type End-fitting Styles for Guard columns Various End-fitting StylesVarious Column manufacturers have employed different types of chromatographic Column connectors. The following explains the differences in each type. mm10-32 UNF T mm10-32 UNF WType UP Type (Parker style) mm ON mm HA Type10-32 UNF4(3) Shipping Solvent(3-1) Various Shipping SolventsGL Sciences hplc columns are shipped in the solvents shown below.

8 Please be reminded that normal-phase columns are shipped in non-aqueous solvents. (3-2) Column Equilibration Prior to UseIt is important to first understand the mobile phase compatibility before changing to a different mobile phase system. This understanding will help to avoid precipitating mobile phase buffers on your Column as well as in your system. Before injecting the sample, thoroughly equilibrate the Column with the mobile phase to be used to ensure stable chromatographic separation. For more details, please refer to the following. Column Equilibration Procedure for Reversed-Phase columns (C18)If the mobile phase does not contain buffers or additives, equilibrate the Column and system with the mobile phase to be used for at least 30 minutes.

9 The Column may be considered equilibrated once a steady Column back pressure and baseline are the mobile phase contain buffers or additives, equilibrate the Column and system with a water/organic solvent mixture, using the same content as in the buffered mobile phase. For example, equilibrate the Column and system with 20 % acetonitrile in water for at least 30 minutes. Then, introduce and equilibrate with 20 % acetonitrile / 80 % buffered mobile phase for at least 30 minutes. The Column may be considered equilibrated once a steady Column back pressure and baseline are observed. Column Equilibration Procedure for Normal-Phase ColumnsIf the mobile phase does not contain buffers or additives, equilibrate the Column and system with the mobile phase to be used for at least 30 minutes.

10 The Column may be considered equilibrated once a constant Column back pressure and baseline are Size, DimensionsShipping SolventReversed-PhaseColumns InertSustain C18, C8, AQ-C18, Phenylhexyl, Phenyl Amide InertSustainSwift C18, C8 Inertsil ODS-3V, ODS-4V, Amide, HILIC, ODS-HL ODS-P ODS-EP ODS-80A C8-4, C8-3, C8, C4, Ph-3, Ph, WP300 C18, WP300 C8, WP300 C4, ODS, C30, Peptides, Acrolein, Sulfa InertCore C18 MonoTower C18, MonoClad C18-HSAll Particle Sizes&All DimensionsAcetonitrile/Water5 m 250 x mm m 150 x mm Particle Sizes, DimensionsAcetonitrile/WaterNormal-Phase columns InertSustain NH2 Inertsil NH2 Diol CN-3 SIL-100A SIL-150A WP300 SIL WP300 Diol, Titansphere TiOAll Particle Sizes&All Dimensionsn-Hexane/EthanolIon-ExchangeCo lumns Inertsil AX CXAll Particle Sizes&All Dimensions100 % MethanolPolymer columns InertSphere Sugar-1 All Particle Sizes&All Dimensions100 mMSodium HydroxideAqueous Solution Inertsil ODS-4, ODS-3, ODS-SP, ODS-2 Methanol/WaterReversed-PhaseColumns5If the mobile phase contain buffers or additives, equilibrate the Column and system with a mixture of solvents.