NUCLEODUR. Professional solutions for HPLC. An optimized phase for every field of application.

Transcription

1 NUCLEODUR Professional solutions for HPLC An optimized phase for every field of application

2 Contents / Customer service NUCLEODUR high purity silica Overview of NUCLEODUR phases Summary of available modifications and properties NUCLEODUR RP phases with.8 μm particle size C 8 Gravity / C 8 Gravity nonpolar high density phases 8 C 8 Isis high steric selectivity C 8 Pyramid for highly aqueous eluents PolarTec RP phase with embedded polar group NEW! PFP hydrophobic pentafluorophenyl phase NEW! 8 Sphinx RP bifunctional RP phase 0 C 8 HTec base-deactivated preparative phase NEW! C 8 ec / C 8 ec nonpolar phases for routine analyses HILIC zwitterionic ammonium sulfonic acid phase for HILIC CN / CN-RP cyano-modified high purity silica 8 NH / NH -RP amino-modified high purity silica 0 unmodified high purity NUCLEODUR silica Applications Pharmaceuticals and drugs Food analysis Biological and natural compounds Pollutants and miscellaneous organics Alphabetical index of analytes Index of application numbers / Trademarks 70 Ordering information of packed columns 7 Meeting your needs If you have any questions concerning our NUCLEODUR program or other chromatography products, please feel free to contact us: Technical support and customer service phone tech-chroma@mn-net.com The MACHEREY-NAGEL internet catalog with integrated webshop is full of useful information about our wide product range. In addition our online database offers more than 000 applications which might actually already solve your analytical questions. Germany and international Phone Toll-free Fax or -98 info@mn-net.com Ask for our application guides USA Phone Toll-free Fax (MACH) sales-us@mn-net.com France Phone Fax sales-fr@mn-net.com Switzerland Phone Fax sales-ch@mn-net.com MN

3 High purity silica NUCLEODUR NUCLEODUR is a fully synthetical type B silica (silica of rd generation) offering highly advanced physical properties like totally spherical particle shape, outstanding surface smoothness, high pressure stability and low metal content. NUCLEODUR as a state-of-the-art silica is the ideal base material for modern HPLC phases. It is the result of MACHEREY-NAGEL s pioneering research in chromatography for more than 0 years and worthy successor of MN s world famous NUCLEOSIL silica. In RP liquid chromatography the efficiency of the packing is strongly affected by the quality of the base silica itself. Shortcomings in the surface geometry of the particles or metal contaminants are the main reasons for inadequate coverage with the covalently bonded alkylsilanes in the subsequent derivatization steps. It is well known, that poor surface coverage and, in consequence, high activity of residual free silanols often results in peak tailing or adsorption, particularly with basic compounds. Particle shape and surface symmetry NUCLEODUR is virtually free of metal impurities and low acidic surface silanols. Elemental analysis data of NUCLEODUR µm measured by AAS are listed below. Elementary analysis (metal ions) of NUCLEODUR 00- Aluminium < ppm Iron < ppm Sodium < ppm Calcium < 0 ppm Titanium < ppm Zirconium < ppm Arsenic < 0. ppm Mercury < 0.0ppm Pressure stability The totally spherical and 00 % synthetic silica gel exhibits an outstanding mechanical stability, even at high pressures and elevated eluent flow rates. In addition, after several cycles of repeated packing, no significant drop in pressure can be observed. The latter is of prime importance for preparative and process-scale applications. Physical data of NUCLEODUR Surface area (BET) 0 m /g Pore size 0 Å Pore volume 0.9 ml/g NUCLEODUR modifications NUCLEODUR silicas are synthesized in a unique and carefully controlled manufacturing process which provides silica particles, which are totally spherical. The picture shows the outstanding smoothness of the NUCLEODUR surface. Purity As already mentioned above, a highly pure silica is required for achieving symmetric peak shapes and maximum resolution. Inclusions of, e.g., iron or alkaline earth metal ions on the silica surface are largely responsible for the unwanted interactions with ionizable analytes, e.g., amines or phenolic compounds (see appl. 80 on page ). Several different surface modifications based on NUCLEODUR silica have been developed over the last years providing a full range of specified HPLC phases and an ideal tool for every separation: NUCLEODUR C 8 Gravity and C 8 Gravity NUCLEODUR C 8 Isis NUCLEODUR C 8 Pyramid NUCLEODUR PolarTec NUCLEODUR PFP NUCLEODUR Sphinx RP NUCLEODUR C 8 HTec NUCLEODUR C 8 ec and C 8 ec NUCLEODUR HILIC NUCLEODUR CN and CN-RP NUCLEODUR NH and NH -RP unmodified NUCLEODUR All phases are described in detail on the following pages.

4 Applications Overview of NUCLEODUR HPLC phases Phase Specification Characteristics* Stability Structure octadecyl phase, high density coating, multi-endcapping 8 % C USP L A B C ph stability, suitable for LC/MS NUCLEODUR (Si-O ) n octyl phase, high density coating, multi-endcapping % C USP L7 A B C ph stability, suitable for LC/MS NUCLEODUR (Si-O ) n octadecyl phase with specially crosslinked surface modification, endcapping 0 % C USP L A B C ph stability 0, suitable for LC/MS NUCLEODUR (Si-O ) n C 8 modification with polar endcapping % C USP L A B C stable against 00 % aqueous eluents, ph stability 9, suitable for LC/MS NUCLEODUR (Si-O ) n OH OH octadecyl phase with embedded polar group, endcapping. % C USP L and L0 A B C stable against 00 % aqueous eluents, ph stability 9, suitable for LC/MS NUCLEODUR (Si-O ) n Pol Si OH Pol Si O Si(CH ) pentafluorophenyl-propyl modification with multiendcapping 8 % C USP L A B C ph stability 9, suitable for LC/MS NUCLEODUR (Si-O ) n Si OH F F F F F Si O Si(CH ) bifunctional RP phase, propylphenyl and C 8 ligands, endcapping % C USP L and L A B C ph stability 0, suitable for LC/MS NUCLEODUR (Si-O ) n octadecyl phase with high capacity, high density coating, multi-endcapping 8 % C USP L A B C ph stability, suitable for LC/MS NUCLEODUR (Si-O ) n * A = hydrophobic selectivity, B = polar / ionic selectivity, C = steric selectivity

5 An optimized phase for every Applications separation Application Similar phases** Separation principle Retention mechanism in general compounds with ionizable functional groups such as basic pharmaceuticals and pesticides NUCLEOSIL C 8 HD XTerra RP8 / MS C 8 ; Luna C8(), Gemini, Synergi Max RP; Zorbax Extend-C8; Inertsil ODS III; Purospher STAR RP-8; Hypersil BDS only hydrophobic interactions (van der Waals interactions) Si(CH ) H C CH N O like C 8 Gravity, however generally shorter retention times for nonpolar compounds NUCLEOSIL C 8 HD XTerra RP8 / MS C 8 ; Luna C8; Zorbax Eclipse XDB-C8 only hydrophobic interactions (van der Waals interactions) Si(CH ) OH OH CH N CH O high steric selectivity, thus suited for separation of positional and structural isomers, planar/nonplanar molecules NUCLEOSIL C 8 AB Inertsil ODS-P; Pro C8 RS; Zorbax SB steric interactions and hydrophobic interactions basic pharmaceutical ingredients, very polar compounds, organic acids Aqua, Synergi Hydro-RP; AQ; Atlantis dc 8 hydrophobic CH OH interactions and N polar interactions H C (H bonds) O basic pharmaceuticals, organic acids, pesticides, amino acids, water-soluble vitamins NUCLEOSIL C 8 Nautilus ProntoSIL C8; Zorbax Bonus-RP, Polaris Amide-C8; Ascentis RP Amide; SymmetryShield RP8; SUPELCOSIL LC- ABZ+; HyPURITY ADVANCE hydrophobic interactions and polar interactions (H bonds) Pol Si(CH ) Pol HO aromatic and unsaturated compounds, halogen compounds, phenols, isomers, polar pharmaceuticals, antibiotics ACQUITY CSH Fluoro-Phenyl; Hypersil GOLD PFP; Luna PFP(); Discovery HS F; Allure PFP Propyl, Ultra II PFP Propyl polar interactions (H bonds), dipole-dipole interactions π-π interactions and hydrophobic interactions F F O O F H F F compounds with aromatic and multiple bond systems no similar phases π-π interactions and hydrophobic interactions NO robust and well base deactivated C 8 phase; all separation tasks with preparative potential XTerra RP8 / MS C 8 / SunFire C 8 ; Luna C8(), Gemini, Synergi Max RP; Zorbax Extend-C8; Inertsil ODS III; Purospher STAR RP-8; Hypersil BDS only hydrophobic interactions (van der Waals interactions) Si(CH ) H C CH N O ** phases which provide a similar selectivity based on chemical and physical properties

6 Applications Overview of NUCLEODUR HPLC phases Phase Specification Characteristics* Stability Structure octadecyl phase, medium density coating endcapping 7. % C USP L A B C ph stability 9 NUCLEODUR (Si-O ) n Si OH Si O Si(CH ) octyl phase, medium density coating endcapping 0. % C USP L7 A B C ph stability 9 NUCLEODUR (Si-O ) n Si OH Si O Si(CH ) zwitterionic ammonium sulfonic acid modification 7 % C A B C ph stability 8., suitable for LC/MS NUCLEODUR (Si-O ) n Si Si CH + N SO OH CH CH + N SO OH CH cyano (nitrile) phase for NP and RP separations 7 % C USP L0 A B C ph stability 8, stable towards highly aqueous mobile phases NUCLEODUR (Si-O ) n C N Si OH C N Si O Si(CH ) amino phase for NP and RP separations. % C USP L8 A B C ph stability 8, stable towards highly aqueous mobile phases NUCLEODUR (Si-O ) n Si Si NH OH NH OH unmodified high purity silica USP L A B n.a. C ph stability 8 NUCLEODUR (Si-O ) n Si Si OH OH * A = hydrophobic selectivity, B = polar / ionic selectivity, C = steric selectivity

7 An optimized phase for every Applications separation Application Similar phases** Separation principle Retention mechanism robust C 8 phase for routine analyses NUCLEOSIL C 8 Spherisorb ODS II, Symmetry C 8 ; Hypersil ODS; Inertsil ODS II; Kromasil C 8 ; LiChrospher RP-8 only hydrophobic interactions (van der Waals interactions) some residual silanol interactions Si(CH ) SiOH H C CH N O robust C 8 phase for routine analyses NUCLEOSIL C 8 ec / C 8 Spherisorb C 8, Symmetry C 8 ; Hypersil MOS; Kromasil C 8 ; LiChrospher RP-8 only hydrophobic interactions (van der Waals interactions) some residual silanol interactions Si(CH ) SiOH H C N N O CH N N O CH hydrophilic compounds such as organic polar acids and bases, polar natural compounds SeQuant ZIC -HILIC; Obelisc ionic / hydrophilic interactions, electrostatic interactions H C N + SO CH O CH HO N NH H C NH N + SO CH polar organic compounds (basic drugs), molecules containing π electron systems NUCLEOSIL CN / CN-RP π-π interactions, polar interactions (H bonds), hydrophobic interactions C N C N HO O sugars, sugar alcohols and other hydroxy compounds, DNA bases, polar compounds in general NUCLEOSIL NH / NH -RP polar / ionic interactions, hydrophobic interactions + NH O OH polar compounds in general unmodified NUCLEOSIL polar / ionic interactions SiOH O N ** phases which provide a similar selectivity based on chemical and physical properties

8 Applications.8 μm particle size Key features Decrease of analysis time (ultra fast HPLC) Shorter columns with high separation efficiency Significant improvement of resolution and detection sensitivity Suitable for LC/MS due to low bleeding characteristics NUCLEODUR.8 μm particles are fractionated to limit the increase in back pressure NUCLEODUR phases available in.8 μm: C 8 Gravity C 8 Gravity C 8 Isis C 8 Pyramid Sphinx RP C 8 HTec HILIC Advantages of.8 µm particle size Miniaturization in HPLC has a long history. It started in the early stage of HPLC development with the reduction of particle size from 0 µm via 7 μm to standard µm which is still the most widely used particle diameter in analytical HPLC to µm spherical particles which so far was the smallest particle size available for gaining higher theoretical plates and efficiencies. With the introduction of.8 µm NUCLEODUR particles researchers have turned over a new leaf in HPLC column technology. Columns packed with these microspherical particles show extraordinary improvements in terms of plate numbers, column efficiencies and resolution compared with their µm counterparts. Features of.8 µm NUCLEODUR silica particles Increase of separation efficiency by higher number of theoretical plates (N): 0 x. mm NUCLEODUR C 8 Gravity µm: N plates/m (h value 0).8 µm: N 7 plates/m (h value ) Increase of the plate number by app. 7 % offers the possibility of using shorter columns with equal plate numbers resulting in a decrease of analysis time. Significant improvement in resolution Use of.8 μm instead of µm particles leads to an increase of resolution by a factor.9 (9 %) since the resolution is inversely proportional to the square root of the particle size: R s a k i N d P R s = N α α k i k i + = resolution = selectivity (separation factor) = retention = plate number with N /d P = particle size Resolution as a function of particle size Column: 0 x mm NUCLEODUR C 8 Gravity A) μm, B).8 μm Eluent: acetonitrile water (80:0, v/v) Flow rate: ml/min, pressure: A) 80 bar, B) 0 bar Detection: UV, nm. Naphthalene. Ethylbenzene A) B) μm.8 μm R s =. R s =. 80 bar 0 bar min

9 Increase in separation Applications efficiency Column back pressure Due to the smaller particle size the back pressure will increase according to D P = F L C η u d P D P = pressure drop F = flow resistance (nondimensional) L C = column length h = viscosity u = linear velocity = particle diameter d P Because of the high sphericity of the NUCLEODUR particles and the very narrow particle size distribution we were able to keep the back pressure on a moderate level. Nevertheless the use of columns packed with sub µm particles generally makes special demands on the HPLC equipment. Pumps should be designed for pressures of bars and the entire system should feature the lowest possible dead volume. Comparison of back pressures: Eluent: 00 % methanol Flow rate:. ml/min Temperature: C Column dimension: 0 x. mm NUCLEODUR C 8 Gravity Competitor A µm 70 bar.8 µm 0 bar 70 bar HETP [μm] Higher flow rates and shorter run times optimal flow rate for.8 µm particles is higher than for and µm particles (see figures the flow rate should be at the van-deemter minimum) Van-Deemter plot column 0 x. mm, acetonitrile water (0:0, v/v), analyte toluene μm 0 μm 0.8 μm u [mm/s] Technical requirements To gain the best result in ultra fast HPLC based on.8 μm particles certain technical demands on the instrument are made. Pumps for pressures of bar realizing a flow rate of ml are required. The dead volume of the LC system has to be reduced to a minimum. In addition, fast data recording is necessary for an optimum chromatographic result. Currently the following NUCLEODUR premium phases (C 8 Gravity, C 8 Gravity, C 8 Isis, C 8 Pyramid, Sphinx RP, C 8 HTec, HILIC) are available in.8 μm. The description of each phase and its selectivity can be found in the individual chapters. More applications on NUCLEODUR.8 µm can be found in the "Applications" section from page. Reduction of analysis time Column: 0 x mm (for μm x mm) NUCLEODUR C 8 Isis Eluent: 00 % methanol Flow rates and pressures see figure Detection: UV, nm m-terphenyl o-terphenyl Triphenylene p-terphenyl.8 μm,.00 ml/min (0 bar).8 μm,.00 ml/min (0 bar).8 μm,. ml/min ( bar) μm, 0.8 ml/min (0 bar) min 7

10 C 8 Gravity / C 8 Gravity Key features: Suitable for LC/MS and HPLC at ph extremes (ph ) Superior base deactivation Ideal for method development Technical characteristics: Available as octadecyl (C 8 ) and octyl (C 8 ), multi-endcapped; pore size 0 Å; particle sizes.8 μm, μm and μm for C 8,.8 and μm for C 8 ; 7, 0, and μm particles for preparative purposes on request; carbon content 8 % for C 8, % for C 8 Recommended application: Overall sophisticated analytical separations Compound classes separated include: pharmaceuticals, e.g., analgesics, anti-inflammatory drugs, antidepressants; herbicides; phytopharmaceuticals; immunosuppressants USP L (C 8 ) / USP L7 (C 8 ) Base deactivation NUCLEODUR C 8 Gravity and NUCLEODUR C 8 Gravity are based on the ultrapure NUCLEODUR silica. A unique derivatization process generates a homogeneous surface with a high density of bonded silanes (carbon content ~8 % for C 8, ~ % for C 8 ). The following thorough endcapping suppresses any unwanted polar interactions between the silica surface and the sample, which makes Gravity particularly suitable for the separation of basic and other ionizable analytes. The figure on the right shows a comparison study, where the strongly basic amitriptyline is eluted on various highly base deactivated C 8 phases under isocratic conditions. For a discussion of the different retention behavior of octadecyl phases compared to octyl phases see page. Tanaka diagrams Several NUCLEODUR phases have been examined in accordance with Tanaka et al. [J. Chromatogr. Sci. 7 (989) 7] and Johnson et al. [Chromatographia (997) ] with respect to the following parameters: Capacity = k (pentylbenzene) Hydrophobicity = α(pentylbenzene, butylbenzene) Steric selectivity = α(triphenylene, o-terphenyl) Hydrogen bonding capacity (silanol capacity) = α(caffeine, phenol) Ion exchange capacity at different ph values (.7 and 7.) = α(benzylamine, phenol) The resulting Tanaka plots are shown with the respective phases. Tanaka plots of NUCLEODUR C 8 and C 8 Gravity capacity 0 ion exchange capacity ph.7 0. C 8 hydrophobicity C 8 ion exchange capacity ph steric selectivity hydrogen bonding capacity 8

11 Nonpolar high density Applications phases Comparison of different base deactivated phases Columns: 0 x mm, all phases C 8, μm A) NUCLEODUR Gravity B) phase I C) phase L ( and overlap) D) phase P E) phase S Eluent: methanol 0 mm KH PO, ph 7.0 (7:, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm D E. Dibutyl phthalate. Acenaphthene. Amitriptyline When is enhanced ph stability beneficial? The option to work at an expanded ph range is often required in method development. Many nitrogen containing compounds like basic drugs are protonated at acidic or neutral ph and exhibit poor retention on a standard C 8 phase. The retention behavior can be improved by working at a higher ph, where the analyte is no longer protonated, but formally neutrally charged, as a rule between ph 9 0. For acidic analytes it is exactly in inverse proportion, maximum retention can be attained at low ph. Surface silanols at different ph values silica matrix (Si-O ) n O O O O O O O O Si Si Si OH Me OOC Me MeHN OH Me OH OH C B silica matrix (Si-O ) n OH OH O Si OH OH O Si OH O Si + NH Me H + Me Me HOOC H + H + H + Me A 0 0 min MN Appl. No. 080 The figure above shows the extent of protonation of surface silanols and of two exemplary analytes at acidic and alkaline ph. The following graph explains the general correlation between retention and ph. Correlation between retention and ph for basic and acidic compounds Enhanced ph stability One major disadvantage of using silica stationary phases is the limited stability at strongly acidic or basic ph ranges. Cleavage of the siloxane bonding by hydrolysis, or dissolution of the silica will rapidly lead to a considerable loss in column performance. Therefore conventional RP phases are usually not recommended to be run with mobile phases at ph > 8 or ph < for extended periods of time. The special surface bonding technology and the low concentration of trace elements of NUCLEODUR C 8 / C 8 Gravity allow for use at an expanded ph range from ph to. kʼ HA BH ph A B 9

12 Applications C 8 Gravity / C 8 Gravity As it was previously mentioned, ph stability of the stationary phase can be helpful for improving selectivity in method development. The figure below shows the separation of basic drugs under acidic and basic conditions. Separation of basic alkaloids Column: x mm NUCLEODUR C 8 Gravity, μm Eluent A: acetonitrile Eluent B: 0 mm (NH ) HPO, ph. / 0.0 Gradient: 0 % A ( min) 7 % A in 0 min Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Lidocaine. Papaverine. Noscapine. Diphenhydramine ph. ph 0.0 A further example how selectivity can be controlled by the ph value is demonstrated below. The sample mixture consists of an acid (ketoprofen), a base (lidocaine) and benzamide. Under acidic conditions the protonated lidocaine is eluted very fast due to lack of sufficiently strong hydrophobic interactions between analyte and C 8 chains, in contrary to the formally neutral ketoprofen, which is eluted after about minutes. However at ph 0 a reversal of the elution order, with a visibly longer retention time for the basic lidocaine, can be achieved. Influence of the ph value on selectivity Column: x mm NUCLEODUR C 8 Gravity, μm Eluents: A) acetonitrile 0 mm ammonium formate, ph.0 (0:0, v/v) B) acetonitrile 0 mm ammonium bicarbonate, ph 0.0 (0:0, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: μl. Lidocaine. Benzamide. Ketoprofen H C CH NH N(C H ) O O NH 0 min MN Appl. No. 800 At ph. the protonated analytes exhibit poor retention (early elution) and in addition an inadequate resolution for papaverine and noscapine, whilst the formally non ionized molecules can be baseline separated due to the better retention pattern at alkaline ph. O ph.0 H C O OH ph min MN Appl. No

13 Nonpolar high density Applications phases The following chromatograms demonstrate the stability of NUCLEODUR C 8 Gravity under alkaline conditions in comparison with commercially available modern RP8 phases. Again, the ultrapure Gravity with its unique high density surface bonding technology withstands strong alkaline mobile phase conditions. Even after 00 injections no loss of column efficiency, identified, e.g., by peak broadening or decrease in retention times, could be observed. Stability of NUCLEODUR C 8 Gravity under alkaline conditions compared with different C 8 phases Columns: 0 x. mm Eluent: methanol water ammonia (0:80:0., v/v/v), ph Flow rate:. ml/min Temperature: 0 C Detection: UV, nm Injection volume:.0 μl. Theophylline. Caffeine Phase X Phase L 0 0 min The ph stability of silica under alkaline conditions is mainly a kinetic effect and based on the velocity of the dissolution of the silica support. It is worth mentioning, that this phenomenon also depends on type and concentration of buffers, as well as on the temperature. It is well known, that the use of phosphate buffers, particularly at elevated temperatures, can reduce column lifetime even at moderate ph. If possible, phosphate buffers should be replaced by less harmful alternatives. The following chromatograms show the excellent column stability of NUCLEODUR C 8 Gravity in acidic conditions. The retention time of all three compounds in the column performance test remains consistent and virtually unchanged, even after the column is run with 000 ml eluent. Due to the extremely stable surface modification, no cleavage of the Si-O-Si bonding occurs, column deterioration is therefore successfully prevented. Stability of NUCLEODUR C 8 Gravity at ph. Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: acetonitrile % TFA in water (0:0, v/v), ph. Flow rate:.0 ml/min Temperature: 0 C, Detection: UV, 0 nm Injection volume: μl. Pyridine. Toluene. Ethylbenzene Phase K Phase I 0 0 min st injection after 00 injections after 000 ml eluent st injection 0 0 min NUCLEODUR C 8 Gravity 0 min MN Appl. No. 080 MN Appl. No. 080 For comparison of the selectivity of NUCLEODUR C 8 Gravity and C 8 Gravity please also see the application Retention behavior of different NUCLEODUR phases on page. Some general selection criteria and principals of different retention and selectivity of C 8 and C 8 columns can be found on page.

14 C 8 Isis Key features: Exceptional steric selectivity Outstanding surface deactivation Suitable for LC/MS and HPLC at ph 0 Technical characteristics: C 8 phase with special polymeric, crosslinked surface modification; pore size 0 Å; particle sizes.8 μm, μm and μm; carbon content 0 % Recommended application: Steroids, (o, p, m-) substituted aromatics, fat-soluble vitamins USP L Surface modification By use of specific C 8 silanes and appropriate polymeric bonding technologies a dense shield of alkyl chains protects the subjacent silica matrix. Elemental analysis of NUCLEODUR C 8 Isis shows a carbon load of 0 %. The target crosslinking of the C 8 chains on the surface enables the separation of compounds with similar molecular structure but different stereochemical properties. The technical term for this feature is steric selectivity. The chromatograms on the right reveal the improved resolution for positional isomers in a test mixture of aromatic compounds on NUCLEODUR C 8 Isis () in direct comparison with monomerically coated () and polar endcapped () C 8 columns. 0. Tanaka plot of NUCLEODUR C 8 Isis ion exchange capacity ph.7 ion exchange capacity ph capacity 0 0. hydrophobicity steric selectivity hydrogen bonding capacity Steric selectivity of NUCLEODUR C 8 Isis Columns: x mm; NUCLEODUR C 8 Isis, monomerically coated C 8 phase, polar endcapped phase Eluent: methanol water (90:0, v/v) Flow rate: ml/min, temperature: C Detection: UV, nm Injection volume: μl. o-terphenyl. m-terphenyl. p-terphenyl. Triphenylene polar endcapped C 8 monomeric C 8 NUCLEODUR C 8 Isis 0 min The separation of o-terphenyl and triphenylene is a concrete example to evaluate the selectivity potential of a reversed phase column in terms of the different shape of two molecules. The phenyl rings of o-terphenyl are twisted out of plane while triphenylene has a planar geometry.

15 High steric selectivity The separation factor (α-value) is a measure for the steric selectivity. As is shown in the following chromatograms the α-value is considerable larger on NUCLEODUR C 8 Isis compared to a conventional C 8 column. Steric selectivity of NUCLEODUR C 8 Isis Columns: x mm Eluent: methanol water (80:0, v/v) Flow rate: ml/min, temperature: 0 C Detection: UV, nm, injection volume: μl. o-terphenyl,. Triphenylene monomerically coated C 8 phase α =. 0 0 NUCLEODUR C 8 Isis α = min Sander and Wise [LCGC 8 (990) 78 90] proposed a model for the retention of aromatic compounds based on molecular shape, which is referred to as Slot Model. This model pictures the bonded C 8 phase on the silica surface with slots which the analytes have to penetrate during retention. Planar molecules are able to penetrate these slots deeper than nonplanar molecules of similar molecular weight and length-to-breadth ratio. Thus triphenylene is longer retained than o-terphenyl. Slot model Stability The applied special surface bonding technology also provides improved stability features for the NUCLEODUR C 8 Isis phase. The proof for this was given in a longterm test in which the decrease of plate counts for caffeine at ph 0 and 0 C has been observed over a period of 00 hours and 000 sample injections, respectively. In addition retention and peak shape of caffeine and theophylline were compared with the chromatographic performance of the brand new column. Stability of NUCLEODUR C 8 Isis at ph 0 Column: x mm NUCLEODUR C 8 Isis, μm Eluent: methanol 0 mm triethylamine, ph 0 (:80, v/v) Flow rate: ml/min, temperature: 0 C Detection: UV, nm, injection volume: μl. Theophylline,. Caffeine column)0 % N(caffeine) (brand new number of injections after 000 injections (00 hours) st injection min The following chromatograms exhibit the excellent stability of NUCLEODUR C 8 Isis at ph and 80 C. After 700 column runs retention time and peak shape of the three test compounds remain almost unchanged. Stability of NUCLEODUR C 8 Isis at ph Column: x mm NUCLEODUR C 8 Isis, μm Eluent: acetonitrile % TFA, ph (0:0, v/v) Flow rate: ml/min, temperature: 80 C Detection: UV, nm, injection volume: μl. Pyridine,. Toluene,. Ethylbenzene after 700 injections st injection 0 min Surface deactivation The chromatography of basic analytes requires a high density of surface-bonded C 8 silanes combined with a thorough endcapping procedure to keep silanol activity at a minimum. This ensures tailing-free elution of even strongly basic amino-containing compounds (see appl. 0 on page ). NUCLEODUR C 8 Isis does not show any degradation under the applied mobile phase conditions. An enhanced ph stability in the range from ph to 0 can be certified for this phase.

16 C 8 Pyramid Key features: Stable in 00 % aqueous mobile phase systems Interesting polar selectivity features Excellent base deactivation; suitable for LC/MS due to low bleeding characteristics Technical characteristics: Special phase with polar endcapping; pore size 0 Å; particle sizes.8 μm, μm and μm (7 and 0 μm particles for preparative purposes on request); carbon content %; ph stability 9 Recommended application: Analgesics, penicillin antibiotics, nucleic acid bases, water-soluble vitamins, complexing agents, organic acids USP L RP-HPLC with highly aqueous mobile phases The efforts to neutralize unwanted activity of unreacted surface silanols often results in well base-deactivated phases with high carbon load, but a limited scope of selectivity beyond non-polar interactions. In particular polar compounds like carboxylic acids, drug metabolites, etc. show only weak retention on densely bonded reversed phase columns due to distinct hydrophobic properties but low polar interactions. Very polar analytes require highly aqueous mobile phases for solubility and retention. Conventional reversed phase columns often display stability problems in eluent systems with high percentage of water (> 9 %) as evidenced by a sudden decrease of retention time and overall poor reproducibility. This phenomenon is described as phase collapse caused by the mobile phase expelled from the pores due to the fact, that hydrophobic RP phases are incompletely wetted with the mobile phase [U. D. Neue et al., Chromatographia (00) 9 77]. Different approaches can be used to increase column stability with highly aqueous mobile phase systems. The most promising concepts are incorporating a polar group in the hydrophobic alkyl chain, or using hydrophilic endcapping procedures to improve the wettability of the reversed phase modification. NUCLEODUR PolarTec may be taken as an example for the embedded polar group strategy, in which a C 8 silane with a polar function is successfully linked to the silica surface. Stability features NUCLEODUR C 8 Pyramid is a silica phase with hydrophilic endcapping, designed especially for use in eluent systems of up to 00 % water. The figure below shows the retention behavior of tartaric, acetic and maleic acid under purely aqueous conditions on NUCLEODUR C 8 Pyramid in comparison with a conventionally bonded RP phase. C 8 Pyramid Stability test initial injection restart after h stop conventional C 8 0 stop restart after min min 0 min both columns x mm ID; 0 mm KH PO, ph., 0.7 ml/min; C; UV, 0 nm; injection volume μl.. Tartaric acid,. Acetic acid,. Maleic acid MN Appl. No It can be shown that the retention times for NUCLEODUR C 8 Pyramid remain nearly unchanged between initial injection and restart after the flow has been stopped for hours, whilst the performance of the conventional RP column already collapsed totally after min.

17 for highly aqueous Applications eluents Retention characteristics Based on the ultrapure NUCLEODUR silica the polar surface derivatization exhibits retention characteristics, which differentiate the Pyramid from conventional C 8 stationary phases. The chromatogram below shows the improved retention behavior of very polar compounds such as short chain organic acids, which are insufficiently retained on RP columns with predominantly hydrophobic surface properties. For more separations on NUCLEODUR C 8 Pyramid see the "Applications" section from page. Separation of very polar compounds Column: x mm NUCLEODUR C 8 Pyramid, μm Eluent: 0. % H PO Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Injection volume: μl. Formic acid. Acetic acid MN Appl. No. 970 t 0 0 min In addition to the exceptional polar selectivity NUCLEODUR C 8 Pyramid also provides adequate hydrophobic retention (see application 990 at ). The capacity factors of the nonpolar, alkyl-substituted benzenes toluene and ethylbenzene do not go too far in comparison with standard C 8 phases. Tanaka plot of NUCLEODUR C 8 Pyramid ion exchange capacity ph capacity 0 hydrophobicity Base deactivation The perceptible increase in polarity has no impact on the retention behavior of ionizable analytes. Even with the strongly basic compounds of the tricyclic antidepressant drug test mixture, no unwanted interactions or a so-called lack in base deactivation are observed (see application 900 on page ). Retention behavior of polar and non-polar compounds on different NUCLEODUR RP columns Columns: 0 x mm, μm particles Eluent: methanol mm NH H PO, ph 7 (:, v/v) Flow rate: 0.8 ml/min Temperature: 0 C Detection: Injection volume: μl. Chlorpheniramine. Dimethyl phthalate. Benzamide. Ethyl benzoate. Benzophenone 90 UV, nm t R polar compounds. Lidocaine 7. Naphthalene 8. Biphenyl 9. Acenaphthene t R hydrophobic compounds NUCLEODUR C 8 Pyramid min 90 t R hydrophobic compounds 7 8 NUCLEODUR C 8 Gravity t R polar compounds min ion exchange capacity ph steric selectivity 9 NUCLEODUR C 8 Gravity hydrogen bonding capacity min

18 PolarTec Key features: Excellent base deactivation Suitable for LC/MS and stable in 00% aqueous mobile phases Pronounced steric selectivity Technical characteristics: Phase with embedded polar group; pore size 0 Å; particle sizes µm and µm; carbon content 7 %; ph stability 9 Recommended application: Exceptional selectivity for carbocyclic acids, phenols and nitrogen containing compounds, polar compounds like basic pharmaceuticals, organic acids, pesticides, amino acids, water soluble vitamins, etc. USP L and L0 RP-HPLC under 00 % aqueous conditions The dominant form of interactions of conventional C 8 phases are non-polar London dispersion forces. Besides non-polar interactions phases with embedded polar groups possess the ability to show polar interactions (dipol-dipol, hydrogen-bondings, π-π, etc.) These interactions enhance retention and selectivity for polar compounds like carbocyclic acids, phenols and nitrogen containing compounds (see applications). In order to increase retention for polar compounds it is often necessary to decrease the organic ratio of the mobile phase to zero. Under these conditions many conventional C 8 phases display the so-called dewetting effect which means that the mobile phase is expelled from the pores. This phenomenon leads to a dramatic loss in retention. NUCLEODUR PolarTec is stable in 00 % aqueous mobile phases and therefore especially suited for the separation of polar compounds like organic acids (see appl. on page ). Tanaka plot of NUCLEODUR PolarTec capacity 0 ion exchange capacity ph.7 0. hydrophobicity ion exchange capacity ph steric selectivity hydrogen bonding capacity Due to the shielding effect of the embedded group NUCLEODUR PolarTec shows an excellent base deactivation, which is at the top-notch of embedded polar group phases on the market. The pronounced steric selectivity (see Tanaka plot) is an additional tool for the separation of complex mixtures.

19 RP phase with embedded polar group Bleeding of NUCLEODUR PolarTec Columns: 0 x mm NUCLEODUR PolarTec, μm 0 x mm Waters SymmetryShield RP8, μm Eluent: A) acetonitrile, B) water Gradient: 0 % B 90 % B in 0 min Flow rate: 0. ml/min Temperature: C Detection: ESI MS Stability of NUCLEODUR PolarTec Column: 0 x mm NUCLEODUR PolarTec, μm Eluent: 0 mm KH PO, ph.0 Flow rate: 0. ml/min Temperature: 0 C Detection: UV, 0 nm. Cytosine. Uracil. Adenine. Guanine. Thymine measurement every h; in between flow was stopped min MN Appl. No h 0 h Due to low bleeding characteristics NUCLEODUR PolarTec is also suitable for LC/MS. Separation of histidine Column: 0 x mm NUCLEODUR PolarTec, μm Eluent:.0 mm perfluoropentanoic acid in water 0. mm perfluoropentanoic acid in acetonitrile (99.:0., v/v) Flow rate: 0. ml/min Temperature: 0 C Detection: UV, 0 nm. -Methylhistidine R = H, R = CH. Histidine R = R = H. -Methylhistidine R = CH, R = H R N NH O N OH 0 7 min MN Appl. No. 0 In spite of the polar character of the embedded functional group NUCLEODUR exhibits sufficient hydrophobic properties and is very well suited for analyzing basic compounds. Good base deactivation of NUCLEODUR PolarTec is illustrated by the separation of pyridine and phenol. Phase comparison of NUCLEODUR PolarTec Columns: 0 x mm NUCLEODUR PolarTec, μm 0 x mm Waters SymmetryShield RP8, μm Eluent: acetonitrile water (0:0, v/v) Flow rate: 0. ml/min Temperature: C Detection: UV, nm. Pyridine. Phenol R min MN Appl. No. 0 Even after days or weeks of operation in purely aqueous eluents the C 8 chains of NUCLEODUR PolarTec are neither folded nor show any collapsing. A significant reduction of retention time cannot be observed. 0 min MN Appl. No. 7 7

20 PFP Key features: Hydrophobic phase with alternative selectivity in comparison to classical C 8 modifications Separation principle based on retention mechanisms: - polar interactions (H bonds) - dipole-dipole interactions - π-π interactions - hydrophobic interactions Suitable for LC/MS due to low bleeding characteristics Technical characteristics: Phase with pentafluorophenyl-propyl modification and multiendcapping; pore size 0 Å; particle sizes µm and µm; carbon content 8 %; ph stability 9 Recommended application: Aromatic and unsaturated compounds, phenols, halogenated compounds, isomers, polar compounds like pharmaceuticals, antibiotics; high retention of basic compounds USP L Orthogonality in selectivity Fluorinated stationary phases in HPLC have gained increasing interest over the last years. Most common representative of fluorinated silica phases is the pentafluorophenyl modification (PFP or F). Especially the orthogonal selectivity compared to traditional alkyl phases widens the scope in analytical HPLC. Thus NUCLEODUR PFP offers an excellent selectivity especially for highly polar analytes like aromatic and unsaturated compounds, phenols or halogenated hydrocarbons. Halogen substitutes in molecules result often in an increase of their polarity accompanied by a decrease of typical retention characteristics in RP-HPLC. While a typical C 8 phase just provides hydrophobic interactions between stationary phase and analyte NUCLEODUR PFP offers four different retention mechanisms: polar interactions (H bonds), dipole-dipole interactions, π-π interactions and hydrophobic interactions. Especially the pronounced ion exchange capacity and distinct steric selectivity are typical for the character of fluorinated phases. ion exchange capacity ph.7 0. ion exchange capacity ph 7. Tanaka plot of NUCLEODUR PFP capacity hydrogen bonding capacity hydrophobicity steric selectivity Due to low bleeding characteristics NUCLEODUR PFP is also suitable for LC/MS. Bleeding of NUCLEODUR PFP Columns: 00 x. mm NUCLEODUR PFP, μm Phenomenex Luna PFP(), μm Eluent: A) acetonitrile, B) water Gradient: % A 00 % A in 0 min ( min) % A in min ( min) Flow rate: 0. ml/min Temperature: C Detection: ESI MS min MN Appl. No. 70 8

21 Hydrophobic pentafluorophenyl phase Separation of antihistamines Columns: 0 x mm NUCLEODUR PFP, μm 0 x mm NUCLEODUR C 8 Gravity, μm Eluent: acetonitrile 0 mm KH PO (0:70, v/v) Flow rate: 0. ml/min Temperature: 0 C Detection: UV, 0 nm. Maleic acid. Chlorpheniramine. Brompheniramine. Triprolidine. Diphenhydramine. Promethazine 7. Cetirizine 8. Hydroxyzine 7 8 Separation of beta- and dexamethasone Columns: 00 x. mm NUCLEODUR PFP, μm 00 x. mm Phenomenex Luna PFP(), μm Eluent: acetonitrile water (0:80, v/v) Flow rate:. ml/min Temperature: 0 C Detection: UV, 0 nm. Betamethasone. Dexamethasone R S =. R S = min MN Appl. No. 8 Based on a special surface modification procedure NUCLEODUR PFP offers highest stability also at low ph values. Stability of NUCLEODUR PFP Column: x mm NUCLEODUR PFP, μm Eluent: acetonitrile water, 0. % TFA, ph (0:0, v/v) Flow rate: ml/min Temperature: 80 C Detection: UV, nm Injection volume: μl Sample: ethylbenzene % initial retention column volumes MN Appl. No. 70 % initial plates min MN Appl. No. 77 NUCLEODUR PFP offers a completely different retention behavior compared to alkyl modified silica and is often used successfully for separations which provide just insufficient results on traditional C 8 phases. More and more applications in the areas of (bio-) pharma, natural compounds and environment are published and show the broad application field for fluorinated phases. Separation of phenol isomers Columns: x mm NUCLEODUR PFP, μm x mm NUCLEODUR C 8 HTec, μm Eluent: acetonitrile, 0. % formic acid water, 0. % formic acid (:, v/v) Flow rate: ml/min Temperature: C Detection: UV, 80 nm. o-cresol.,-dichlorophenol. m-cresol 7.,-Dichlorophenol.,-Dimethylphenol 8.,-Dichlorophenol.,-Dimethylphenol 9.,-Dichlorophenol.,-Dimethylphenol 0.,-Dibromophenol.,-Dibromophenol Compared to fluorinated HPLC columns of other manufactures NUCLEODUR PFP shows excellent separation capabilities and shorter retention times e.g., for critical isomers like beta- and dexamethasone min MN Appl. No. 9

22 Sphinx RP Key features: Distinct selectivity based on well-balanced bifunctional surface coverage Widens the scope for method development based on additional π-π interactions Suitable for LC/MS due to low bleeding characteristics Technical characteristics: Octadecyl and propylphenyl modified silica; pore size 0 Å; particle sizes.8 μm, μm and μm; carbon content %; ph stability 0; high reproducibility and consistent quality Recommended application: Quinolone antibiotics, sulfonamides, xanthines, substituted aromatics USP L and L Alternative RP selectivity NUCLEODUR Sphinx RP is characterized by exceptional selectivity features generated by a well-balanced ratio of covalently bonded octadecyl and phenyl groups. The combination of classical hydrophobic with π-π interactions (aromatic ring system) expands the scope of selectivity in comparison with conventional reversed phase packings. NUCLEODUR Sphinx RP is particularly suited for the separation of molecules containing aromatic and multiple bonds. For the separation of polar compounds NUCLEODUR Sphinx RP can be especially recommended and can also outperform many customary C 8 phases. In addition, exhaustive endcapping steps minimize unwanted surface silanol activity and guarantee excellent peak shapes even for strong basic analytes. Tanaka plot of NUCLEODUR Sphinx RP capacity ion exchange capacity ph.7 0. ion exchange capacity ph hydrophobicity steric selectivity hydrogen bonding capacity Stability of NUCLEODUR Sphinx RP at ph 0 Column: 0 x. mm NUCLEODUR Sphinx RP, μm Eluent: methanol dil. NH, ph 0 (0:80, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, 7 nm Injection volume: μl. Theophylline. Caffeine after 00 injections (with L eluent) st injection 0 min MN Appl. No

23 Bifunctional RP phase Different from standard phenyl phases, NUCLEODUR Sphinx RP is far more stable towards hydrolysis and is also suggested for LC/MS applications. Due to the additional intermolecular interactions NUCLEODUR Sphinx RP is an interesting replenishment to the high density bonded phases NUCLEODUR C 8 / C 8 Gravity and the polar endcapped NUCLEODUR C 8 Pyramid. Comparison of surface deactivation of different phenyl modified RP phases Columns: 0 x. mm NUCLEODUR Sphinx RP, μm Competitor (column XP) Competitor (column LP) Competitor (column SP) Eluent: methanol water (0:70, v/v) Flow rate: ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. Pyridine. Phenol Sphinx RP Phase XP The selectivity advantage of NUCLEODUR Sphinx RP is impressively shown in the flavonoid application below. While a baseline separation of kaempferol and isorhamnetin can be achieved on NUCLEODUR Sphinx RP, the two compounds are not or just poorly separated on NUCLEODUR C 8 Gravity or C 8 Gravity. The additional π-π interactions of the aromatic ring systems provide the necessary difference in retention to outperform the classical C 8 and C 8 phases. Separation of flavonoids on different NUCLEODUR phases Columns: 0 x. mm NUCLEODUR Sphinx RP, μm NUCLEODUR C 8 Gravity, μm NUCLEODUR C 8 Gravity, μm Eluent: water methanol (0:0, v/v) Flow rate: ml/min Temperature: 0 C Detection: UV, 70 nm Injection volume: μl OH R OH OH HO OH OH. Catechin. Rutin R = R = OH, R = O-rutinose. Fisetin R = R = OH, R = H. Quercetin R = R = R = OH. Kaempferol R = H, R = R = OH. Isorhamnetin R = OCH, R = R = OH O HO R O O R 0 min 0 min Phase LP Phase SP Sphinx RP C 8 Gravity C 8 Gravity 0 min 0 min MN Appl. No min MN Appl. No. 980

24 C 8 HTec Key features: Reliable and durable standard RP phase for up-scaling to preparative scale, suited for LC/MS High loadability and excellent stability Outstanding base deactivation Technical characteristics: High density octadecyl modification (C 8 ); pore size 0 Å; particle sizes.8 μm, μm, μm, 7 μm and 0 μm for analytical and preparative separations; carbon content 8 %; ph stability Recommended application: Sophisticated analytical and preparative separations of basic, neutral and acidic pharmaceuticals, derivatized amino acids, pesticides, fatsoluble vitamins, aldehydes, ketones and phenolic compounds USP L Preparative separations place high demands on silica based HPLC materials. Apart from excellent selectivity and base deactivation, robustness (ph, pressure stability, ) and capacity are vital criteria for optimal and efficient separation at the preparative scale. Selectivity and base deactivation The innovative and special endcapping procedure leads to exceptionally good base deactivation the Engelhardt test demonstrates superb selectivity, peak symmetry and peak shape over the entire polarity range. In addition NUCLEODUR C 8 HTec scores in low bleed characteristics and is therefore highly suitable for LC/MS. Engelhardt test Column: 0 x mm NUCLEODUR C 8 HTec, μm Eluent: methanol water (9:, v/v) Flow rate: ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. Uracil. Aniline. Phenol. -Ethylaniline. N,N-Dimethylaniline. Toluene 7. Ethylbenzene min MN Appl. No. 80 Tanaka plot of NUCLEODUR C 8 HTec capacity 0 ion exchange capacity ph.7 0. hydrophobicity ion exchange capacity ph steric selectivity hydrogen bonding capacity

25 Base-deactivated preparative phase Stability and lifetime Based on fully synthetic and extremely robust totally spherical NUCLEODUR silica, NUCLEODUR C 8 HTec offers outstanding mechanical rigidity and is thus the perfect choice also for self-packing of prep-columns. The special surface modification and endcapping procedure result in high chemical stability even at extreme chromatographic conditions like high flow rates, temperature or critical solvents (DMSO). Furthermore, NUCLEODUR C 8 HTec columns show a remarkably long lifetime in acidic (ph ) as well as basic (ph 0) mobile phases. ph stability test 0 x mm NUCLEODUR C 8 HTec, μm Column: Flow rate: ml/min Detection: UV, nm Injection: μl ph : Eluent: acetonitrile % TFA in water (0:0, v/v); 80 C % initial retention of ethylbenzene 9 injections % 00 ph 0: Eluent: methanol 0 mm triethylamine (:8, v/v); 0 C % initial N of theophylline 0 injections Capacity A vital criterion for efficiency in preparative HPLC is the capacity of the separation medium. NUCLEODUR C 8 HTec is characterized by a notably high loadability under both basic and acidic conditions, while competitor columns show overload effects even at lower loads (x). Loadability under acidic conditions Columns: VP 00 x mm NUCLEODUR C 8 HTec, μm 00 x. mm AXIA Gemini μm C 8 0 Å Eluent: acetonitrile formic acid in H O ph.0 (0:70, v/v) Flow rate: 8 ml/min; temperature C; pressure bar Detection: UV, nm Peaks (total load 0 mg): (sample dissolved in DMSO). -Acetamidophenol ( mg). -Acetamidophenol (0 mg). Acetylsalicylic acid ( mg) 0 min h h Up-scaling Due to highest quality standards in our silica production and phase chemistry combined with optimized packing technology, NUCLEODUR C 8 HTec delivers exceptional transferability from analytical to preparative scale. This doesn t just apply to the use of different particle sizes (e.g.,, 7 or 0 µm) but also for diverse column dimensions (e.g., ID. to mm). Due to innovative surface coating procedures NUCLEODUR C 8 HTec offers excellent analytical separation properties and is the first choice for up-scaling to preparative column dimensions. Please ask for our special NUCLEODUR C 8 HTec brochure for preparative HPLC separation. Up-scaling with NUCLEODUR C 8 HTec Columns: EC 0 x. mm NUCLEODUR C 8 HTec, μm VP 0 x mm NUCLEODUR C 8 HTec, μm Eluent: acetonitrile water (80:0, v/v) Flow rates:. ml/min, 7 ml/min Temperature: C Pressure: 8 bar, 09 bar Detection: UV, nm Inj. volume: μl, 0 μl ( mg/ml of each compound). Phenol. Naphthalene. Anthracene 8 min MN Appl. No. 780

26 C 8 ec / C 8 ec Key features: Ideal and reliable standard RP phase for daily routine analysis and up-scaling for preparative HPLC Medium density octadecyl (C 8 ) and octyl (C 8 ) modification with exhaustive endcapping Wide range of application areas Technical characteristics: Pore size 0 Å; particle sizes μm and μm; 7 μm, 0 μm, μm, μm, 0 μm, 0 μm and 0 μm for preparative separations; carbon content 7. % for C 8, 0. % for C 8 ; ph stability 9, high reproducibility from lot to lot Recommended application: Basic, neutral or acidic drugs derivatized amino acids pesticides fat-soluble vitamins aldehydes and ketones phenolic compounds USP L (C 8 ) / L7 (C 8 ) NUCLEODUR C 8 ec for daily routine analysis and up-scaling for preparative HPLC The efficiency of a separation is controlled by particle size and selectivity of the stationary phase. The exceptional surface coverage of monomeric bonded alkylsilanes, combined with an exhaustive endcapping, results in a surface with lowest silanol activity. This allows the tailing-free elution of polar compounds such as basic drugs. NUCLEODUR C 8 ec is available in 9 different particle sizes (,, 7, 0,,, 0, 0 and 0 μm) which cover the whole range from high speed analytical HPLC up to medium and low pressure prep LC. NUCLEODUR C 8 ec is also an ideal tool for scaleup purposes. Chemical stability The utmost purity of the base silica and the exceptional si- lane bonding chemistry minimizes the risk of dissolution, or hydrolysis at ph extremes. The follow- ing chromatograms show the reten- tion behavior at ph values of. and 0.0 for NUCLEODUR 00- C 8 ec. ph stability of NUCLEODUR C 8 ec Separation of theophylline and caffeine at ph 0 Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: methanol aq. NH (0:80, v/v), ph 0 Flow rate: 0. ml/min Temperature: C Detection: UV, nm 000 Inj. 70 Inj. 00 Inj. 0 Inj. Start 0 min Separation of uracil, veratrol, toluene and ethylbenzene at ph. Column: 0 x mm NUCLEODUR 00 C 8 ec Eluent: acetonitrile H O (:, v/v), TFA, ph. Flow rate:.0 ml/min Temperature: C Detection: UV, nm 000 Inj. 00 Inj. 0 min Loadability Loadability, probably the most important feature for preparative LC applications, is determined by pore size, pore volume and surface area of the packing. However, it can also be influenced by the molecular weight of the analytes. In the figure below the mass loading curve for acetophenone and butyrophenone on a NUCLEODUR

27 Nonpolar phases for routine analyses 00-0 C 8 ec column describes the correlation between the increase of column loading and the decrease of separation efficiency. Loading curve Column: 0 x. mm NUCLEODUR 00-0 C 8 ec, eluent: acetonitrile H O 80:0 (v/v), flow rate:.0 ml/min, temperature: C, detection: UV, nm 000 theoret. plates acetophenone butyrophenone μg / compound ion exchange capacity ph.7 0. ion exchange capacity ph 7. Tanaka plot of NUCLEODUR C 8 ec capacity 0 0. hydrogen bonding capacity hydrophobicity steric selectivity NUCLEODUR octyl phases In addition to the program of NUCLEODUR C 8 phases MACHEREY-NAGEL offers the corresponding octyl modified NUCLEODUR C 8 Gravity and NUCLEODUR C 8 ec columns to expand the reversed phase tool box effectively. Based on the same totally spherical and highly pure silica the C 8 phases exhibit the same excellent chemical and mechanical stability features as the C 8 counterparts. Indeed NUCLEODUR C 8 Gravity can also be run at ph extremes (ph ) by choosing appropriate elution parameters. Due to the shorter chain and less hydrophobic properties of the stationary phase the retention of non-polar compounds is decreased, and in consequence a reduction in time of analysis can be achieved. Moreover a stronger polar selectivity, particularly with the separation of ionizable analytes is frequently observed (as distinct from the C 8 phases). NUCLEODUR C 8 ec and NUCLEODUR C 8 Gravity are most suitable for the development of new methods but also for robust routine analysis. C 8 or C 8 the best of both worlds Chromatographers now might wonder about the differences between C 8 and C 8 phases and the preferred range of application. Indeed there are no general guidelines which could make the choice easier but it will always be beneficial to add both phases to the existing pool of reversed phase columns in the laboratory. However, comparative studies reveal some different selectivity patterns of NUCLEODUR C 8 ec and NUCLEODUR C 8 ec. The separation of phenols below shows baseline separation for -ethoxyphenol and dimethoxybenzene (veratrol) and in addition a reversal of the elution order of phenol and -methoxyphenol can be shown on the octyl phase. Separation of phenols Columns: 0 x mm NUCLEODUR 00- C 8 ec 0 x mm NUCLEODUR 00- C 8 ec Eluent: A) water, B) methanol Gradient for C 8 : 0 % B ( min) 0 % B in min Gradient for C 8 : % B ( min) % B in min Flow rate:.0 ml/min, temperature C Detection: UV, 7 nm, injection volume: 0 μl. Resorcinol. -Ethoxyphenol. Pyrocatechol 7. Veratrol. -Methoxyphenol 8. Biphenyl--ol. Phenol 9. Phenetole. -Methoxyphenol 7 C 8 ec 0 0 min C 8 ec MN Appl. No / 089 Some general principles are: High density C 8 and C 8 phases allow tailing-free elution, also for very polar compounds Octyl phases (C 8 ) show superior polar selectivity Octadecyl phases (C 8 ) show superior hydrophobic selectivity Hydrophobic compounds show shor ter retention times on C 8 phases

28 HILIC Key features: Ideal for reproducible and stable chromatography of highly polar analytes Suitable for analytical and preparative applications as well as LC/MS Very short column conditioning period Technical characteristics: Ammonium - sulfonic acid modified silica; pore size 0 Å; particle sizes.8, and μm; carbon content 7 %; ph stability 8. Recommended application: Hydrophilic compounds such as organic polar acids and bases, polar natural compounds, nucleosides, oligonucleotides, amino acids, peptides, water soluble vitamins NUCLEODUR HILIC Separation science is always looking for new and effective strategies to accomplish the tasks of modern analytics. Especially for polar compounds reversed phase HPLC the most common analytical method is often limited. Here, hydrophilic stationary phases provide an additional tool for the separation of polar analytes in HPLC. The expression HILIC (Hydrophilic Interaction LIquid Chromatography) was firstly published by Andrew Alpert in 990 since then it took quite some efforts to develop robust and reproducible hydrophilic HPLC phases for HILIC chromatography [A. Alpert, J. Chromatography 99 (990), 77 9]. HILIC combines the characteristics of the major methods in liquid chromatography reversed phase (RPC), normal phase (NPC) and ion chromatography (IC): stationary phases (adsorbents) are mostly polar modifications of silica or polymers (SiOH, NH, Diol, (zwitter) ions, ) like in NPC mobile phases (eluents) are mixtures of aqueous buffer systems and organic modifier like acetonitrile or methanol - like in RPC fields of application include quite polar compounds as well as organic and inorganic ions like in IC HILIC is NP chromatography of polar and ionic compounds under RP conditions. NPC adsorbent HILIC eluent RPC analyte NUCLEODUR HILIC is a special zwitterionic modified stationary phase based on ultra spherical NUCLEODUR particles. The betaine character of the ammonium-sulfonic acid ligands results in total charge equalization and in an overall neutrally charged but highly polar surface. SiO + N CH CH SO Retention characteristic Commonly HILIC is described as partition chromatography or liquid/liquid extraction system between the mobile and stationary phase. Versus a water-poor mobile phase a water-rich layer on the surface of the polar stationary phase is formed. Thus, a distribution of the analytes between these two layers will occur. Furthermore HILIC includes weak electrostatic mechanisms as well as hydrogen donor interactions between neutral polar molecules under high organic elution conditions. This distinguishes HILIC from ion exchange chromatography - main principle for HILIC separation is based on compound s polarity and degree of solvation. More polar compounds will have stronger interaction with the stationary aqueous layer than less polar compounds resulting in a stronger retention. Nonpolar IC

29 Zwitterionic phase compounds exhibit faster elution profiles due to minor hydrophobic interactions. Thus, as shown for the separation of uracil and naphthalene the elution order is quite often inverse on HILIC columns compared to RP columns. Separation of uracil and naphthalene Columns: x mm NUCLEODUR C 8 Pyramid, μm x mm NUCLEODUR HILIC, μm Eluent: acetonitrile water (90:0, v/v) Flow rate:.0 ml/min, temperature C Detection: UV, nm. Uracil. Naphthalene 0 min MN Appl. No. 9/9 In comparison with medium polar aminopropyl phases or modification with less balanced charge equalization NUCLEODUR HILIC shows a superb separation and peak shape for critical compounds like adenosine phosphates. Stability features Due to an advanced and unique surface modification procedure (pat. pend.) NUCLEODUR HILIC columns provide short equilibration times after just 0 min equilibration already the nd injection shows stable and reproducible results. Beyond this, NUCLEODUR HILIC columns are characterized by an outstanding column life time - even after nearly 800 runs the columns show no loss of pristine performance - peak shape and retention are still immaculate. Column: Eluent: Flow rate: Temperature: C Detection: UV, nm Inj. Inj. 78 Inj. 79 Inj. Stability and equilibration 0 x mm NUCLEODUR HILIC, μm acetonitrile mm ammonium acetate (80:0, v/v) 0. ml/min min. Thymine. Uracil. Adenine. Cytosine. Guanosine MN Appl. No. 00 Separation of adenosine and phosphates Columns: x mm NUCLEODUR HILIC, μm x mm zwitterionic phase with quat. ammonium sulfonic acid ratio :0.9 x mm amino-modified silica Eluent: acetonitrile 00 mm ammonium acetate, ph. (70:0, v/v) Flow rate:. ml/min, temperature C Detection: UV, 9 nm. Adenosine. camp. AMP. ADP. ATP NUCLEODUR HILIC zwitterionic phase less balanced charge equalization amino modification 0 0 min Separation of growth regulators Column: x mm NUCLEODUR HILIC, μm Eluent: acetonitrile mm ammonium acetate, ph.8 (80:0, v/v) Flow rate: 0. ml/min, temperature C Detection: MS (SIM). Chlormequat. Mepiquat MN Appl. No min Due to its high loadability NUCLEODUR HILIC is absolutely suitable for preparative and semi-preparative applications. Overall NUCLEODUR HILIC provides excellent chromatographic features and is hereby the perfect choice for separation of polar or charged compounds. 7

30 CN / CN-RP Key features: High retention capacity especially for very polar and unsaturated compounds Multi-mode column (RP and NP) widens scope of selectivity Stable against hydrolysis at low ph (working range ph 8) Technical characteristics: Cyanopropyl-modified high purity silica; pore size 0 Å; particle sizes μm and μm; carbon content 7 %; special endcapping; high reproducibility from lot to lot; different retention characteristics in comparison to C 8 and C 8 Recommended application: Tricyclic antidepressants, steroids, organic acids USP L0 Alternative bonded-phase functionality In reversed phase HPLC it is fairly common to start with C 8 or C 8 columns, if new methods have to be developed. However, superior polarity and selectivity properties often required for more sophisticated separations, are not always sufficiently provided by classical RP phases, which are usually characterized by a hydrophobic layer of monomeric or polymeric bonded alkylsilanes. One approach to improve the resolution of compounds poorly separated on nonpolar stationary phases, is to change bonded-phase functionality. The fully endcapped and highly reproducible (see figure top right) NUCLEODUR CN-RP phase has cyanopropyl groups on the surface able to generate a clearly recognizable different retention behavior compared to purely alkyl-functionalized surface modifications (see figure down right). The polarity of the NUCLEODUR CN-RP phase can be classified as intermediate based on multiple retention mechanisms such as dipole-dipole, π-π, and also hydrophobic interactions [C. S. Young and R. J. Weigand, LCGC 0 (00) 7]. Therefore, this phase shows a distinct selectivity for polar organic compounds as well as for molecules containing π-electron systems (e.g., analytes with double bonds, tricyclic antidepressants) [V. R. Meyer, Practical High Performance Liquid Chromatography (John Wiley & Sons, New York, rd. ed., 999)]. Reproducibility of NUCLEODUR CN-RP Column: 0 x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile water (0:0, v/v) Flow rate:.0 ml/min, temperature 0 C Detection: UV, nm, injection volume: μl. Benzamide. Dimethyl phthalate. Phenetole. o-xylene. Biphenyl lot D lot C lot B lot A 0 min MN Appl. No. 080 Separation of cold medicine ingredients on two different NUCLEODUR phases Columns: 0 x mm NUCLEODUR 00- C 8 ec 0 x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile 00 mm sodium citrate, ph. (:8, v/v) Flow rate:.0 ml/min, temperature C Detection: UV, 70 nm, injection volume: 0 μl. Maleic acid. Norephedrine. Ephedrine. Paracetamol. Chlorpheniramine. Brompheniramine 0 8 min MN Appl. No. 90 8

31 Cyano-modified high purity silica Interactions on cyano-modified silica O O H H N N C hydrogen bonding O C O π π N C O H hydrophobic Short-chain bonded phases are sometimes suspected of revealing shortcomings in stability towards hydrolysis at low ph [J. J. Kirkland, LCGC (99) 8 00]. The following chromatograms show that even after 00 sample injections and four weeks storage at ph (blue curve), neither a considerable shift in retention, nor a visible change in peak symmetry could be noticed (green curve = new column). Si O Si Si Due to the exceptional polarity features the cyano phase can also be run in the normal phase mode. NUCLEODUR CN columns for normal phase applications are shipped in n-heptane. The drastic change in selectivity and order of elution for a mixture of various steroids in normal and reversed phase mode is displayed in following figure. Moreover the high coverage combined with a thorough endcapping makes NUCLEODUR 00- CN-RP suitable for the separation of ionizable compounds such as basic drugs. Separation of steroids in normal phase and reversed phase mode Normal phase mode Column: 0 x mm NUCLEODUR 00- CN Eluent: n-heptane -propanol (90:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: 0 μl. Methyltestosterone. Testosterone. Norgestrel. Medrysone. Cortisone. Hydrocortisone 7. Prednisolone Stability of NUCLEODUR CN-RP at ph Column: x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile water, % TFA, ph (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Benzamide. Dimethyl phthalate. Phenetole. o-xylene. Biphenyl min Reversed phase mode Column: 0 x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile water (:7, v/v) other conditions as for normal phase mode + 7 after 00 injections + weeks at ph st injection 0 min MN Appl. No min MN Appl. Nos. 97 / 97 9

32 NH / NH -RP Amino-modified silica Key features: Multi-mode columns (for RP, NP and IC) Stable against hydrolysis at low ph (working range ph 8), 00 % stable in water; suitable for LC/MS Widens scope of analytical HPLC into the polar range Technical characteristics: Aminopropyl-modified high purity silica; pore size 0 Å; particle sizes, and 7 μm; carbon content. %; not endcapped Recommended application: Polar compounds under RP conditions (sugars, DNA bases), hydrocarbons under NP conditions USP L8 Normal phase chromatography (NP) with hexane, dichloromethane or -propanol as mobile phase for polar compounds such as substituted anilines, esters, chlorinated pesticides Reversed phase chromatography (RP) of polar compounds in aqueous-organic eluent systems Ion exchange chromatography of anions and organic acids using conventional buffers and organic modifiers Some compounds, especially polar substances, cannot be sufficiently resolved on C 8 phases. Polar-modified silica phases offer alternative selectivities such expanding the spectrum of analytical HPLC into the polar range. Multi-mode columns Besides cyano modifications, amino modifications belong to the most frequently used polar silica phases both feature the important advantage, that they can be run in the RP mode using aqueous-organic eluent mixtures as well as in the NP mode e.g., with hexane as mobile phase. NUCLEODUR NH, too, belongs to the so-called multi-mode columns. It can be used for reversed phase chromatography (RP) of polar compounds such as sugars in aqueous-organic eluent systems, for normal phase chromatography (NP) of substituted aromatics or chlorinated pesticides with organic mobile phases such as hexane, dichloromethane or -propanol, but also for ion exchange chromatography of anions and organic acids using conventional buffers and organic modifiers. Main field of application of NUCLEODUR NH is the separation of simple and complex sugars, sugar alcohols and other hydroxy compounds under RP conditions as well as hydrocarbons under NP conditions. Reversed phase separation of sugars Column: 0 x mm NUCLEODUR 00- NH -RP Eluent: acetonitrile water (79:, v/v) Flow rate: ml/min Detection: RI. Fructose. Glucose. Saccharose. Maltose. Lactose min MN Appl. No. 0 Normal phase separation of middle distillates in accordance with DIN EN 9 Columns: 0 x mm NUCLEODUR 00- NH conventional aminopropyl phase Eluent: heptane Flow rate: ml/min Detection: RI. Cyclohexane. -Phenyldodecane.,-Dimethylbenzene. Hexamethylbenzene. Naphthalene. Dibenzothiophene 7. 9-Methylanthracene min MN Appl. No. 80 0

33 SiOH Unmodified high Applications purity silica Even at lower flow rates than for C 8 phases, NUCLEODUR NH achieves good separations of polar compounds such as DNA bases this reduces the back pressure as well as the solvent consumption. Even very polar compounds like streptomycin are retained sufficiently for quantitative and qualitative analysis. The example below proves the enhanced ph stability of the NUCLEODUR amino phase and also the outstanding suitability of this column for the separation of total herbicides (AMPA, glyphosate, glufonisate, ) - you may find the complete application 90 in the "Applications" section on page 0. Separation of DNA bases Column: 0 x mm NUCLEODUR 00- NH -RP Eluent: acetonitrile water (80:0, v/v) Flow rate: 0. ml/min Temperature: C Pressure: 0 bar Detection: UV, nm. Thymine. Uracil. Cytosine. Adenine Resistance towards hydrolysis for NUCLEODUR NH -RP Column: 0 x mm NUCLEODUR 00- NH -RP Eluent: acetonitrile 0 mmol KH PO, ph.7 (0:0, v/v) Flow rate: 0. ml/min Detection: UV, nm. Aminomethyl-phosphonic acid (AMPA) after 87 min st injection min 8 min MN Appl. No. 70 One of the main problems with conventional amino phases is insufficient resistance towards hydrolysis. Due to a special method of surface modification NUCLEODUR NH features a pronounced stability at higher as well as at lower ph values. The figure at right shows, that even after several days of exposure of the column material at ph.7 good separation efficiency and peak symmetry are maintained. The resulting high column life allows cost reduction due to lower column consumption. Based on the superspherical silica NUCLEODUR this amino phase like all other members of the NUCLEODUR family features a very good pressure stability, which makes it the perfect choice for preparative separations as well as for LC-MS applications. Additionally, the high batch-to-batch reproducibility of NUCLEODUR NH offers the advantage of reliable analyses especially for routine work. SiOH Key features: Totally spherical high purity silica Pressure stable up to 00 bar Suitable for analytical and preparative separation of polar and midpolar compounds Technical characteristics: Unmodified high purity silica; pore size 0 Å; particle sizes to 0 μm; pore volume 0.9 ml/g, surface area (BET) 0 m /g; ph stability 8; metal content < 0 ppm (see table on page ) Recommended application: Polar and midpolar compounds under normal phase conditions USP L

34 Applications Anesthetics MN Appl. No. 90 Column: x mm NUCLEODUR C 8 Pyramid, μm Eluent: methanol 0 mm KH PO, ph.9 (:, v/v) Flow rate: ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. Benzocaine. Lidocaine. Tetracaine. Butacaine Analgesics MN Appl. No. 800 Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: methanol 0. % phosphoric acid (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm a) Thomapyrin tablet; b) standard Thomapyrin is a trademark of Boehringer Ingelheim Pharma KG. Paracetamol. Caffeine. -Acetamidophen. Acetanilide. Acetylsalicylic acid. Phenactin a) b) min 0 min Analgesics MN Appl. No Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile 0 mm KH PO, ph. (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Injection volume: μl. Paracetamol. Acetylsalicylic acid. Salicylic acid. Ketoprofen. Diclofenac. Ibuprofen Analgesics MN Appl. No. 90 Column: 0 x mm NUCLEODUR C 8 Pyramid, μm Eluent: acetonitrile 0. % TFA (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Paracetamol. Acetylsalicylic acid. Methyl--hydroxybenzoate. Ketoprofen. Flurbiprofen. Ibuprofen 0 0 min For a fast separation (< min) of acetylsalicylic acid and salicylic acid on NUCLEODUR 00- C 8 ec see application 7780 at. 0 8 min

35 Pharmaceuticals and drugs Anti-inflammatory drugs MN Appl. No. 0 Column: 0 x mm NUCLEODUR C 8 Pyramid,.8 μm Eluent: phosphate buffer, ph. acetonitrile methanol (:7:00, v/v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: μl. Chlorocresol. Clobetasol 7-propionate. Beclometasone dipropionate Anti-inflammatory drugs MN Appl. No / 088 Columns: 0 x mm NUCLEODUR 00- C 8 ec 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile water, % acetic acid (8:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Injection volume: 0 μl. Piroxicam. Suprofen. Ketoprofen. Carprofen. Fenoprofen. Diclofenac + C 8 ec min + Analgesic and anti-inflammatory drugs MN Appl. No. 890 Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile % acetic acid (8:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Injection volume: 0 μl. Acetylsalicylic acid (. μg/ml). Tolmetin ( μg/ml). Piroxicam ( μg/ml). Suprofen ( μg/ml). Naproxen (0. μg/ml). Diflunisal (. μg/ml) 7. Fenoprofen ( μg/ml) 8. Flurbiprofen ( μg/ml) 9. Indomethacin ( μg/ml) 9 0. Ibuprofen ( μg/ml) 7 8 C 8 ec 0 0 min This separation of various non-steroidal anti-inflammatory drugs illustrates the differences in polarity between C 8 and C 8 and the resulting impact on efficiency. NUCLEODUR C 8 ec exhibits enhanced selectivity and excellent resolution for the polar compounds piroxicam and suprofen which co-elute on the C 8 column. However due to the longer alkyl chain NUCLEODUR C 8 ec shows a distinct hydrophobic selectivity that leads to baseline separation of the more non-polar analytes carprofen and fenoprofen with superior peak shapes min

36 Applications Anti-inflammatory drugs MN Appl. No. 780 Column: x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile 0 mm KH PO, ph. (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Injection volume: μl. Acetylsalicylic acid. Sulindac. Tolmetin. Ketoprofen. Flurbiprofen. Diclofenac 7. Ibuprofen 8. Meclofenamic acid Barbiturates MN Appl. No. 780 Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile water (0:0, v/v) Flow rate: 0.7 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Phenobarbital. Pentobarbital. Hexobarbital. Mephobarbital. Thiamylal min 0 0 min MN App. No. 0 Column: 0 x. mm NUCLEODUR C 8 Gravity, μm Flow rate:. ml/min other conditions as above. Acetylsalicylic acid. Sulindac. Piroxicam. Suprofen. Tolmetin. Naproxen 7. Diflunisal 8. Fenoprofen 9. Flurbiprofen 0. Niflumic acid. Indomethacin. Ibuprofen Tricyclic antidepressants MN Appl. No. Columns: 0 x mm NUCLEODUR PolarTec, μm 0 x mm Waters SymmetryShield RP8, μm Eluent: methanol mm KH PO, ph 7 (70:0, v/v) Flow rate: 0. ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. Protryptiline. Doxepin. Imipramine. Amitriptyline. Trimipramine min 0 0 min Excellent endcapping of NUCLEODUR PolarTec displays significantly better peak shapes and less tailing for strong basic components compared to other phases with embedded polar group.

37 Pharmaceuticals and drugs Tricyclic antidepressants MN Appl. No Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: acetonitrile 0 mm KH PO, ph 7.0 (:, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. Protriptyline. Nortriptyline. Doxepin. Imipramine. Amitriptyline. Trimipramine Tricyclic antidepressants MN Appl. No. 0 Column: 0 x mm NUCLEODUR C 8 Isis, μm Eluent: methanol 0 mm KH PO, ph 7 (7:, v/v) Flow rate: ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: 8 μl. Protriptyline. Maprotiline. Nortriptyline. Imipramine. Amitriptyline. Clomipramine 0 min MN Appl. No. 80 Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: methanol 0 mm KH PO, ph 7.0 (:, v/v) Temperature: C Peaks and other conditions as above min Peak symmetry at 0 % of peak height: A s (imipramine):.9 A s (amitriptyline):. A s (clomipramine):. MN Appl. No. 980 Column: 0 x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile 0 mm KH PO, ph. (:, v/v) Flow rate: ml/min Temperature: 0 C Detection: UV, nm Injection volume:. μl ( μg/ml). Doxepin. Nortriptyline. Trimipramine. Clomipramine 0 0 min MN Appl. No. 900 Column: x mm NUCLEODUR C 8 Pyramid, μm Eluent: methanol 0 mm NH H PO, ph.9 (70:0, v/v) Temperature: 0 C Injection volume: μl Peaks and other conditions as above 0 0 min 0 min

38 Applications Neuroleptics MN Appl. No. Column: 0 x mm NUCLEODUR C 8 Gravity, μm Eluent: acetonitrile.0 g/l KH PO,.9 g/l sodium dodecylsulfate, 9.0 g/l tetra-n-butylammonium bromide ph 8 (0:0, v/v) Flow rate:. ml/min Temperature: 0 C Detection: 7 nm Injection volume: μl. Chloroprothixene hydrochloride a. Additives b. Impurities a b Cold medicine MN Appl. No. 780 Column: x mm NUCLEODUR C 8 Gravity, μm Eluents: A) 0 mm KH PO + mm pentanesulfonate (Na salt), ph.; B) methanol % B % B in min Flow rate:.0 ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: μl. Maleic acid. Paracetamol. Pseudoephedrine. Benzoic acid. Chlorpheniramine. Dextromethorphan a b b b b b min For separation on NUCLEODUR C 8 Gravity see application at. 0 min Gastric acid inhibitors MN Appl. No. 0 Column: 7 x. mm NUCLEODUR C 8 Gravity, μm Eluent: methanol 0 mm KH PO, ph 7 with TEA (0:80, v/v) Flow rate:. ml/min Temperature: C Detection: UV, nm Injection volume: 0 μl. Famotidine. Cimetidine. Nizatidine. Pirenzepine hydrochloride Cold medicine ingredients MN Appl. No. 90 / 90 Column: 0 x mm NUCLEODUR C 8 Pyramid, μm Eluent: A) 0 mm NH H PO, ph.; B) acetonitrile 0 % B 0 % B in min Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm for. min, then nm Injection volume: a) μl, b) μl. Ascorbic acid. Paracetamol. Caffeine. Chlorpheniramine. Dextromethorphan. Diphenhydramine a) OTC drug b) standard min 0 8 min

39 Pharmaceuticals and drugs Antihistamines MN Appl. No. 8 Columns: 00 x. mm NUCLEODUR PFP, μm 00 x. mm Phenomenex Luna PFP(), μm Eluent: acetonitrile 0 mm KH PO, ph.0 (0:70, v/v) Flow rate:. ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: μl Maleic acid. Chlorpheniramine. Brompheniramine. Triprolidine. Diphenhydramine. Promethazine 7. Cetirizine 8. Hydroxyzine NUCLEODUR PFP b -Agonists in human urine by LC-MS / MS MN Appl. No. 970 Column: 7 x mm NUCLEODUR C 8 Pyramid, μm Sample prep.: please refer to Thevis et al., J. Mass Spectrom 8 (00) 97 0 Eluents: A) mm ammonium acetate with 0. % acetic acid, ph.; B) acetonitrile; 0 % B 00 % B in min, reequilibration at 00 % A for. min Flow rate: 0.8 ml/min Temperature: C Detection: electrospray ionization / multiple reaction monitoring (MRM) on an Applied Biosystems API 000 Injection volume: 0 μl LC-MS / MS chromatogram ml urine aliquot fortified with 00 ng each. Reproterol (. min). Fenoterol (. min). Ritodrine (. min). Ractopamine (. min). Clenbuterol (.8 min). Bambuterol (.97 min) 7. Mapenterol (. min) 7 Luna PFP() min Xylometazoline in nasal spray MN Appl. No. 090 Column: x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile 0 mm Na citrate, ph.0 (0:0, v/v) Flow rate: 0.8 ml/min Temperature: 0 C Detection: UV, nm Injection volume: 00 μl. Xylometazoline.. Benzalkonium chlorides with different chain lengths (C 8, C 0, C, C ) min Courtesy of M. Thevis and W. Schänzer, Institute of Biochemistry, German Sport University, Cologne, Germany. a) nasal spray ( mg/ml, diluted :0 in eluent) b) standard 0 min 7

40 Applications Basic drugs MN Appl. No. 90 Column: x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile 0 mm KH PO, ph. (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume:.0 μl Tailing factor. Procainamide ( ng/μl).. Clonidin (0 ng/μl).. Clenbuterol ( ng/μl). Benzodiazepine midazolam and metabolite from plasma MN Appl. No. 870 Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: 7 ml KH PO (9. g/l H O) + 09 ml Na HPO (.9 g/l H O) + 8 ml methanol + 0. g octanesulfonic acid, ph. Flow rate: 0.7 ml/min Temperature: C Detection: UV, DAD. a-hydroxymidazolam (metabolite). Midazolam (0 ng/ml). Internal standard 0. min 0 8 min Courtesy of Mrs. Richter, Institute of Anesthetics, Biochemical Laboratory, University of Erlangen, Germany Benzodiazepines MN Appl. No. 780 Column: x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile 0 mm KH PO, ph. (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Bromazepam. Oxazepam. Lorazepam. Temazepam Sedative drugs MN Appl. No. 900 Column: x mm NUCLEODUR 00- CN-RP Eluent: A) methanol B) 0 mm ammonium acetate, ph.0 70 % B 0 % B in 0 min (0 min) Flow rate:. ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl ( + : 70 μg/ml, : μg/ml). Promethazine. Promazine. Chlorpromazine 0 min 0 0 min 8

41 Pharmaceuticals and drugs Coronary therapeutic drugs (Ca-antagonists) MN Appl. No. 90 Column: x mm NUCLEODUR 00- CN-RP Eluent: A) acetonitrile, B) 0 mm KH PO, ph. 0 % B 0 % B in 7. min (7. min) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume:. μl ( μg/ml each). Nifedipine. Nitrendipine. Nimodipine. Nisoldipine. Nicardipine Antibacterial drugs MN Appl. No Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile water (0:0, v/v) 0.0 % TFA Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Ofloxacin. Ciprofloxacin. Cinoxacin. Penicillin G. Penicillin V. Cloxacillin 0 8 min 0 8 min Cardiovascular drugs MN Appl. No. 0 Column: 7 x. mm NUCLEODUR C 8 Gravity, μm Eluent: A) 0 mm KH PO + Na pentanesulfonate ph. B) methanol % B 90 % B in min Flow rate:. ml/min Temperature: C Detection: UV, 0 nm Injection volume: μl. Dipyridamole. Nifedipine. Flunarizine hydrochloride Gyrase inhibitors MN Appl. No. 000 Column: 0 x mm NUCLEODUR Sphinx RP, μm Eluent: A) 0.0 M H PO, B) acetonitrile % B 0 % B in 0 min Flow rate: 0. ml/min Detection: UV DAD, 0 nm, 8 nm and 00 nm Injection volume: 0 μl (0. ng/μl of each compound). Marbofloxacin. Ciprofloxacin. Enrofloxacin. Sarafloxacin 7. Oxolinic acid. Nalidixic acid 0 nm 7. Flumequine 8 nm 00 nm 0 min 8 0 min Courtesy of R. Lippold, Chemical and Veterinary Research Agency, Freiburg, Germany. 9

42 Applications Quinolone antibiotics MN Appl. No. 00 / 070 Columns: x mm NUCLEODUR C 8 Gravity, μm x mm NUCLEODUR C 8 Gravity, μm x mm NUCLEODUR C 8 Pyramid, μm Eluent: methanol 0. % formic acid (0:0, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. Cinoxacin. Oxolinic acid. Nalidixic acid Penicillin antibiotics MN Appl. No. 780 Column: x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile 0 mm KH PO, ph.0 (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Amoxicillin. Penicillin G. Penicillin V. Cloxacillin. Nafcillin. Dicloxacillin C 8 Gravity C 8 Gravity 0 min C 8 Pyramid min MN Appl. No. 90 Column: 0 x mm NUCLEODUR C 8 Pyramid, μm Eluent: acetonitrile 0. % TFA (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl Peaks as listed above MN Appl. No Column: 0 x. mm NUCLEODUR Sphinx RP, μm Eluent: methanol 0 mm KH PO, ph. (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl Peaks as listed above min 0 0 min 0

43 Pharmaceuticals and drugs b-lactam antibiotics MN Appl. No. 80 Column: 0 x mm NUCLEODUR PFP, μm Eluents: A) acetonitrile, B) mm KH PO, ph.7 0 % A 0 % A in min A) acetonitrile, B) 0. % TFA 0 % A % A in min ( min) Flow rate: 0. ml/min Temperature: 0 C, 0 C Detection: UV, 0 nm, UV, 0 nm Injection volume: μl. Amoxicillin. Cephalexin. Ampicillin. Cefamandole 7. Cefalotin 8. Piperacillin. Cloxacillin. Nafcillin. Dicloxacillin. Cefotaxime. Cefoxitin 9. Penicillin V 0. Oxacillin * Impurity Anticoccidial drugs (polyether antibiotics) MN Appl. No. 870 Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: methanol 0 mmol phosphate buffer ph.0 methylheptylamine (900:99:, v/v/v) Flow rate: 0.7 ml/min Temperature: C Detection: UV/VIS, 00 nm after post column derivatization with dimethylaminobenzaldehyde (0. ml/min) Injection volume: 00 μl. Monensin sodium. Salinomycin sodium 7 0 a) * min b) 0 0 min a): Sample spiked with monensin sodium and content of salinomycin sodium b): Standard of monensin sodium and salinomycin sodium Courtesy of J. Schönherr, Saxon State Institute for Agriculture, Leipzig, Germany MN Appl. No. 70 Column: x mm NUCLEODUR C 8 HTec, μm Eluent: A) acetonitrile, B) 0.0 % TFA in water 70 % B ( min) 0 % B in 0. min (. min) 0 % B in min 7. % B in min Flow rate: 0.9 ml/min Temperature: C Detection: UV, nm Injection volume: 0 μl Concentration: 00 μg/ml. Amoxicillin. Enrofloxacin. Cinoxacin. Oxolinic acid. Nalidixic acid. Penicillin V 7. Cloxacillin Cephalosporin antibiotics MN Appl. No. 80 / 90 Column: 0 x. mm NUCLEODUR C 8 Gravity, μm Eluent: acetonitrile mm KH PO (0:80, v/v) ph with H PO, ph 7 Flow rate: 0.8 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Cefotaxime. Cefoxitin. Cefamandole. Cephalothin 7 ph 7 ph 0 8 min 0 0 min Protonation causes a drastic increase in retention time, but an improved peak symmetry.

44 Applications Antibacterial drugs MN Appl. No. 70 Column: 0 x mm NUCLEODUR C 8 Gravity, μm Eluent: A) acetonitrile, B) water % TFA 0 % B ( min) 0 % B in min ( min) Flow rate: 0.9 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Amoxicillin. Enrofloxacin. Cinoxacin. Oxolinic acid. Nalidixic acid. Penicillin V 7. Cloxacillin Determination of chloramphenicol residues in honey by microbore HPLC MN Appl. No. 980 Column: Eluent: Flow rate: Detection: Injection volume: μl Peaks. Chloramphenicol (CAP) 00 x mm NUCLEODUR C 8 Gravity, μm A) methanol, B) water % A 80 % A in 9 min ( min) % A in min; injection after 7 min 0 μl/min MS c) Honey spiked with 0. μg/kg CAP TIC m/z m/z 7 b) Blind value of a honey sample a) Chloramphenicol standard 0 min 0 0 min S. Oepkemeier, H.D. Winkler, GIT (00) Sulfonamides MN Appl. No Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: methanol 0. % TFA (0:80, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Injection volume: μl. Sulfanilamide. Sulfadiazine. Sulfathiazole. Sulfamerazine. Sulfadimidine. Succinylsulfathiazole Separation of theobromine, vanillin and caffeine MN Appl. No. 990 Column: x mm NUCLEODUR Sphinx RP, μm Eluent: methanol. % acetic acid (0:80, v/v) Flow rate: ml/min Temperature: C Detection: UV, nm Injection volume: 0.8 μl. Theobromine. Caffeine. Vanillin 0 8 min min

45 Food analysis Column: Eluent: Flow rate: Detection: Injection volume: 0 μl. Gallic acid Alkaline tannic acid mixture MN Appl. No x. mm NUCLEODUR C 8 Pyramid, μm A) 0. % H PO, ph., B) acetonitrile % B % B in min ( min) % B in min ( min) 0.8 ml/min UV, 7 nm (optimized for gallic acid) Sweeteners MN Appl. No. 70 Column: 0 x. mm NUCLEODUR C 8 HTec, μm Eluent: A) acetonitrile, B) mm KH PO, ph. % A (. min) % A in 9. min ( min) Flow rate:. ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: μl Concentration: 0. mg/ml each. Acesulfame K. Saccharin. Aspartame. Benzoic acid. Sorbic acid. Dehydroacetic acid 0 0 min Soft drink additives MN Appl. No. 80 Column: 0 x. mm NUCLEODUR 00- C 8 ec Eluent: 0 mm KH PO, ph acetonitrile (:, v/v) Flow rate:.9 ml/min Temperature: C Detection: UV, 0 nm Injection volume: 0 μl. Ascorbic acid (acidic). Acesulfame K. Saccharin. Caffeine (basic). Aspartame. Quinine (strongly basic) 7. Vanillin 8. Sorbic acid (acidic) 9. Benzoic acid (acidic) Soft drink samples were degassed for min and injected undiluted. Coca Cola light Coca Cola decaffeinated 0 0 min Preservatives MN Appl. No. 90 Column: 0 x mm NUCLEODUR PolarTec, μm Eluent: A) acetonitrile, 0. % TFA, B) water, 0. % TFA 0 % A 0 % A in min % A in min 9 % A in min Flow rate: 0.9 ml/min Temperature: C Detection: UV, 0 nm Injection volume: μl. Benzyl alcohol 8. Ethyl paraben. Phenoxyethanol 9. Propyl paraben. Dehydroacetic acid 0. Isobutyl paraben. p-anisic acid. Butyl paraben. Methyl paraben. Irgasan. Salicylic acid.,,-triclocarbanilid 7. Benzoic acid Schweppes Bitter Lemon min standard For fast separation of sweeteners on NUCLEODUR 00- C 8 ec see appl. 790 at min

46 Applications Food dyes MN Appl. No. 00 / 0 Column: 0 x mm NUCLEODUR C 8 Gravity, μm Eluent: A) acetonitrile, B) 0 mm KH PO, ph 9 % B 0 % B in 0 min 0 % B in min 9 % B in min ( min) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl Peaks application 0. Ponceau R (E ). Ponceau R (E ). Azorubine (E ). Erythrosine (E 7). Fast Red E Peaks application 00.,. Tartrazine (E 0). Fast Yellow.. Quinoline yellow (E 0) 7. Yellow orange S (sunset yellow CFC, E 0) Acrylamide, methacrylamide and methacrylic acid MN Appl. No. 00 Column: x mm NUCLEODUR HILIC, μm Eluent: acetonitrile 0. % formic acid (98:, v/v) Flow rate: 0. ml/min Temperature: C Detection: UV, 0 nm Injection volume: 0. μl. Methacrylamide. Acrylamide. Methacrylic acid 0 min 7 Appl. 0 Appl min Patulin and hydroxymethylfurfural in apple juice MN Appl. No. 800 Column: 0 x mm NUCLEODUR C 8 Gravity, μm + 8 x mm guard column Sample prep.: see appl. 800 at Eluent: water acetonitrile (9:, v/v) Flow rate:. ml/min Detection: UV, 7 nm Injection volume: 0 μl. Hydroxymethylfurfural. Patulin juice standard 0 8 min Courtesy of A. Gessler, Wesergold Getränkeindustrie GmbH & Co. KG, Rinteln, Germany.

47 Food analysis Melamine and cyanuric acid MN Appl. No. 070 Column: x mm NUCLEODUR HILIC, μm Eluent: acetonitrile 0 mm ammonium formate, ph (90:0, v/v) Flow rate: 0. ml/min Temperature: C Detection: UV, 0 nm and MS (TIC) Injection volume: μl. Melamine (0 μg/ml). Cyanuric acid (90 μg/ml) Creatinine and creatine MN Appl. No. 000 Column: x mm NUCLEODUR HILIC, μm Eluent: acetonitrile 0 mm ammonium acetate, ph (70:0, v/v) Flow rate: 0. ml/min Temperature: C Detection: MS Injection volume: μl (0 ng/μl). Creatinine. Creatine MS (TIC) UV (0 nm) 0 7 min 0 min MN Appl. No. 090 Column: x mm NUCLEODUR HILIC, μm Eluent: acetonitrile 0 mm ammonium formate, ph (90:0, v/v) Flow rate: 0. ml/min Temperature: C Detection: MS Injection volume: μl. Melamine in milk (00 pg/injection) 0 7 min For UV detection see appl. 990 at. Separation of urea, biuret and cyanuric acid MN Appl. No. 00 Column: 0 x mm NUCLEODUR C 8 Pyramid, μm Eluent: water (00 %) Flow rate: ml/min Detection: UV, 90 nm Injection volume: 0 μl. Urea (0. mg/ml) urea O. Biuret (0.09 mg/ml). impurity in biuret. Cyanuric acid (0.0 mg/ml) H N NH cyanuric acid biuret HO H N O N OH N NH N OH cyanuric acid hydrolase O NH 0 min Courtesy of C. Greve, Institute of Chemical Engineering, University of Clausthal, Germany.

48 Applications Amino acids as OPA derivatives MN Appl. No. 80 Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: A) methanol acetonitrile (0:0, v/v), B) Na acetate buffer ph. + % A) 00 % B (.9 min) 9 % B in. min 8 % B in min 8 % B in min 70 % B in 0 min % B in 8 min % B in 7 min 0 % B in min ( min) 80 % B in 0. min (. min) 00 % B in 0. min Flow rate: ml/min Detection: fluorescence, l ex 0 nm, l em 0 nm. Aspartic acid. Glutamic acid. Asparagine. Serine. Glutamine. Histidine 7. Glycine 8. Alanine 9. Arginine 0. g-aminobutyric acid. Tyrosine. Valine. Methionine. Norvaline (int. std.). Tryptophan. Phenylalanine 7. Isoleucine 8. Leucine 9. Lysine min Courtesy of Mr. Zürcher, Technical University of Munich, Chair of Brewing Technology, Freising Weihenstephan, Germany. For separation of amino acids also see appl. 00 at. 8 Determination of physiological amino acids from supernatants of cell cultures MN Appl. No Column: 0 x mm NUCLEODUR C 8 Gravity, μm Sample preparation: supernatants from cell cultures are deproteinated, derivatized with phenylisothiocyanate and filtered Eluent: A) 70 mm sodium acetate, ph.,. % acetonitrile, ppm EDTA; B) acetonitrile water methanol (:0:, v/v/v) % B ( min) 7. % B in min (8 min) % B in 9 min (washing: 00 % B for 0 min; equilibration: % B for min) Flow rate: 0.8 ml/min Temperature: 7 ± C Detection: UV, nm Injection volume: 0 μl. -Hydroxyproline 9. Serine. Glutamine. Histidine 7 8. Citrulline (.90 μmol/l). Threonine 7. Arginine (0. μmol/l) R 8. Tyrosine R 9. Valine 0. Methionine 0. Isoleucine. Leucine. Phenylalanine. Tryptophan. Ornithine (. μmol/l) R = reagents min Citrulline, arginine and ornithine were determined quantitatively. Courtesy of Dr. J. Weinreich, Center for Medical Research, Clinic for General Surgery, University Clinical Center, Tübingen, Germany.

49 Biological and natural compounds Aromatic amino acids and histamine MN Appl. No. 980 Column: x mm NUCLEODUR HILIC, μm Eluent: acetonitrile 00 mm ammonium acetate, ph (7:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 8 nm Injection volume: 0. μl. Phenylalanine. Phenylglycine. Tyrosine. Histamine. Histidine Nucleic acid bases MN Appl. No. 90 Column: 0 x mm NUCLEODUR C 8 Pyramid, μm Eluent: A) 0 mm NH H PO, ph., B) acetonitrile 00 % A (. min) 90 % A in 0 min Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Cytosine. Adenine. Uracil. Thymine 0 8 min 0 8 min Nucleic acid bases MN Appl. No. 7 Columns: 0 x mm NUCLEODUR PolarTec, μm 0 x mm Waters SymmetryShield RP8, μm Eluent: 0 mm KH PO, ph.0 Flow rate: 0. ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: μl, μl. Cytosine. Uracil. Adenine. Guanine. Thymine MN Appl. No. 90 Column: x mm NUCLEODUR HILIC, μm Eluent: acetonitrile mm ammonium acetate (80:0, v/v) Flow rate: 0. ml/min Temperature: C Detection: UV, nm. Thymine. Uracil. Adenine. Cytosine. Guanosine 0 7 min min Separation of all five nucleic acid bases just on NUCLEODUR PolarTec 7

50 Applications Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: A) 0 mm NH H PO, ph B) acetonitrile Gradient: 0 % B 0 % B in min Flow rate: ml/min Temperature: 0 C Detection: UV, 0 nm. Quinine. Scopolamine. Brucine. Strychnine. Atropine. Papaverine 7. Noscapine Determination of quinine in cinchona bark MN Appl. No. 880 cinchona bark 7 standard 0 0 min Quinine alkaloids MN Appl. No. 790 Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: A) methanol, B) 0 mm KH PO, ph. 90 % B 70 % B in min 0 % B in 7 min Flow rate:. ml/min Temperature: C Detection: UV, 0 nm Injection volume: 0 μl. Chloroquine. Quinine. Mefloquine Steroids MN Appl. No. 80 Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: acetonitrile water (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm. Cortisone. Hydrocortisone. Hydrocortisone -acetate. a-methyl-bhydroxyprogesterone. a-methyl-7ahydroxyprogesterone. a-methyl-7ahydroxyprogesterone acetate 0 min 0 0 min 8

51 Biological and natural compounds Steroids MN Appl. No. 0 Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: A) acetonitrile, B) water 70 % B (7 min) 0 % B in min 70 % B in min Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Injection volume: μl. Cortisone. Prednisolone. a-methylprednisolone. Dexamethasone. Corticosterone 9. Hydrocortisone 7. 7a-Hydroxycortisone 8. Progesterone 9. Deoxycorticosterone Steroids MN Appl. No. 70 Column: x mm NUCLEODUR C 8 HTec, μm Eluent: A) acetonitrile, B) water 70 % B 0 % B in min ( min) % B in min ( min) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: 0 μl. Estriol. Estrone. Prednisolone 7. a-methyl-b-hydroxyprogesterone. Cortisone 8. a-methyl-7a-hydroxyprogesterone. Estradiol 9. Progesterone. Testosterone min min Steroids MN Appl. No. 80 Column: x mm NUCLEODUR 00- C 8 ec Eluent: A) water, B) methanol 0 % B ( min) % B in 0 min ( min) 0 % B in min ( min) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: 0 μl (each ~0 0 μg/ml). Estriol. Prednisolone. Cortisone 8. Testosterone. a-methyl-bhydroxyprogesterone. a-methyl-7ahydroxyprogesterone 0 7. a-methyl-7a- hydroxyprogesterone 9 acetate 8. Estradiol 9. Estrone 0. Progesterone 7 Hydroxytestosterones from cytochrome P0 MN Appl. No. 0 Colum: 0 x mm NUCLEODUR C 8 Isis,.8 μm Eluent: A) water + 0. % formic acid; B) acetonitrile methanol + 0. % formic acid 7 % A 0 % A in. min 0 % A in 0.0 min, 7 % A in 0.0 min (0.9 min) Flow rate: 0.9 ml/min Temperature: 70 C Detection: LC-MS/MS. b-hydroxytestosterone. 7a-Hydroxytestosterone min min 9

52 Applications Catecholamines MN Appl. No. 00 Column: 0 x mm NUCLEODUR HILIC, μm Eluent: acetonitrile mm ammonium formate, ph (7:, v/v) Flow rate: 0.8 ml/min Temperature: C Detection: UV, 8 nm Injection: μl, 0 ng/μl. Norephedrine. Dopamine. Adrenaline. L-DOPA Analysis of aflatoxins from baby food MN Appl. No Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: methanol acetonitrile water (:7:7, v/v/v) with 9 mg KBr and 00 μl HNO ( %) per liter Flow rate:.0 ml/min Detection: fluorescence, l ex nm, l em 0 nm, post column derivatization in a CoBrA cell (Dr. Weber Consulting Kft) Injection volume: 00 μl. Aflatoxin G. Aflatoxin G. Aflatoxin B. Aflatoxin B min 0 min Catecholamines MN Appl. No. 790 Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: 00 mm NaH PO, ph.0 Flow rate: 0.8 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Norephedrine. Adrenaline. Dihydroxyphenylalanine. Hydroxytyramine. Tyrosine Analysis of mycotoxins MN Appl. No Column: 0 x mm NUCLEODUR C 8 Gravity, μm Guard column: 8 x mm NUCLEODUR C 8 Gravity, μm Eluent: acetonitrile water (:, v/v), ml conc. H PO /L, adjusted to ph. with NaOH Flow rate: 0.9 ml/min Detection: fluorescence 7 nm and nm Injection volume: 0 μl (7. ng of each substance). b-zearalenol. a-zearalenol. Zearalenone 0 min 0 0 min Courtesy of K.H. Ueberschär, Federal Agricultural Research Centre, Institute of Animal Feed, Celle, Germany. 0

53 Biological and natural compounds Dexa- and betamethasone MN Appl. No. 70 Column: 0 x mm NUCLEODUR C 8 Isis, μm Eluent: acetonitrile water (0:70, v/v) Flow rate: ml/min Temperature: C Detection: UV, 0 nm Injection volume: μl. Betamethasone. Dexamethasone Sunscreen ingredients MN Appl. No. 00 Column: x mm NUCLEODUR 00- C 8 ec Eluent: methanol 0. % H PO (8:8, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 00 nm Injection volume: 0 μl. Benzimidazolecarboxylic acid. Benzophenone-. -Methylbenzylidine camphor. Octocrylene. Ethylhexyldimethyl PABA. Ethylhexyl methoxycinnamate 7. Butyl methoxydibenzoylmethane (BMDBM) 8. Ethylhexyl salicylate min 0 0 min For separation on C 8 Gravity see appl. 0 at. Determination of iso-alpha-acids, alpha- and betaacids in isomerized hop pellets MN Appl. No. 00 Column: x mm NUCLEODUR 00- C 8 ec Eluent: A) methanol, B) methanol water H PO (7::, v/v/v); 00 % B (7 min) % B in 8 min 00 % B in min Flow rate:.0 ml/min Temperature: C Detection: UV, 9 min 70 nm, then nm. Isocohumulone. Isohumulone. Isoadhumulone. Cohumulone. ad- + n-humulone. Colupulone 7. ad- + n-lupulone 7 Sunscreen ingredients MN Appl. No. 0 Column: x mm NUCLEODUR C 8 HTec, μm Eluent: methanol 00 mm ammonium acetate, ph. (80:0, v/v) Flow rate: 0.9 ml/min Temperature: C Detection: UV, 7 nm Injection volume: μl. Benzophenone. -Methylbenzylidine camphor. Uvinul Plus. Octocrylene. Ethylhexyldimethyl PABA. Ethylhexyl methoxycinnamate 7. Ethylhexyl salicylate min M. Biendl et al., European Brewery Convention, J. of the Institute of Brewing 0 (00). 0 0 min

54 Applications Water-soluble vitamins MN Appl. No. 70 Column: 0 x mm NUCLEODUR PolarTec, μm Eluent: A) mm KH PO, ph.0, B) acetonitrile 0 % B ( min) 0 % B in min Flow rate: 0.7 ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: μl. Vitamin B. Vitamin B. Panthotenic acid. p-aminobenzoic acid. Vitamin B min Water-soluble vitamins MN Appl. No Column: x mm NUCLEODUR C 8 Pyramid, μm Eluent: A) water, mm heptanesulfonic acid (Na salt), mm NaH PO, 0. % CH COOH, 0.00 % triethylamine (ph.), B) acetonitrile water (0:0, v/v), mm heptanesulfonic (Na salt), 0. % CH COOH, 0.00 % triethylamine (ph ~.); multistep gradient: 0 % B ( min) 0 % B in. min % B in. min 0 % B in 8 min 70 % B in 7 min 0 % B in min Flow rate:.0 ml/min Temperature: C Detection: UV,, 7 and nm Injection volume: 0 μl. Nicotinic acid (0. mg/ml). Nicotinamide (0. mg/ml). -Aminobenzoic acid (0.0 mg/ml). Folic acid (0. mg/ml). Vitamin B (pyridoxine hydrochloride, 0.0 mg/ml). Vitamin B (riboflavin, 0.0 mg/ml) 7. Vitamin B (thiamine hydrochloride, 0.0 mg/ml) 8. Rutin (0.0 mg/ml) orange curves: vitamin test mixture (in eluent A) blue curves: multivitamin juice (undiluted) both detected at three different wave lengths Water-soluble vitamins MN Appl. No. 970 Column: x mm NUCLEODUR HILIC, μm Eluent: A) acetonitrile, B) mm ammonium acetate, ph 80 % A ( min) 70 % A in min ( min) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: 0 μl. Nicotinamide. Vitamin B 7 (vitamin B 8, vitamin H, biotin). Vitamin B (pyridoxine). Vitamin C (ascorbic acid). Vitamin B (cyanocobalamine). Vitamin B (thiamine) 7 8 nm 7 nm nm 0 min min

55 Biological and natural compounds Water-soluble vitamins MN Appl. No. 0 Column: x mm NUCLEODUR C 8 Pyramid, μm Eluent: A) 0 mm KH PO, ph, B) methanol acetonitrile (70:0, v/v) 0 % B ( min) % B in min % B in 0 min ( min) Flow rate: 0. ml/min Temperature: 0 C Detection: UV, 8, and 7 nm Injection volume: 0 μl. Vitamin B (thiamine). Pyridoxamine. Vitamin C (ascorbic acid). Pyridoxal. Vitamin B (pyridoxine). Vitamin B 9 (viamin M, folic acid) 7. Vitamin B (cyanocobalamine) 8. Vitamin B 7 (vitamin B 8, vitamin H, (+)-biotin) 9. Vitamin B (riboflavin) Fat-soluble vitamins and tocopherols MN Appl. No Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile Flow rate:. ml/min Temperature: 0 C Detection: UV, 80 nm Injection volume: μl. Vitamin K. Vitamin A. Vitamin A acetate. Vitamin D. Vitamin D. Vitamin E (a-tocopherol) 7. Vitamin E acetate (a-tocopherol acetate) 8. Vitamin K min 0 nm nm MN Appl. No. 0 Column: x mm NUCLEODUR C 8 Isis, μm Eluent: acetonitrile water (00:, v/v) Flow rate: 0. ml/min Temperature: C Detection: UV, 7 nm Injection volume: μl. Vitamin A. g-tocopherol. Vitamin A acetate 7. Vitamin E (a-tocopherol). Vitamin K 8. Vitamin E acetate (a-tocopherol acetate). Vitamin D 9. Vitamin K. Vitamin D 7 nm min min For separation of tocopherols on NUCLEODUR 00- C 8 ec see appl. 790 at.

56 Applications Complexing agents acc. to DIN 8-8 MN Appl. No Column: 0 x mm NUCLEODUR C 8 Pyramid, μm Eluent: 0. mm HNO, 7. mm N(C H 9 ) HSO,. mm N(C H 9 ) OH, 7 μm Fe + Flow rate: 0. ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: 0 μl. NTA (nitrilotriacetic acid). EDTA (ethylenediaminetetraacetic acid). DTPA (diethylenetrinitrilopentaacetic acid) Courtesy of H. Albrich, C. Geis, GIU; Commercial Institute for Environmental Analysis, Teningen, Germany. Aromatic acids MN Appl. No. 80 Column: x mm NUCLEODUR C 8 Isis, μm Eluent: methanol 0 mm KH PO, ph (0:90, v/v) Flow rate: 0. ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. Gallic acid.,-dihydroxybenzoic acid.,-dihydroxybenzoic acid. -Hydroxybenzoic acid. Syringic acid. Vanillic acid 0 8 min 0 0 min Ascorbic acid and dehydroascorbic acid MN Appl. No. 90 Column: 0 x mm NUCLEODUR HILIC, μm Eluent: acetonitrile 00 mm ammonium acetate (70:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm. Dehydroascorbic acid. Ascorbic acid Organic acids MN Appl. No. 990 Column: 0 x mm NUCLEODUR 00- CN-RP Eluent: mm KH PO, ph.0 Flow rate: 0. ml/min Temperature: 0 C Detection: UV, 0 nm Injection volume: μl. Aspartic acid. Fumaric acid. Maleic acid min 0. min

57 Pollutants and miscellaneous organics Organic acids MN Appl. No. 90 Column: x mm NUCLEODUR HILIC, μm Eluent: acetonitrile 00 mm ammonium acetate, ph.8 (70:0, v/v) Flow rate: ml/min Temperature: C Detection: UV, 0 nm Injection volume: 0. μl. Fumaric acid. Oxalic acid. Citric acid Organic acids MN Appl. No. Columns: 0 x mm NUCLEODUR PolarTec, μm 0 x mm Waters SymmetryShield RP8, μm Eluent: A) acetonitrile, 0. % TFA, B) water, 0. % TFA 0 % A 0 % A in min Flow rate: 0.7 ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. Dihydroxymandelic acid. Vanillic acid. Gallic acid 7. -Hydroxybenzoic acid. Dihydroxyphenylacetic acid 8.,-Dihydroxybenzoic acid.,-dihydroxybenzoic acid 9.,-Dihydroxybenzoic acid. Syringic acid min MN Appl. No. 000 Column: 0 x. mm NUCLEODUR C 8 Pyramid, μm Eluent: 0 mm KH PO, ph. Flow rate: 0.7 ml/min Detection: UV, 0 nm Injection volume: 0 μl. Tartaric acid. Malic acid. Shikimic acid. Lactic acid. Acetic acid. Citric acid 7. Fumaric acid 7, min 0 0 min Also see appl. 980 at.

58 Applications Nitrophenols MN Appl. No. 0 Column: 0 x mm NUCLEODUR C 8 Gravity, μm Eluent: methanol 0 mm NaH PO, ph (9:, v/v) Flow rate: 0.8 ml/min Temperature: 0 C Detection: UV, nm Injection volume:.0 μl. p-nitrophenol. o-nitrophenol. -Nitro-m-cresol. m-nitrophenol. -Nitro-p-cresol. -Nitro-m-cresol 7. -Nitro-o-cresol min 7 Substituted aromatics MN Appl. No. 980 / 980 Columns: 0 x. mm NUCLEODUR C 8 Gravity, μm 0 x. mm NUCLEODUR Sphinx RP, μm Eluent: methanol water (:, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. Uracil. Benzamide 7. Phenol 8 0. Benzaldehyde 9. Acetophenone. -Nitrophenol C 8 Gravity 7. Nitrobenzene 9 0 Sphinx RP 8. Propyl -hydroxybenzoate 9. Toluene 0. Benzophenone. Xylene min Nitroaromatics EPA 80 MN Appl. No. 90 / 00 Column: 0 x mm NUCLEODUR PolarTec, μm Eluent: Mix A: water methanol (0:0, v/v) Mix B: water, 0. % formic acid methanol (:, v/v) Flow rate: 0. ml/min Temperature: 0 C, 0 C Detection: UV, nm Injection volume: μl Peaks Mix A: Peaks Mix B:. Octogen (HMX) 9. N-Methyl-N-,,-tetranitroaniline (Tetryl). Hexogen (RDX).,,-Trinitrobenzene 0. -Amino-,-dinitrotoluene.,-Dinitrobenzene.,-Dinitrotoluene. Nitrobenzene. -Nitrotoluene.,,-Trinitrotoluene. -Nitrotoluene 7.,-Dinitrotoluene. -Nitrotoluene 8. -Amino-,-dinitrotoluene 9 0 Selectivity comparison of NUCLEODUR C 8 Gravity and Sphinx RP shows lower hydrophobicity and retention of Sphinx RP for substituted aromatics like toluene and xylene. Phenolic compounds MN Appl. No Column: x mm NUCLEODUR 00- C 8 ec Eluent: methanol water, 0. % H PO (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl. Resorcinol. Pyrocatechol. -Methoxyphenol. Phenol. -Methoxyphenol. -Ethoxyphenol 7 8 A B min For separation of Mix A and Mix B in a single run see appl. 0 at. 0 min

59 Pollutants and miscellaneous organics Phenolic compounds MN Appl. No. 70 Column: 0 x mm NUCLEODUR PFP, μm Eluent: A) acetonitrile + % acetic acid B) water + % acetic acid Gradient: % A (7 min) 70 % A in min ( min) Flow rate: 0. ml/min Temperature: C Detection: UV, nm Injection volume: μl. Phenol. -Nitrophenol. -Nitrophenol.,-Dinitrophenol. -Chlorophenol 7. -Chloro--methylphenol 8.,-Dichlorophenol 9.,-Dimethylphenol 0.,,-Trichlorophenol. Pentachlorophenol. -Methyl-,-dinitrophenol 9 Separation of phenol isomers MN Appl. No. Columns: 00 x. mm NUCLEODUR PFP, μm 00 x. mm Phenomenex Luna PFP(), μm Eluent: acetonitrile, 0. % formic acid water, 0. % formic acid (:, v/v) Flow rate:. ml/min Temperature: C Detection: UV, 80 nm. o-cresol 7.,-Dichlorophenol. m-cresol 8.,-Dichlorophenol.,-Dimethylphenol 9.,-Dichlorophenol.,-Dimethylphenol 0.,-Dibromophenol.,-Dimethylphenol.,-Dibromophenol.,-Dichlorophenol min Phenolic compounds MN Appl. No. 70 Column: 00 x mm NUCLEODUR PFP, μm Eluent: A) methanol, B) 0 mm ammonium acetate, ph.8 % A 80 % A in min Flow rate: 0. ml/min Temperature: 8 C Detection: UV, 0 nm Injection volume: μl. Uracil. Pyrogallol. Phloroglucinol. Resorcinol. Benzamide. Catechol 7. Phenol 8. Nitrobenzene min NUCLEODUR PFP provides under identical conditions a better separation than Luna PFP(). While on Luna PFP() for peaks 0 and only a resolution of.7 is obtained, on NUCLEODUR PFP the peaks are baseline separated (R S =.) min 7

60 Applications Amines MN Appl. No. 00 Column: 0 x. mm NUCLEODUR C 8 Isis, μm Eluent: acetonitrile 0 mm K HPO (0:0, v/v), ph 8 Flow rate: ml/min Temperature: C Detection: UV, nm Injection volume: 8 μl. Uracil. Pyridine. Desethylatrazine. -Acetylpyridine. -Ethylaniline. N,N-Dimethylaniline 7. -Aminoanthraquinone 8.,-Dinitro-(-phenylethylbenzamide) Aromatic aldehydes MN Appl. No Column: x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile water, ph.0 (:78, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl (~0 0 μg/ml). p-carboxybenzaldehyde. p-hydroxybenzaldehyde. Vanillin. -Ethoxyvanillin. Benzaldehyde min 0 8 min Aromatic amines MN Appl. No. 800 Column: 0 x mm NUCLEODUR PFP, μm Eluent: A) methanol, B) 0 mm K HPO, ph 0 % A ( min) 70 % A in 0 min (7 min) Flow rate: ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. -Chloroaniline. -Nitroaniline. -Nitroaniline. -Chloroaniline. -Chloroaniline. -Nitroaniline 7.,-Dichloroaniline Chloro--nitroaniline Aromatic ketones MN Appl. No. 7 Columns: 0 x mm NUCLEODUR PFP, μm 0 x mm NUCLEODUR C 8 Gravity, μm Eluent: methanol water (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Injection volume: μl. o-methylacetophenone. p-methylacetophenone. m-methylacetophenone min min Distinct steric selectivity of NUCLEODUR PFP provides separation of all three regioisomers, while on NUCLEODUR C 8 Gravity baseline separation can't be achieved. 8

61 Pollutants and miscellaneous organics Aromatic ketones MN Appl. No. 70 / 70 Column: x mm NUCLEODUR C 8 Gravity, μm 0 x mm NUCLEODUR C 8 Gravity,.8 μm Eluent: acetonitrile water (0:0, v/v) Flow rate: 0. ml/min,. ml/min Temperature: C Detection: UV, 0 nm. Acetophenone. Eugenol. Propiophenone. Butyrophenone. Benzophenone. Valerophenone Furfurol and related compounds in transformer oil in accordance with DIN EN 98-B MN Appl. No. Column: x mm NUCLEODUR 00- C 8 ec Sample prep.: SPE see appl. 080 at Eluent: methanol water (0:90, v/v) Flow rate:.0 ml/min Detection: UV, 0, 7, 8 and 9 nm. -Hydroxymethyl--furfurol. -Furfuryl alcohol. -Furfurol. -Acetylfuran. -Methyl--furfurol 9 nm 8 nm 7 nm 0 nm.8 μm μm 0 min 0 0 min A. Heiseler et al., GIT (00) 0 0 MN Appl. No Column: x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile water (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Injection volume: μl (~0 0 μg/ml). Acetophenone. Propiophenone. Butyrophenone. Benzophenone. Valerophenone Column: Sample prep.: Eluent: Flow rate: Detection:. Naphthalene. Fluorene. Acenaphthylene. Phenanthrene. Anthracene. Fluoranthene 7. Pyrene 8. Chrysene PAHs from tar MN Appl. No x. mm NUCLEODUR C 8 Gravity, μm see Appl. 070 at A) water, B) acetonitrile 0 % B ( min) 00 % B in 7 min.0 ml/min UV, 0 nm 9. Benz[a]anthracene 0. Benzo[b]fluoranthene. Benzo[k]fluoranthene. Benzo[a]pyrene. Dibenz[ah]anthracene. Indeno[,,-cd]pyrene. Benzo[ghi]perylene min min 9

62 Applications Column: Sample prep.: Organophosphorus herbicides MN Appl. No x mm NUCLEODUR C 8 Gravity, μm for SPE see Appl at ; derivatization with FMOC-Cl Eluent: A) acetonitrile, B) H PO, ph. 0 % A % A in 7 min 90 % A in min ( min) 0 % A in min (7 min) Flow rate: 0. ml/min Temperature: 0 C Detection: fluorescence, l ex nm, l em 7 nm. Glyphosate. AMPA (aminomethylphosphonic acid). Glufosinate min Courtesy of Mr. Schüssler, Mrs. Mikler, Bavarian State Agency for Water Management, Munich. MN Appl. No. 90 Column: 0 x mm NUCLEODUR 00- NH -RP Sample prep.: derivatization with FMOC, concentration of each pesticide 0. mg/ml Eluent: acetonitrile 0 mm KH PO, ph. (0:0, v/v) Flow rate: 0.8 ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. AMPA. Glyphosate. Glufosinate Pesticides from soil MN Appl. No Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: A) water, B) acetonitrile, 0 % B % B in 0 min 0 % B in 0 min ( min) 0 % B in 0 min 0 % B in 0 min (0 min) Flow rate: 0.8 ml/min Temperature: C Detection: UV, 0 nm. Metamitron. Desethylatrazine. Hexazinone. Metoxuron. Simazine. Cyanazine 7. Methabenzthiazuron (Tribunil ) 8. Chlortoluron 9. Atrazine 0. Monolinuron. Isoproturon. Diuron. Metobromuron. Metazachlor. Sebuthylazine. Dichlobenil 7. Terbuthylazine 8. Linuron 9. Chloroxuron 0. Propyzamid. Terbutryn. Metolachlor a) Pesticide standard b) Soil sample min Courtesy of E. Marek, LUFA Center for Analyses, Münster, Germany min Also see appl. 800 at. 0

63 Pollutants and miscellaneous organics Pesticides MN Appl. No. 08: triazines MN Appl. No. 08: phenylurea derivatives MN Appl. No. 08: phenoxycarboxylic acids MN Appl. No. 08: pesticides Column: 0 x mm NUCLEODUR 00- C 8 ec with 8 x mm guard column Eluent: A) acetonitrile, B) 0 mm KH PO + ml conc. H PO, 8 % B ( min) 7 % B in 0 min Flow rate: 0.7 ml/min Temperature: C Detection: UV, 8 nm Injection volume: 0 μl 8 9. Desisopropylatrazine.,-Dichlorobenzamide. Desethylatrazine 7. Hexazinone 0. Metoxuron. Dicamba 7. Bromacil 8. Simazine 9. Desethylterbuthylazine 0. Cyanazine 7. Methabenzthiazuron. Chlortoluron. Bentazone. Atrazine.,-D. Metalaxyl 9 7. Monolinuron 8. MCPA 9. Isoproturon 0. Diuron. Metobromuron. Metazachlor. Sebuthylazine. Dichlorprop. Mecoprop. Propazine 7. Dimefuron 8. Terbuthylazine 9. Linuron 8 0. Metolachlor Appl. 08 Appl. 08 Courtesy of C. Geis, GIU; Commercial Institute for Environmental Analysis, Teningen, Germany. + Appl Appl min

64 Applications Herbicides MN Appl. No. 00 / 00 Column: x mm NUCLEODUR HILIC, μm Eluent: acetonitrile 0 mm ammonium formate, ph. (80:0, v/v) Flow rate: 0. ml/min Temperature: C Detection: UV, and 08 nm; MS Injection volume: μl, 0. mg/ml. Paraquat. Diquat Perfluorinated surfactants in water MN Appl. No. 90 Column: x mm NUCLEODUR Sphinx RP, μm Sample prep.: see appl. 90 at Eluent: A) 0 mm NH acetate in water methanol (7:, v/v); B) 0 mm NH acetate in acetonitrile methanol (7:, v/v); 0 % B 0 % B in min % B in 8 min 70 % B in min Flow rate: 0. ml/min; temperature 0 C Detection: LC-MS-MS; injection volume 0 μl. Perfluorobutanoic acid 7. Perfluorooctanoic acid. Perfluoropentanoic acid 8. Perfluorononanoic acid. K perfluorobutanesulfonate 9. K perfluorooctanesulfonate. Perfluorohexanoic acid 0. Perfluorodecanoic acid. Perfluoroheptanoic acid. Perfluoroundecanoic acid. K perfluorohexansulfonate. Perfluorododecanoic acid 7 9 Appl. 00 MS min D. Skutlarek et al., Environ Sci Pollut Res (00) nm Appl. 00 nm 0 0 min Selectivity test MN Appl. No Column: x mm NUCLEODUR Sphinx RP, μm Eluent: methanol mm NH H PO, ph 7 (:, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl. Uracil.,7-Dihydroxynaphthalene.,-Dihydroxynaphthalene. Ethyl benzoate. Lidocaine. Biphenyl 7. Acenaphthene 7 0 min

65 Test for metal ions in silica adsorbent MN Appl. No. 80 Column: x mm NUCLEODUR C 8 Gravity, μm Eluent: methanol 0 mm KH PO, ph 7 (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm.,7-dihydroxynaphthalene.,-dihydroxynaphthalene Pollutants and miscellaneous organics HO OH OH OH M n+ 0 min The ratio of the asymmetry factors of,-dihydroxynaphthalene () and,7-dihydroxynaphthalene () is a measure for the metal ion content of the silica phase, because () can form complexes with metal ions, resulting in broad peaks for this compound. Dihydroxynaphthalenes MN Appl. No. 90 Column: x mm NUCLEODUR C 8 Isis, μm Eluent: methanol 0. % H PO (0:70, v/v) Flow rate: ml/min Temperature: 0 C Detection: UV, nm Injection volume: μl.,-dihydroxynaphthalene.,-dihydroxynaphthalene.,7-dihydroxynaphthalene.,-dihydroxynaphthalene.,-dihydroxynaphthalene 0 0 min

66 Substance index A Acenaphthene C 8 Gravity, μm 9 C 8 Pyramid, C 8 Gravity, C 8 Gravity Sphinx RP, μm Acenaphthylene C 8 Gravity, μm 9 Acesulfame K 00- C 8 ec C 8 HTec, μm -Acetamidophen C 8 Gravity, μm Acetamidophenol isomers C 8 HTec, μm Acetaminophen see Paracetamol Acetanilide C 8 Gravity, μm Acetic acid C 8 Pyramid, μm,, Acetophenone 00- C 8 ec 9 C 8 Gravity,.8 vs. μm 9 C 8 Gravity, Sphinx RP, μm -Acetylfuran 00- C 8 ec 9 -Acetylpyridine C 8 Isis, μm 8 Acetylsalicylic acid 00- C 8 ec 00- C 8 ec, C 8 Gravity, μm C 8 Gravity, μm C 8 HTec, μm C 8 Pyramid, μm Acrylamide HILIC, μm Adenine 00- NH -RP C 8 Pyramid, μm 7 HILIC, μm 7, 7 PolarTec, μm 7 PolarTec, μm 7 Adhumulone, adlupulone 00- C 8 ec Adrenaline C 8 Gravity, μm 0 HILIC, μm 0 Aflatoxins 00- C 8 ec 0 Alanine 00- C 8 ec -Amino-,-dinitrotoluene PolarTec, μm -Amino-,-dinitrotoluene PolarTec, μm -Aminoanthraquinone C 8 Isis, μm 8 -Aminobenzoic acid C 8 Pyramid, μm p-aminobenzoic acid PolarTec, μm g-aminobutyric acid 00- C 8 ec Aminomethylphosphonic acid see AMPA Amitriptyline C 8 Gravity, C 8 Isis, C 8 Pyramid C 8 Gravity, μm 9, PolarTec, μm Amoxicillin C 8 Gravity, μm C 8 HTec, μm C 8 Pyramid, μm 0 PFP, μm AMPA 00- NH -RP, 0 C 8 Gravity, μm 0 Ampicillin PFP, μm Aniline C 8 HTec, μm p-anisic acid PolarTec, μm Anthracene C 8 Gravity, μm 9 C 8 HTec, μm; EC vs. VP Arginine 00- C 8 ec C 8 Gravity, μm Ascorbic acid 00- C 8 ec C 8 Pyramid, μm, HILIC, μm HILIC, μm Asparagine 00- C 8 ec Aspartame 00- C 8 ec C 8 HTec, μm Aspartic acid 00- C 8 ec 00- CN-RP Atrazine 00- C 8 ec 0, Atropine C 8 Gravity, μm 8 Azorubine C 8 Gravity, μm B Bambuterol C 8 Pyramid, μm 7 Beclometasone dipropionate C 8 Pyramid,.8 μm Bentazone 00- C 8 ec Benzaldehyde 00- C 8 ec 8 C 8 Gravity, Sphinx RP, μm Benzalkonium chlorides 00- CN-RP 7 Benzamide 00- CN-RP 8, 9 C 8 Gravity, μm 0,, C 8 Pyramid, C 8 Gravity, μm PFP, μm 7 Sphinx RP, μm Benz[a]anthracene C 8 Gravity, μm 9 Benzocaine C 8 Pyramid, μm Benzo[b]fluoranthene C 8 Gravity, μm 9 Benzo[k]fluoranthene C 8 Gravity, μm 9 Benzoic acid 00- C 8 ec C 8 Gravity, μm C 8 HTec, μm PolarTec, μm Benzo[ghi]perylene C 8 Gravity, μm 9 Benzophenone 00- C 8 ec 9 C 8 Gravity,.8 vs. μm 9 C 8 Gravity, μm, C 8 HTec, μm C 8 Pyramid, C 8 Gravity, μm Sphinx RP, μm Benzophenone- 00- C 8 ec Benzo[a]pyrene C 8 Gravity, μm 9 Benzyl alcohol PolarTec, μm Betamethasone C 8 Isis, μm PFP, μm 9 Biotin C 8 Pyramid, μm HILIC, μm Biphenyl 00- CN-RP 8, 9 C 8 Pyramid, C 8 Gravity, C 8 Gravity Sphinx RP, μm Biphenyl--ol 00- C 8 ec / C 8 ec Biuret C 8 Pyramid, μm Bromacil 00- C 8 ec Bromazepam 00- C 8 ec 8 Brompheniramine 00- C 8 ec / 00- CN-RP 8 PFP, μm 7 PFP, C 8 Gravity, μm 9 Brucine C 8 Gravity, μm 8 Butacaine C 8 Pyramid, μm Butyl methoxydibenzoylmethane 00- C 8 ec Butyl paraben PolarTec, μm Butyrophenone 00- C 8 ec 9 C 8 Gravity,.8 vs. μm 9 C Caffeine 00- C 8 ec 00- C 8 ec C 8 Gravity, μm C 8 Gravity, μm C 8 Isis, μm C 8 Pyramid, μm Sphinx RP, μm 0, -Carboxybenzaldehyde 00- C 8 ec 8 Carprofen 00- C 8 ec / C 8 ec Catechin Sphinx RP, C 8 Gravity, C 8 Gravity Catechol PFP, μm 7

67 Alphabetical index of analytes Cefalotin PFP, μm Cefamandole C 8 Gravity, PFP, μm Cefotaxime C 8 Gravity, PFP, μm Cefoxitin C 8 Gravity, PFP, μm Cephalexin PFP, μm Cephalothin C 8 Gravity, μm Cetirizine PFP, μm 7 PFP, C 8 Gravity, μm 9 Chloramphenicol C 8 Gravity, μm Chlormequat HILIC, μm 7 -Chloro--nitroaniline PFP, μm 8 -Chloro--methylphenol PFP, μm 7 Chloroaniline isomers PFP, μm 8 Chlorocresol C 8 Pyramid,.8 μm -Chlorophenol PFP, μm 7 Chloroprothixene hydrochloride C 8 Gravity, μm Chloroquine C 8 Gravity, μm 8 Chloroxuron 00- C 8 ec 0 Chlorpheniramine 00- C 8 ec / 00- CN-RP 8 C 8 Gravity, μm C 8 Gravity, μm, C 8 Pyramid, μm, PFP, μm 7 PFP, C 8 Gravity, μm 9 Chlorpromazine 00- CN-RP 8 Chlortoluron 00- C 8 ec 0, Chrysene C 8 Gravity, μm 9 Cimetidine C 8 Gravity, μm Cinoxacin 00- C 8 ec 9 C 8 Gravity, μm C 8 Gravity, C 8 Gravity, C 8 Pyramid 0 C 8 HTec, μm Sphinx RP, μm 0 Ciprofloxacin 00- C 8 ec 9 Sphinx RP, μm 9 Citric acid C 8 Pyramid, μm HILIC, μm Citrulline C 8 Gravity, μm Clenbuterol 00- CN-RP 8 C 8 Pyramid, μm 7 Clobetasol 7-propionate C 8 Pyramid,.8 μm Clomipramine 00- CN-RP; C 8 Isis, μm Clonidin 00- CN-RP 8 Cloxacillin 00- C 8 ec 9, 0 C 8 Gravity, μm C 8 HTec, μm PFP, μm Cohumulone, colupulone 00- C 8 ec Corticosterone C 8 Gravity, μm 9 Cortisone 00- C 8 ec CN / CN-RP 9 C 8 Gravity, μm 8 C 8 Gravity, μm 9 C 8 HTec, μm 9 Creatine HILIC, μm Creatinine HILIC, μm o-, m-cresol PFP, μm 7 PFP, C 8 HTec, μm 9 Cyanazine 00- C 8 ec 0, Cyanocobalamine C 8 Pyramid, μm HILIC, μm Cyanuric acid C 8 Pyramid, μm HILIC, μm Cyclohexane 00- NH 0 Cytosine 00- NH -RP C 8 Pyramid, μm 7 HILIC, μm 7, 7 PolarTec, μm 7 PolarTec, μm 7 D,-D 00- C 8 ec Dehydroacetic acid C 8 HTec, μm PolarTec, μm Dehydroascorbic acid HILIC, μm Deoxycorticosterone C 8 Gravity, μm 9 Desethylatrazine 00- C 8 ec 0, C 8 Isis, μm 8 Desethylterbuthylazine 00- C 8 ec Desisopropylatrazine 00- C 8 ec Dexamethasone C 8 Gravity, μm 9 C 8 Isis, μm PFP, μm 9 Dextromethorphan C 8 Gravity, μm C 8 Pyramid, μm Dibenz[ah]anthracene C 8 Gravity, μm 9 Dibenzothiophene 00- NH 0 Dibromophenol isomers PFP, μm 7 PFP, C 8 HTec, μm 9 Dibutyl phthalate C 8 Gravity, μm 9 Dicamba 00- C 8 ec Dichlobenil 00- C 8 ec 0,-Dichloroaniline PFP, μm 8,-Dichlorobenzamide 00- C 8 ec,-dichlorophenol PFP, μm 7 Dichlorophenol isomers PFP, μm 7 PFP, C 8 HTec, μm 9 Dichlorprop 00- C 8 ec Diclofenac 00- C 8 ec 00- C 8 ec,, Dicloxacillin 00- C 8 ec, C 8 Pyramid, μm 0 PFP, μm Diethylenetrinitrilopentaacetic acid C 8 Pyramid, μm Diflunisal 00- C 8 ec C 8 Gravity, μm Dihydroxybenzoic acid isomers C 8 Isis, μm PolarTec, μm Dihydroxymandelic acid PolarTec, μm Dihydroxynaphthalene isomers C 8 Gravity, μm C 8 Isis, μm Sphinx RP, μm Dihydroxyphenylacetic acid PolarTec, μm Dihydroxyphenylalanine C 8 Gravity, μm 0 Dimefuron 00- C 8 ec N,N-Dimethylaniline C 8 HTec, μm C 8 Isis, μm 8,-Dimethylbenzene 00- NH 0,-Dimethylphenol PFP, μm 7 Dimethylphenol isomers PFP, μm 7 PFP, C 8 HTec, μm 9 Dimethyl phthalate 00- CN-RP 8, 9 C 8 Pyramid, C 8 Gravity, C 8 Gravity,-Dinitrobenzene PolarTec, μm,-dinitrophenol PFP, μm 7,-Dinitro-(-phenylethylbenzamide) C 8 Isis, μm 8 Dinitrotoluene isomers PolarTec, μm

68 Substance index Diphenhydramine C 8 Gravity, μm 0 C 8 Pyramid, μm PFP, μm 7 PFP, C 8 Gravity, μm 9 Dipyridamole C 8 Gravity, μm 9 Diquat HILIC, μm Diuron 00- C 8 ec 0, L-DOPA HILIC, μm 0 Dopamine HILIC, μm 0 Doxepin 00- CN-RP; C 8 Gravity, C 8 Gravity, C 8 Pyramid, μm PolarTec, μm DTPA C 8 Pyramid, μm E E 0 / 0 / 0 / / / / 7 C 8 Gravity, μm EDTA C 8 Pyramid, μm Enrofloxacin C 8 Gravity, μm C 8 HTec, μm Sphinx RP, μm 9 Ephedrine 00- C 8 ec / 00- CN-RP 8 Erythrosine C 8 Gravity, μm Estradiol 00- C 8 ec; C 8 HTec, μm 9 Estriol 00- C 8 ec; C 8 HTec, μm 9 Estrone 00- C 8 ec; C 8 HTec, μm 9 -Ethoxyphenol 00- C 8 ec 00- C 8 ec, -Ethoxyvanillin 00- C 8 ec 8 -Ethylaniline C 8 HTec, μm C 8 Isis, μm 8 Ethylbenzene 00- C 8 ec C 8 Gravity,.8 vs. μm C 8 Gravity, μm C 8 HTec, μm C 8 Isis, μm Ethyl benzoate C 8 Pyramid, C 8 Gravity, C 8 Gravity Sphinx RP, μm Ethylenediaminetetraacetic acid C 8 Pyramid, μm Ethylhexyldimethyl PABA 00- C 8 ec; C 8 HTec, μm Ethylhexyl methoxycinnamate 00- C 8 ec; C 8 HTec, μm Ethylhexyl salicylate 00- C 8 ec; C 8 HTec, μm Ethyl paraben PolarTec, μm Eugenol C 8 Gravity,.8 vs. μm 9 F Famotidine C 8 Gravity, μm Fast Red E C 8 Gravity, μm Fast Yellow C 8 Gravity, μm Fenoprofen 00- C 8 ec; 00- C 8 ec C 8 Gravity, μm Fenoterol C 8 Pyramid, μm 7 Fisetin Sphinx RP, C 8 Gravity, C 8 Gravity Flumequine Sphinx RP, μm 9 Flunarizine hydrochloride C 8 Gravity, μm 9 Fluoranthene C 8 Gravity, μm 9 Fluorene C 8 Gravity, μm 9 Flurbiprofen 00- C 8 ec 00- C 8 ec C 8 Gravity, μm C 8 Pyramid, μm Folic acid C 8 Pyramid, μm, Formic acid C 8 Pyramid, μm Fructose 00- NH -RP 0 Fumaric acid 00- CN-RP C 8 Pyramid, μm HILIC, μm -Furfurol 00- C 8 ec 9 -Furfuryl alcohol 00- C 8 ec 9 G Gallic acid C 8 Isis, μm C 8 Pyramid, μm PolarTec, μm Glucose 00- NH -RP 0 Glufosinate 00- NH -RP 0 C 8 Gravity, μm 0 Glutamic acid 00- C 8 ec Glutamine 00- C 8 ec C 8 Gravity, μm Glycine 00- C 8 ec Glyphosate 00- NH -RP 0 C 8 Gravity, μm 0 Guanine PolarTec, μm 7 PolarTec, μm 7 Guanosine HILIC, μm 7, 7 H Hexamethylbenzene 00- NH 0 Hexazinone 00- C 8 ec 0, Hexobarbital 00- C 8 ec Hexogen (RDX) PolarTec, μm Histamine HILIC, μm 7 Histidine 00- C 8 ec C 8 Gravity, μm HILIC, μm 7 PolarTec, μm 7 n-humulone 00- C 8 ec Hydrocortisone 00- CN / CN-RP 9 C 8 Gravity, μm 8 C 8 Gravity, μm 9 Hydrocortisone acetate C 8 Gravity, μm 8 -Hydroxybenzaldehyde 00- C 8 ec 8 -Hydroxybenzoic acid C 8 Isis, μm -Hydroxybenzoic acid PolarTec, μm 7a-Hydroxycortisone C 8 Gravity, μm 9 Hydroxymethylfurfural C 8 Gravity, μm -Hydroxymethyl--furfurol 00- C 8 ec 9 a-hydroxymidazolam C 8 Gravity, μm 8 -Hydroxyproline C 8 Gravity, μm Hydroxytestosterone isomers C 8 Isis,.8 μm 9 Hydroxytyramine C 8 Gravity, μm 0 Hydroxyzine PFP, μm 7 PFP, C 8 Gravity, μm 9 I Ibuprofen 00- C 8 ec 00- C 8 ec, C 8 Gravity, μm C 8 Pyramid, μm Imipramine C 8 Gravity, C 8 Gravity, C 8 Isis, C 8 Pyramid, μm PolarTec, μm Indeno[,,-cd]pyrene C 8 Gravity, μm 9 Indomethacin 00- C 8 ec C 8 Gravity, μm Irgasan PolarTec, μm Isoadhumulone 00- C 8 ec Isobutyl paraben PolarTec, μm Isocohumulone 00- C 8 ec Isohumulone 00- C 8 ec

69 Alphabetical index of analytes Isoleucine 00- C 8 ec C 8 Gravity, μm Isoproturon 00- C 8 ec 0, Isorhamnetin Sphinx RP, C 8 Gravity, C 8 Gravity K Kaempferol Sphinx RP, C 8 Gravity, C 8 Gravity Ketoprofen 00- C 8 ec 00- C 8 ec,, C 8 Gravity, μm 0 C 8 Pyramid, μm L Lactic acid C 8 Pyramid, μm Lactose 00- NH -RP 0 Leucine 00- C 8 ec C 8 Gravity, μm Lidocaine C 8 Gravity, μm C 8 Gravity, μm 0, C 8 Pyramid, μm, Sphinx RP, μm Linuron 00- C 8 ec 0, Lorazepam 00- C 8 ec 8 n-lupulone 00- C 8 ec Lysine 00- C 8 ec M Maleic acid 00- C 8 ec CN-RP 8, C 8 Gravity, μm C 8 Pyramid, μm PFP, μm 7 PFP, C 8 Gravity, μm 9 Malic acid C 8 Pyramid, μm Maltose 00- NH -RP 0 Mapenterol C 8 Pyramid, μm 7 Maprotiline C 8 Isis, μm Marbofloxacin Sphinx RP, μm 9 MCPA 00- C 8 ec Meclofenamic acid 00- C 8 ec Mecoprop 00- C 8 ec Medrysone 00- CN / CN-RP 9 Mefloquine C 8 Gravity, μm 8 Melamine HILIC, μm Mephobarbital 00- C 8 ec Mepiquat HILIC, μm 7 Metalaxyl 00- C 8 ec Metamitron 00- C 8 ec 0 Metazachlor 00- C 8 ec 0, Methabenzthiazuron 00- C 8 ec 0, Methacrylamide HILIC, μm Methacrylic acid HILIC, μm Methionine 00- C 8 ec C 8 Gravity, μm Methoxyphenol isomers 00- C 8 ec 00- C 8 ec, -Methyl-,-dinitrophenol PFP, μm 7 o-, m-, p-methylacetophenone PFP, C 8 Gravity, μm 8 9-Methylanthracene 00- NH 0 -Methylbenzylidine camphor 00- C 8 ec C 8 HTec, μm -Methyl--furfurol 00- C 8 ec 9 Methylhistidine isomers PolarTec, μm 7 Methyl--hydroxybenzoate C 8 Pyramid, μm a-methyl-b-hydroxyprogesterone 00- C 8 ec; C 8 HTec, μm 9 C 8 Gravity, μm 8 a-methyl-7a-hydroxyprogesterone / + acetate 00- C 8 ec; C 8 HTec, μm 9 C 8 Gravity, μm 8 Methyl paraben PolarTec, μm a-methylprednisolone C 8 Gravity, μm 9 Methyltestosterone 00- CN / CN-RP 9 Metobromuron 00- C 8 ec 0, Metolachlor 00- C 8 ec 0, Metoxuron 00- C 8 ec 0, Midazolam C 8 Gravity, μm 8 Monensin sodium C 8 Gravity, μm Monolinuron 00- C 8 ec 0, N Nafcillin 00- C 8 ec, C 8 Pyramid, μm 0 PFP, μm Nalidixic acid C 8 Gravity, μm C 8 Gravity, C 8 Gravity, C 8 Pyramid 0 Sphinx RP, μm 0 C 8 HTec, μm Sphinx RP, μm 9 Naphthalene 00- NH 0 C 8 Gravity,.8 vs. μm C 8 Gravity, μm, 9 C 8 HTec, μm; EC vs. VP C 8 Pyramid, C 8 Gravity HILIC, μm 7 Naproxen 00- C 8 ec C 8 Gravity, μm Nicardipine 00- CN-RP 9 Nicotinamide C 8 Pyramid, μm HILIC, μm Nicotinic acid C 8 Pyramid, μm Nifedipine 00- CN-RP 9 C 8 Gravity, μm 9 Niflumic acid C 8 Gravity, μm Nimodipine 00- CN-RP 9 Nisoldipine 00- CN-RP 9 Nitrendipine 00- CN-RP 9 Nitrilotriacetic acid C 8 Pyramid, μm Nitroaniline isomers PFP, μm 8 Nitrobenzene C 8 Gravity, Sphinx RP, μm PFP, μm 7 PolarTec, μm Nitrocresol isomers C 8 Gravity, μm -Nitrophenol C 8 Gravity, Sphinx RP, μm Nitrophenol isomers C 8 Gravity, μm PFP, μm 7 Nitrotoluene isomers PolarTec, μm Nizatidine C 8 Gravity, μm N-Methyl-N-,,-tetranitro-aniline (Tetryl) PolarTec, μm Norephedrine 00- C 8 ec / 00- CN-RP 8 C 8 Gravity, μm 0 HILIC, μm 0 Norgestrel 00- CN / CN-RP 9 Nortriptyline 00- CN-RP; C 8 Gravity, C 8 Gravity, C 8 Isis, C 8 Pyramid, μm Norvaline 00- C 8 ec Noscapine C 8 Gravity, μm 8 C 8 Gravity, μm 0 NTA C 8 Pyramid, μm 7

70 Substance index O Octocrylene 00- C 8 ec; C 8 HTec, μm Octogen (HMX) PolarTec, μm Ofloxacin 00- C 8 ec 9 Ornithine C 8 Gravity, μm Oxacillin PFP, μm Oxalic acid HILIC, μm Oxazepam 00- C 8 ec 8 Oxolinic acid C 8 Gravity, C 8 Pyramid, μm 0 C 8 Gravity, μm 0, C 8 HTec, μm Sphinx RP, μm 9, 0 P Panthotenic acid PolarTec, μm Papaverine C 8 Gravity, μm 8 C 8 Gravity, μm 0 Paracetamol 00- C 8 ec 8, 00- CN-RP 8 C 8 Gravity, μm C 8 Gravity, μm C 8 Pyramid, μm, Paraquat HILIC, μm Patulin C 8 Gravity, μm Penicillins G + V 00- C 8 ec 9, 0 C 8 Pyramid, μm 0 Penicillin V C 8 Gravity, μm C 8 HTec, PFP, μm Pentachlorophenol PFP, μm 7 Pentobarbital 00- C 8 ec Perfluorinated surfactants Sphinx RP, μm Phenactin C 8 Gravity, μm Phenanthrene C 8 Gravity, μm 9 Phenetole 00- C 8 ec / C 8 ec 00- CN-RP 8, 9 Phenobarbital 00- C 8 ec Phenol 00- C 8 ec 00- C 8 ec, C 8 Gravity, μm C 8 HTec, μm C 8 HTec, μm; EC vs. VP PFP, μm 7 PFP, μm 7 PolarTec, μm 7 Sphinx RP, μm, Phenoxyethanol PolarTec, μm Phenylalanine 00- C 8 ec; C 8 Gravity, μm HILIC, μm 7 -Phenyldodecane 00- NH 0 Phenylglycine HILIC, μm 7 Phloroglucinol PFP, μm 7 Piperacillin PFP, μm Pirenzepine hydrochloride C 8 Gravity, μm Piroxicam 00- C 8 ec, 00- C 8 ec C 8 Gravity, μm Ponceau R and R C 8 Gravity, μm Prednisolone 00- C 8 ec CN / CN-RP 9 C 8 Gravity, μm 9 C 8 HTec, μm 9 Procainamide 00- CN-RP 8 Progesterone 00- C 8 ec; C 8 Gravity, C 8 HTec, μm 9 Promazine 00- CN-RP 8 Promethazine 00- CN-RP 8 PFP, μm 7 PFP, C 8 Gravity, μm 9 Propazine 00- C 8 ec Propiophenone 00- C 8 ec 9 C 8 Gravity,.8 vs. μm 9 Propyl -hydroxybenzoate C 8 Gravity, Sphinx RP, μm Propyl paraben PolarTec, μm Propyzamid 00- C 8 ec 0 Protriptyline C 8 Gravity, C 8 Gravity, C 8 Isis, C 8 Pyramid, μm Protryptiline PolarTec, μm Pseudoephedrine C 8 Gravity, μm Pyrene C 8 Gravity, μm 9 Pyridine C 8 Gravity, μm C 8 Isis, μm, 8 PolarTec, μm 7 Sphinx RP, μm Pyridoxal C 8 Pyramid, μm Pyridoxamine C 8 Pyramid, μm Pyridoxine C 8 Pyramid, μm HILIC, μm Pyridoxine hydrochloride C 8 Pyramid, μm Pyrocatechol 00- C 8 ec 00- C 8 ec, Pyrogallol PFP, μm 7 Q Quercetin Sphinx RP, C 8 Gravity, C 8 Gravity Quinine 00- C 8 ec C 8 Gravity, C 8 Gravity, μm 8 Quinoline yellow C 8 Gravity, μm R Ractopamine C 8 Pyramid, μm 7 Reproterol C 8 Pyramid, μm 7 Resorcinol 00- C 8 ec 00- C 8 ec, PFP, μm 7 Riboflavin C 8 Pyramid, μm, Ritodrine C 8 Pyramid, μm 7 Rutin C 8 Pyramid, μm Sphinx RP, C 8 Gravity, C 8 Gravity S Saccharin 00- C 8 ec; C 8 HTec, μm Saccharose 00- NH -RP 0 Salicylic acid 00- C 8 ec PolarTec, μm Salinomycin sodium C 8 Gravity, μm Sarafloxacin Sphinx RP, μm 9 Scopolamine C 8 Gravity, μm 8 Sebuthylazine 00- C 8 ec 0, Serine 00- C 8 ec C 8 Gravity, μm Shikimic acid C 8 Pyramid, μm Simazine 00- C 8 ec 0, Sorbic acid 00- C 8 ec; C 8 HTec, μm Strychnine C 8 Gravity, μm 8 Succinylsulfathiazole C 8 Gravity, μm Sulfadiazine C 8 Gravity, μm Sulfadimidine C 8 Gravity, μm Sulfamerazine C 8 Gravity, μm Sulfanilamide C 8 Gravity, μm Sulfathiazole C 8 Gravity, μm Sulindac 00- C 8 ec; C 8 Gravity, μm Sunset yellow CFC C 8 Gravity, μm Suprofen 00- C 8 ec, 00- C 8 ec C 8 Gravity, μm Syringic acid C 8 Isis, μm PolarTec, μm 8

71 Alphabetical index of analytes T Tartaric acid C 8 Pyramid, μm, Tartrazine C 8 Gravity, μm Temazepam 00- C 8 ec 8 Terbuthylazine 00- C 8 ec 0, Terbutryn 00- C 8 ec 0 o-, m-, p-terphenyl C 8 Isis,.8 vs. μm 7 C 8 Isis, μm o-terphenyl C 8 Isis, μm Testosterone 00- C 8 ec CN / CN-RP 9 C 8 HTec, μm 9 Tetracaine C 8 Pyramid, μm Theobromine Sphinx RP, μm Theophylline 00- C 8 ec C 8 Gravity, μm C 8 Isis, μm Sphinx RP, μm 0 Thiamine C 8 Pyramid, μm HILIC, μm Thiamine hydrochloride C 8 Pyramid, μm Thiamylal 00- C 8 ec Threonine C 8 Gravity, μm Thymine 00- NH -RP C 8 Pyramid, μm 7 HILIC, μm 7, 7 PolarTec, μm 7 PolarTec, μm 7 a-tocopherol see Vitamin E Tocopherols C 8 Isis, μm Tolmetin 00- C 8 ec 00- C 8 ec C 8 Gravity, μm Toluene 00- C 8 ec C 8 Gravity, μm, C 8 HTec, μm C 8 Isis, μm Sphinx RP, μm Tribunil see Methabenzthiazuron,,-Trichlorophenol PFP, μm 7,,-Triclocarbanilid PolarTec, μm Trimipramine 00- CN-RP; C 8 Gravity, C 8 Gravity, μm PolarTec, μm,,-trinitrobenzene PolarTec, μm,,-trinitrotoluene PolarTec, μm Triphenylene C 8 Isis,.8 vs. μm 7 C 8 Isis, μm, Triprolidine PFP, μm 7 PFP, C 8 Gravity, μm 9 Tryptophan 00- C 8 ec C 8 Gravity, μm Tyrosine 00- C 8 ec C 8 Gravity, μm C 8 Gravity, μm 0 HILIC, μm 7 U Uracil 00- C 8 ec 00- NH -RP C 8 Gravity, μm C 8 HTec, μm C 8 Isis, μm 8 C 8 Pyramid, μm 7 HILIC, μm 7 HILIC, μm 7, 7 PFP, μm 7 PolarTec, μm 7 PolarTec, μm 7 Sphinx RP, μm, Urea C 8 Pyramid, μm Uvinul Plus C 8 HTec, μm V Valerophenone 00- C 8 ec 9 C 8 Gravity,.8 vs. μm 9 Valine 00- C 8 ec C 8 Gravity, μm Vanillic acid C 8 Isis, μm PolarTec, μm Vanillin 00- C 8 ec 00- C 8 ec 8 Sphinx RP, μm Veratrol 00- C 8 ec 00- C 8 ec, Vitamin A + acetate 00- C 8 ec; C 8 Isis, μm Vitamin B / B / B PolarTec, μm Vitamin D / D 00- C 8 ec; C 8 Isis, μm Vitamin E + acetate 00- C 8 ec; C 8 Isis, μm Vitamin K 00- C 8 ec; C 8 Isis, μm Vitamin K C 8 Isis, μm Vitamin K 00- C 8 ec Vitamins, water-soluble (B, B, B, B 7, B 9, B, C, H) HILIC, μm C 8 Pyramid, μm, X o-xylene 00- CN-RP 8, 9 Xylene C 8 Gravity, Sphinx RP, μm Xylometazoline 00- CN-RP 7 Y Yellow orange S C 8 Gravity, μm Z Zearalenol C 8 Gravity, μm 0 Zearalenone C 8 Gravity, μm 0 Image credits Ewa Walicka (), Filipebvarela (cover,, 9), Habman8 (cover), Ioana Davies (Drutu) (cover, ), isma (cover, ), lightmoon (cover), lunamarina (), MAK (cover, ), Marcel Nijhuis (), maxi (cover, ), Otmar Smit (cover, ), Ramón López (cover, ), Sebastian Duda (cover), Sven Hoppe (cover, ), Tomislav (cover, ), Valcho (cover) fotolia.de 9

72 Appl. no. index / Trademarks Index of application numbers MN appl. page MN appl. page MN appl. page MN appl. page MN appl. page Trademarks MACHEREY-NAGEL trademarks ALUGRAM coated aluminium sheets for TLC CHROMABOND columns for solid phase extraction (SPE) ChromCart cartridge system for HPLC NUCLEODUR spherical high purity silica for HPLC Registered trademarks of other companies Acclaim Dionex Corp. (USA) ACQUITY Waters Corp. (USA) Allure Restek Corp. (USA) Atlantis Waters Corp. (USA) Ascentis Sigma-Aldrich Co. (USA) Discovery Sigma-Aldrich Co. (USA) Gemini Phenomenex Inc. (USA) Inertsil GL Sciences, Inc. (USA) Kromasil Eka Chemicals AB (Sweden) LiChrospher Merck KGaA (Germany) Luna Phenomenex Inc. (USA) NUCLEOSIL OPTIMA Polaris ProntoSIL Purospher Spherisorb Symmetry Synergi Xterra YMC ZIC Zorbax spherical standard silica for HPLC high performance fused silica capillary columns with immobilized phases Agilent Technologies Inc. (USA) Bischoff Chromatography (Germany) Merck KGaA (Germany) Waters Corp. (USA) Waters Corp. (USA) Phenomenex Inc. (USA) Waters Corp. (USA) YMC Co. Ltd. (Japan) Merck SeQuant AB (Sweden) Agilent Technologies Inc. (USA) Common law trademarks of other companies Hypersil Thermo Fisher Scientific Inc. (USA) SymmetryShield Waters Corp. (USA) HyPURITY Thermo Fisher Scientific Inc. (USA) SunFire Waters Corp. (USA) Obelisc Sielc Technologies (USA) SUPELCOSIL Sigma-Aldrich Co. (USA) SeQuant Merck SeQuant AB (Sweden) Disclaimer All used names and denotations can be brands, trademarks or registered labels of their respective owner also if they do not have a special denotation. To mention products and brands is only a kind of information, i.e. it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment. Regarding these products or services we can not grant any guarantees regarding selection, efficiency or operation. 70

73 Ordering information NUCLEODUR C 8 Gravity Pore size 0 Å; high density octadecyl phase, endcapped, 8 % C; eluent in column acetonitrile water Length 0 mm 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR C 8 Gravity,.8 µm particle size.8 µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: NUCLEODUR C 8 Gravity, µm particle size µm Microbore analytical columns mm ID EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: CC guard columns** 8 x mm: x mm: 7.0 NUCLEODUR C 8 Gravity, µm particle size µm Microbore analytical columns mm ID EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: CC guard columns** 8 x mm: x mm: 7.0 VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: 70.0 x mm: 7.0 NUCLEODUR C 8 Gravity, 0 µm particle size 0 µm VarioPrep preparative columns mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: 70.0 x mm: 7.0 Microbore, EC, and VarioPrep columns in packs of, guard columns see page 7 MN 7

74 Packed columns NUCLEODUR C 8 Gravity Pore size 0 Å; high density octyl phase, endcapped, % C; eluent in column acetonitrile water Length 0 mm 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR C 8 Gravity,.8 µm particle size.8 µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: NUCLEODUR C 8 Gravity, µm particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: CC guard columns** 8 x mm: x mm: 77.0 VarioPrep preparative columns 0 mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: EC and VarioPrep columns in packs of, guard columns see right, Microbore columns with NUCLEODUR C 8 Gravity on request! HPLC column systems from MACHEREY-NAGEL Microbore columns: On request available in lengths of 0, 0, 00,, 0, 00, 0 and 00 mm and with 0.0, 0.07, 0., 0., 0., 0., 0., 0.7,.0 and. mm ID. EC columns: Analytical ready-to-use columns; available dimensions see page 8. VarioPrep columns: Preparative columns with axially adjustable endfitting; available dimensions see page MN

75 NUCLEODUR C 8 Isis Pore size 0 Å; octadecyl phase with high steric selectivity, polymer modification, 0 % C; eluent in column acetonitrile water Ordering information Length 0 mm 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR C 8 Isis,.8 µm particle size.8 µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: NUCLEODUR C 8 Isis, µm particle size µm Microbore analytical columns mm ID EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: NUCLEODUR C 8 Isis, µm particle size µm Microbore analytical columns mm ID EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: 70.0 VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: 70.0 x mm: 7.0 Microbore, EC, and VarioPrep columns in packs of, guard columns see below Guard column systems Guard columns for EC columns with ID mm mm mm. mm Guard column holder * Column Protection System (pack of) EC / () / () / () / () 789 ** ChromCart guard columns (pack of) CC 8/ () 8/ () 8/ () 8/ () 79 Guard columns for VarioPrep columns with ID 8, 0 mm, mm, 0 mm 0 mm *** VarioPrep guard columns (pack of) VP 0/8 () 0/ () / () /0 () VP guard column holder MN 7

76 Packed columns NUCLEODUR C 8 Pyramid Pore size 0 Å; octadecyl phase with hydrophilic endcapping, % C; eluent in column acetonitrile water Length 0 mm 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR C 8 Pyramid,.8 µm particle size.8 µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 NUCLEODUR C 8 Pyramid, µm particle size µm Microbore analytical columns mm ID EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: 78.0 NUCLEODUR C 8 Pyramid, µm particle size µm Microbore analytical columns mm ID EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: CC guard columns** 8 x mm: x mm: VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: 79.0 x mm: 79.0 Microbore, EC, and VarioPrep columns in packs of, guard columns see right 7 MN

77 NUCLEODUR PolarTec Pore size 0 Å; octadecyl phase with embedded polar group, endcapped, 7 % C; eluent in column acetonitrile water Ordering information Length 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR PolarTec, µm particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: CC guard columns** 8 x mm: x mm: 70.0 NUCLEODUR PolarTec, µm particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: CC guard columns** 8 x mm: x mm: 7.0 VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: 7.0 x mm: 7.0 EC and VarioPrep columns in packs of, guard columns see below Guard column systems Guard columns for EC columns with ID mm mm mm. mm Guard column holder * Column Protection System (pack of) EC / () / () / () / () 789 ** ChromCart guard columns (pack of) CC 8/ () 8/ () 8/ () 8/ () 79 Guard columns for VarioPrep columns with ID 8, 0 mm, mm, 0 mm 0 mm *** VarioPrep guard columns (pack of) VP 0/8 () 0/ () / () /0 () VP guard column holder MN 7

78 Packed columns NUCLEODUR PFP Pore size 0 Å; pentafluorophenyl-propyl modification, multi-endcapped, 8 % C; eluent in column acetonitrile water Length 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR PFP, µm particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: CC guard columns** 8 x mm: x mm: 7.0 NUCLEODUR PFP, µm particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: CC guard columns** 8 x mm: x mm: 7.0 VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: 7.0 x mm: 7.0 EC and VarioPrep columns in packs of, guard columns see right Online Application Database /apps MACHEREY-NAGEL, one of the leading companies for chromatography products and the manufacturer of well known brands like NUCLEODUR, NUCLEOSIL, CHROMABOND, and OPTIMA presents its free Online Application Database! More than 000 applications covering: HPLC (NUCLEODUR, NUCLEOSIL, ) GC (OPTIMA, ) SPE (CHROMABOND ) TLC (ADAMANT, ALUGRAM, ) Applications of various fields: Drugs and pharmaceutical compounds Environmental analysis and pollutants Biological samples and natural compounds Food and beverages Petrochemical products, solvents and chemicals Organic compounds Fragrances and cosmetic components Chiral separations by HPLC and GC Standard protocols for SPE and derivatization As one of the pioneers in chromatography MACHEREY-NAGEL is a leading producer of HPLC, GC, SPE and TLC products resulting in this highly informative and helpful collection of applications. 7 MN

79 Ordering information NUCLEODUR Sphinx RP Pore size 0 Å; special bifunctional RP phase, % C; eluent in column acetonitrile water Length 0 mm 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR Sphinx RP,.8 µm particle size.8 µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: NUCLEODUR Sphinx RP, µm particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: 77.0 NUCLEODUR Sphinx RP, µm particle size µm Microbore analytical columns mm ID EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: 70.0 VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: x mm: 79.0 Microbore, EC, and VarioPrep columns in packs of, guard columns see below Guard column systems Guard columns for EC columns with ID mm mm mm. mm Guard column holder * Column Protection System (pack of) EC / () / () / () / () 789 ** ChromCart guard columns (pack of) CC 8/ () 8/ () 8/ () 8/ () 79 Guard columns for VarioPrep columns with ID 8, 0 mm, mm, 0 mm 0 mm *** VarioPrep guard columns (pack of) VP 0/8 () 0/ () / () /0 () VP guard column holder MN 77

80 Packed columns NUCLEODUR C 8 HTec Pore size 0 Å; high density octadecyl phase, endcapped, 8 % C; eluent in column acetonitrile water Length 0 mm 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR C 8 HTec,.8 µm particle size.8 µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 NUCLEODUR C 8 HTec, µm particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: 70.0 NUCLEODUR C 8 HTec, µm particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: CC guard columns** 8 x mm: x mm: 70.0 VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: 79.0 x mm: 79.0 x 0 mm: NUCLEODUR C 8 HTec, 7 µm particle size 7 µm VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: 79.0 x mm: 79.0 x 0 mm: MN

81 Ordering information Length 0 mm 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR C 8 HTec, 0 µm particle size 0 µm VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: 79.0 x mm: 79.0 x 0 mm: EC and VarioPrep columns in packs of, guard columns see below Guard column systems Guard columns for EC columns with ID mm mm mm. mm Guard column holder * Column Protection System (pack of) EC / () / () / () / () 789 ** ChromCart guard columns (pack of) CC 8/ () 8/ () 8/ () 8/ () 79 Guard columns for VarioPrep columns with ID 8, 0 mm, mm, 0 mm 0 mm *** VarioPrep guard columns (pack of) VP 0/8 () 0/ () / () /0 () VP guard column holder Scale up factors and parameters for typical MN column dimensions ID x length [mm] x 0 8 x 0 0 x 0 x 0 x 0 x 0 0 x 0 0 x 0 80 x 0 Linear scale-up factor Typical sample mass* [mg] Typical flow rate [ml/min] * For RP material; the maximum amounts given here always depend on the separation problem and on the sample composition. In some cases even half of the amounts given can cause drastic overload, in other cases the maximum amounts can be even higher still giving acceptable separations. MN 79

82 Packed columns NUCLEODUR C 8 ec Pore size 0 Å; octadecyl phase, endcapped, 7. % C; eluent in column acetonitrile water Length 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR 00- C 8 ec particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: NUCLEODUR 00- C 8 ec particle size µm Microbore analytical columns mm ID EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: x mm: 7.0 x 0 mm: 7.00 NUCLEODUR 00-0 C 8 ec particle size 0 µm VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: x mm: 7.0 x 0 mm: 7.00 Microbore, EC, and VarioPrep columns in packs of, guard columns see right 80 MN

83 Ordering information NUCLEODUR C 8 ec Pore size 0 Å; octyl phase, endcapped, 0. % C; eluent in column acetonitrile water Length 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR 00- C 8 ec particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: 70.0 NUCLEODUR 00- C 8 ec particle size µm EC analytical columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: CC guard columns** 8 x mm: x mm: VarioPrep preparative columns 0 mm ID mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: x mm: 7.0 EC and VarioPrep columns in packs of, guard columns see below, Microbore columns with NUCLEODUR C 8 ec on request! Guard column systems Guard columns for EC columns with ID mm mm mm. mm Guard column holder * Column Protection System (pack of) EC / () / () / () / () 789 ** ChromCart guard columns (pack of) CC 8/ () 8/ () 8/ () 8/ () 79 Guard columns for VarioPrep columns with ID 8, 0 mm, mm, 0 mm 0 mm *** VarioPrep guard columns (pack of) VP 0/8 () 0/ () / () /0 () VP guard column holder MN 8

84 Packed columns NUCLEODUR HILIC Pore size 0 Å; zwitterionic phase for HILIC chromatography, 7 % C; eluent in column acetonitrile water 80:0 Length 0 mm 0 mm 7 mm 00 mm mm 0 mm 0 mm NUCLEODUR HILIC,.8 µm particle size.8 µm EC columns mm ID mm ID mm ID mm ID EC guard columns* x mm: x mm: NUCLEODUR HILIC, µm particle size µm EC columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: NUCLEODUR HILIC, µm particle size µm EC columns mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: Columns in packs of, guard columns see right, Microbore columns and preparative columns with NUCLEODUR HILIC on request! Our HPLC QC policy Highest production standard our facilities are EN ISO 900:008 certified Strict quality specifications for outstanding reliability Perfect reproducibility within each batch and from lot to lot Each column is individually tested and supplied with test chromatogram and test conditions Test mixture for reversed phase columns Designation Pack of REF Test mixture for reversed phase ml 79 columns in acetonitrile 8 MN

85 Ordering information NUCLEODUR CN and CN-RP Pore size 0 Å; cyano phase (nitrile), 7 % C Length 0 mm mm 0 mm 0 mm NUCLEODUR 00- CN-RP particle size µm; eluent in column acetonitrile water EC columns mm ID mm ID mm ID mm ID 70. EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: 70.0 NUCLEODUR 00- CN-RP particle size µm; eluent in column acetonitrile water EC columns mm ID mm ID EC guard columns* x mm: 79.0 CC guard columns** 8 x mm: 70.0 NUCLEODUR 00- CN particle size µm; eluent in column n-heptane EC columns mm ID mm ID EC guard columns* x mm: 79.0 CC guard columns** 8 x mm: 79.0 Columns in packs of, guard columns see below, Microbore columns and preparative columns with NUCLEODUR CN / CN-RP on request! Guard column systems Guard columns for EC columns with ID mm mm mm. mm Guard column holder * Column Protection System (pack of) EC / () / () / () / () 789 ** ChromCart guard columns (pack of) CC 8/ () 8/ () 8/ () 8/ () 79 Guard columns for VarioPrep columns with ID 8, 0 mm, mm, 0 mm 0 mm *** VarioPrep guard columns (pack of) VP 0/8 () 0/ () / () /0 () VP guard column holder MN 8

86 Packed columns NUCLEODUR NH and NH -RP Pore size 0 Å; amino phase,. % C Length 00 mm mm 0 mm 0 mm NUCLEODUR 00- NH -RP EC columns mm ID particle size µm; eluent in column acetonitrile water. mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: 70.0 NUCLEODUR 00- NH -RP EC columns particle size µm; eluent in column acetonitrile water mm ID mm ID mm ID mm ID EC guard columns* x mm: 79.0 x mm: 79.0 CC guard columns** 8 x mm: x mm: 77.0 NUCLEODUR 00- NH EC columns particle size µm; eluent in column n-heptane mm ID mm ID EC guard columns* x mm: 79.0 CC guard columns** 8 x mm: 70.0 Columns in packs of, guard columns see page 8, Microbore and preparative columns with NUCLEODUR NH / NH -RP on request! Unmodified NUCLEODUR Pore size 0 Å; unmodified; eluent in column n-heptane Length 0 mm mm 0 mm 0 mm NUCLEODUR 00- particle size µm EC analytical columns. mm ID EC guard columns* x mm: 79.0 CC guard columns** 8 x mm: NUCLEODUR 00- particle size µm EC analytical columns mm ID mm ID EC guard columns* x mm: CC guard columns** 8 x mm: 70.0 VarioPrep preparative columns 0 mm ID mm ID mm ID VP guard columns*** 0 x 8 mm: x mm: x mm: 70.0 Columns in packs of, guard columns see page 8, Microbore columns with unmodified NUCLEODUR on request! 8 MN

87 Ordering information EC standard columns for analytical HPLC Features Analytical column system manufactured from stainless steel M 8 outer threads on both ends Combination of sealing element and very fine-meshed stainless steel screen, PTFE ring and fitting adapter Column heads SW, with inner threads M8 x 0.7 and UNF 0- (= / fitting) As screw-on guard column system we recommend the Column Protection System used with EC guard column cartridges with mm length (see page 8). As built-in guard columns ChromCart guard column cartridges with 8 mm length can be used with the guard column adapter EC (see page 87). Supplied with NUCLEODUR, NUCLEOSHELL and NUCLEOSIL spherical silicas Available standard dimensions of EC columns ID [mm] Length [mm] End fitting design Please ask for availability of certain phases Guard columns for EC columns EC columns with ID (pack of each) mm mm mm. mm Use guard column holder EC guard columns for Column Protection System guard column holder ChromCart guard columns for EC guard column holder EC / / / / REF 789 CC 8/ 8/ 8/ 8/ REF 79 Guard column systems, accessories and replacement parts for EC columns Ordering information Description Pack of REF Column Protection System kit 789 see figures and description on page 8 Guard column adapter EC 79 see figures and description on page 87 EC fitting adapter EC column head (nut) EC PTFE sealing ring part sealing combination for EC columns kits MN 8

88 Packed columns Column Protection System Innovative and universal screw-on guard column holder system Suitable for all analytical HPLC columns with / fittings Features Cartridges filled with specified NUCLEODUR, NUCLEOSIL and NUCLEOSHELL HPLC adsorbents Ideal protection for your analytical main column significant increase in column lifetime Minimized void volume suitable also for ultra fast HPLC Special ferrules pressure stability up to 0 bar ( 000 psi) Visual contamination check in-time changing of the guard column Guard column length mm, ID mm (for main columns with mm ID) or ID mm (for main columns with, and. mm ID) UNIVERSAL RP guard columns suitable for all HPLC columns under RP conditions Content of the Column Protection System Description REF Column Protection System 789 Details Content Cartridge Holder Capillaries Ferrules Wrenches Manual Replacement parts for the Column Protection System Ordering information Description Pack of REF Ferrules 7897 Replacement connector including O-ring 7898 Capillary tubes, nuts and metal ferrules 7899 Wrench (size and mm) EC / UNIVERSAL RP guard column (for main columns with mm ID) EC / UNIVERSAL RP guard column (for main columns with, and. mm ID) Visual Contamination Check The cartridge is fitted with a white filter membrane: A discoloration of the filter membrane usually indicates that the cartridge should be replaced. In case of colorless contaminants, a rising back pressure and / or loss of chromatographic performance advise to change the guard column. 8 MN

89 Ordering information EC guard column adapter Standard guard column built-in adapter system Suitable for EC columns Features Cartridges filled with specified NUCLEODUR and NUCLEOSIL HPLC adsorbents Ideal protection for your analytical EC main column significant increase of column lifetime Guard column length 8 mm, ID mm (for main columns with and mm ID) or ID mm (for main columns with and. mm ID) EC guard column adapter Ordering information Description Pack of REF EC guard column adapter 79 Installation of the EC guard column adapter. Unscrew the column head. Remove the fitting. Unscrew the EC guard column adapter. Screw the adapter sleeve onto the column. Insert the CC guard column. Screw the nut of the guard column adapter in place MN 87

90 Packed columns VarioPrep (VP) columns for preparative HPLC Features Column system for preparative HPLC manufactured from stainless steel with two adjustable end fittings, e.g. for frequent use of backflushing techniques Allows compensation of a dead volume, which could result at the column inlet after some time of operation, without need for opening the column Supplied with all NUCLEODUR and NUCLEOSIL spherical silicas Available standard dimensions of VarioPrep columns with axially adjustable end fittings ID [mm] Length [mm] Length [mm] End fitting design 0* * * 0 x 8, 0 x, x and x 0 mm ID columns are used as guard columns and require adequate holders, see page 89. The VarioPrep principle A B C D dead volume Readjustment of fitting Based on our special packing procedure VarioPrep columns are produced with highest packing quality and bed density (A). Due to intensively chemical and/or mechanical exposure of the column adsorbent, shrinking of the column bed can occur (B; orange gap). In this even unlikely case readjustment of the VarioPrep column fitting (C; turning the nut at column inlet clockwise) will eliminate the emerged dead volume (D). The performance of the VarioPrep column is completely reconstituted and column lifetime is significantly extended. Column Reconstitution 80 th injection just after readjustment of fitting Reconstitution of VarioPrep column performance Slight peak broadening and deformation after 800 injections under strongly demanding conditions (ph ; 0 C; sample in DMSO) Readjustment of the column fitting restores the column performance and prolongs column lifetime noticeably 800 injections 70 injections approx. 00 injections 88 MN

91 Ordering information The new guard column system for (semi-) preparative HPLC VP / for and 0 mm ID columns VP 0/ for and mm ID columns VP 0/8 for 8 and 0 mm ID columns VP /0 for 0 mm ID columns Easy handling and cartridge exchange Robust hardware Free rotary plunger fittings low O-ring abrasion Cost-efficient cartridges Minimally invasive / no disturbance of the separation efficiency of main column Low back pressure Designed for pressures up to 00 bar Performance of preparative column when used with VarioPrep guard column Columns: x mm NUCLEODUR C 8 HTec, μm x mm NUCLEODUR C 8 HTec, μm + 0 x mm NUCLEODUR C 8 HTec guard column Eluent: acetonitrile water (80:0, v/v) Flow rate: ml/min Temperature: C H-value:. Asymmetry:. Pressure: bar H-value:.9 Asymmetry:.0 Pressure: 7 bar. Phenol. Naphtaline. Anthracene min min Using VarioPrep guard columns provides ideal protection of your main column symmetry, pressure and retention stay almost constant. Technical data / thread free rotary plunger fittings low O-ring abrasion stainless steel Guard cartridge Holder Holder ID Replacement O-ring ( pcs) Recommended for column ID Preferred capillary ID Typical flow rate VP 0/8 REF 78 8 mm REF and 0 mm ID 0.7 and 0. mm ml/min VP 0/ REF 78 mm REF 7897 and mm ID 0.7, 0. and 0. mm ml/min VP / REF 78 mm REF and 0 mm ID 0., 0. and.0 mm 0 ml/min VP /0 REF 78 0 mm REF mm ID 0. and.0 mm 0 0 ml/min Guard columns for VarioPrep columns Ordering information VP columns with ID 8, 0 mm, mm, 0 mm 0 mm Use guard column holder Holder ID VP guard columns for VarioPrep guard column holder (pack of) VP 0/8 () REF 78 8 mm VP 0/ () REF 78 mm VP / () REF 78 mm VP /0 () REF 78 0 mm MN 89