Classical Phases TM Hypersil Thermo Electron Corporation ...

1 Innovators in life and laboratory sciences, Thermo Electron Corporation providesadvanced analytical technologies, scientific instrumentation, laboratory informaticssolutions, and laboratory consumables to help scientists and clinicians to discovernew drugs, improve manufacturing processes, and diagnose illness and in our capabilities, we can help you every step of the way from samplepreparation and sample analysis through interpretation of Electron Corporationhas direct subsidiary offices inNorth America, Europe and complement thesedirect subsidiaries, we maintaina network of representativeorganizations throughout theworld. Use this reference list orvisit our web site to locate therepresentative nearest (33) 1 60 92 48 00 GermanyTel. (49) 6103 4080 United KingdomTel. (44) 1928 581000 United StatesTel. (01) 800 437 2999 For more information on our products and services,please visit our website at: information contained in this publication is forreference purposes only and is subject to change withoutnotice.

2 Every effort has been made to supply complete andaccurate information; however, Thermo Electron assumes noresponsibility and will not be liable for any errors, omissions,damage, or loss that might result from any use of theinformation contained therein (even if this information isproperly followed and problems still arise).Reference to specifications supersede all previous informationand are subject to change without , BetaBasic, BetaMax, BETASIL, BioBasic, DASH,DELTABOND, Duet, Fluophase, Hyperbond, Hypercarb, KAPPA,HOT POCKET, HyperGEL, HyperREZ, HyperSEP, Hypersil ,HyPURITY, HyPURITY AQUASTAR, Javelin, KEYSTONE, MultiSEP,PIONEER, PRISM, Retain, SLIPFREE, UNIGUARD, UNIPHASE, andVerify are trademarks of Thermo Electron Corporation and Siliconizing Fluid for treating glass surfaces is sold byPierce Chemical Co., Rockford, other trademarks are the property of their respective owners. 2003 Thermo Electron , Printed in USA 05 TM Classical Phasesincluding Hypersil ODSC olumnsTechnical GuideExceptionally reliable and reproducible columnsfor neutral and polar compounds2 Silica3, 5, (C18)3, 5, (C8)3, 5, (C8)3, 5, , 5, , 5, (C1)3, 5, (NH2)3, 5, (Cyano)3, 5, (Cyano)3, 5, (-NMe3) PhaseParticle SizePore SizePore VolumeSurface Area%CarbonEnd-Capped( )(cc/gm)(m2/gm) Hypersil Classical Phases and Hypersil ODS ColumnsIntroduction to Hypersil Classic Column RangeThermo Electron columns offer exceptional performance and documentation of quality, batchand colum QA information and validation going back to 1978.

3 Accredited under ISO9001:2000,the Thermo Electron HPLC column manufacturing plants insure strict adherence to quality,through the initial silica production, bonded phase production and finally to the manufactureof the HPLC columns themselves. In this Technical Guide we review the different HypersilClassical columns in terms of physical properties and usage, and then focus in greater detailon the quality assurance protocols associated with Hypersil ODS Classical ColumnsExceptionally reliable and reproducible columns for neutral and polar compoundsAll columns supplied with test certificatesProven, reproducible column efficiencyLong column lifetimes, even under basic conditionsWide range of bonded Phases with very low pressure dropOne of the world s most widely referenced column packing materials with a proventrack recordSpecifications:Urine Metabolites12345 Sample:1. p-Hydroxymandelic Acid2.

4 Vanilmandelic Acid3. 7-Methyluric Acid4. 1-Methyluric Acid5. Internal Standard02468 MINH301-5035H301-5055 Nitrofurazones in Shrimp036 MIN036 MIN(a) Shrimp Control(b) Shrimp fortified withNitrofurazone (NFZ) andFurazolidone (FZD)NFZFZDP eptides1234567 Sample:1. Cycloguanil2. 4-Chlorophenyl-Biguanide3. Quinine4. Proguanil5. Desethylchloroquine6. Chloroquine7. ChlorproguanilHypersil ODS, : 50% ACN / 50% KH2PO4, pH 60mM SLS and 10 mM TBAFlow: mL/minDetector: UV @ 254 Ref: Taylor et al Biom. An 10(1992) 867H301-504202550 MINC omparison of Retention on Hypersil ODS and MOS Columns24681012 MINH ypersil ODS , 5 m, : 60% ACN / 40% H2 OFlow: mL/minDetector: UV @ 254 Hypersil MOS, 5 m, : 60% ACN / 40% H2 OFlow: mL/minDetector: UV @ 2544532112354 Sample:1. Uracil2. Ethyl paraben3. Butyl paraben4. Naphthalene5. AnthraceneHypersil MOSH ypersil ODST echnical GuideHypersil Classical & ODS Columns7 Hypersil ODS, : EtOH in 10mM KH2PO4, pH : mL/minDetector: UV @ 280 Hypersil ODS, 5 m, : 25% ACN / 75% 1% aq.

5 Acetic AcidFlow: mL/minDetector: UV @ 375 Hypersil is a registered Trademark of Thermo Electron and its subsidiaries. 2002 Thermo Electron . All Rights MIN12 Sample:1. 4-Acetylbiphenyl2. StanozololHypersil ODS, 5 m, : 85% MeOH / 15% AmmoniumPhosphateFlow: mL/minDetector: UV @ 230 Courtesy of Cavrini V. et al Analyst 112 (1987) 1671 Figure 5 m ODS ColumnsPeak AsymmetryPeak asymmetry (peak tailing)gives a useful measure of the qual-ity of a column. Peak tailing is usu-ally observed when a column de-teriorates, but may also be ob-served if the columns are not wellpacked. The asymmetry ratio fora given peak is the width of thetail to the width of the front at 10%of the peak height. To pass speci-fication the asymmetry measure-ment must fall within a range Figure 5 demonstratesasymmetry measurements thatare used to monitor the effective-ness of the packing (N/m)AsymmetryIsosorbideH301-50600510 MIN12 Sample:1.

6 Isosorbide-2-Mononitrate2. Isosorbide-5-Mononitrate3. IsosorbidedinitrateHypersil ODS, 5 m, : 80% H2O / 20% MeOHFlow: mL/minDetector: UV @ 210 Courtesy of Azcona T. et al J. Pharm Biom An 09(1991) 7283 FMOC Amino AcidsHypersil ODS, 5 m, : A: Acetic Acid pH : MethanolFlow: 1 mL/minGradient: Time%B05020703090 Detector: Fluorescence151015202530 MIN12367121018171691911581541320142221 FluorescenceH301-5014standardsplant extractSamples:1. Asparagine2. Glutamine3. Citruline4. Aspartic Acid5. Serine6. Glutamic Acid7. FMOC-OH8. Glycine9. Ammonia10. Threonine11. Aminobutyric Acid12. Arginine13. Proline14. Methionine15. Valine16. Tryptophan17. Isoleucine18. Leucine19. Phenylalanine20. Ornithine21. Lysine22. HistidinePhase SummaryHypersil Silica is the base media for the Hypersil Classical range of bonded phase columns . The silicaitself is also an HPLC media that is a powerful and efficient tool in thechromatography of non-polar and moderately polar organic compounds.

7 Designed and first introduced tothe HPLC market in the mid 1970 s, the Classical Hypersil silica is a Type A silica having the followingcharacteristics:Made from aqueous sol with controlled amounts of metal ionActive toward bases; requires basic additives in the mobile phaseOne of the most stable silicas, even at high pHA number of bonded phase chemistries are available in the Classic Hypersil APS-2 has propyl amino groups bondedto the silica surface. This versatile phase exhibitsexcellent chromatographic properties in threemodes: weak anion exchange, reversed phase andnormal phase. The APS-2 phase can be used withcommon buffers and an organic modifier as a weakion-exchange material for the analysis of anionsand organic acids. When used as a normal phasematerial, APS-2 offers a different selectivity thansilica with less sensitivity to water in the mobilephase.

8 APS-2 used as a reversed phasematerialexcels for carbohydrate CPS is a cyanopropyl phase that can beused for both normal and reversed phase normal phase conditions this materialoffers different selectivity than Hypersil Silica andAPS-2. In addition to complementaryselectivity,CPS equilibrates very rapidly with the mobilephase, and is not sensitive to small quantities ofwater, making it the ideal choice for separationswhere gradient elution is a reversed phase material, the selectivity ofHypersil CPS complements the alkyl chain bondedphases. Short equilibration times make it an idealchoice for analytes needing gradient elution. TheCPS-2 phase is endcapped to produce a highquality stationary SAX is a silica based strong anion ex-changer, which is highly stable and robust, work-ing well in aqueous and low pH mobile Phases . Itis suitable for the analysis of smaller organicmolecules including nucleotides and organic SCX is a silica based strong cation ex-changer, which is highly stable and robust, work-ing well in aqueous and low pH mobile Phases .

9 Itis suitable for the analysis of smaller organicmolecules including nucleosides, and organic GuideHypersil Classical & ODS ColumnsHypersil ODS is an excellent reversed phase pack-ing for a large range of applications and is one ofthe world s most popular packings. Made withHypersil Silica as a base, a monolayer of octadecylsilane (C18) is bonded on to the silica surface. It isthen fully endcapped to minimize secondary inter-actions of analytes with residual silanol ODS is a highly efficient chromatographicmedium, showing the quality and reproducibilitytypical of the Hypersil Classical family. ODS is suit-able for the analysis of non-polar to moderatelypolar acids, neutrals and lipophilic compounds. Itis available in a range of sizes including narrow borecolumns ( and ). Narrow bore columnsprovide the proven characteristics of Hypersil ODSin a format that allows higher sensitivity for criticalapplications, together with a dramatic reduction insolvent Electron manufacturing prides itself onquality and stability.

10 The ODS bonded phase has adocumented history of achievement in reproduc-ibility and column efficiency. A full review of thequality assurance protocols for Hypersil ODS col-umns is MOS -This phase has a monolayer cov-erage of octylsilane (C8 alkyl chain) chemicallybonded onto the Hypersil silica surface. The MOS-2phase is end-capped to produce a high qualitystationary phase. MOS Phases are highly efficientreversed phase materials that exhibit similarselectivity to ODS, but are less SAS has a short alkyl chain (C1 ortrimethyl)chemically bonded onto the silica material is the least retentive of all the alkylgroup bonded Phases for non-polar solutes. SAShas unique selectivity for polar and multi-functionalcompounds and has been successfully used forion-pair GuideHypersil Classical & ODS ColumnsHypersil ODS columns inDetailHypersil ODS is an excellentpacking for a wide range of re-versed phase applications, andis one of the world s most popu-lar stationary phase with the renownedHypersil as a silica backbone, amonolayer of octadecyl silane iscovalently bonded to the silicasurface.