NUCLEDUR (Macherey-Nagel) Ultrapure silica Excellent mechanical and chemical stability Low metal content Enhanced ph stability Manufactured by Macherey-Nagel GmbH & Co. KG, Düren, Germany, NUCLEDUR is a fully synthetic type silica offering highly advanced physical properties. The special surface bonding technology and the low concentration of metal ions lead to high efficiency symmetrical peaks. NUCLEDUR Phase Functional Group Endcapped Particle ze (µm) Pore ze (Å) Surface rea (m 2 /g) Carbon Load (%) C18 Gravity ctadecyl Yes 1.8, 3, 5 110 340 18 C18 ec ctadecyl Yes 3, 5 110 340 17.5 C8 Gravity ctyl Yes 1.8, 5 110 340 11 C8 ec ctyl Yes 3, 5 110 340 10.5 C18 Pyramid ctadecyl Yes 1.8, 3, 5 110 340 14 C18 Isis ctadecyl Yes 1.8, 3, 5 110 340 20 C18 HTec ctadecyl Yes 1.8, 3, 5, 7, 10 110 340 18 CN / CN-RP Cyano Yes 3, 5 110 340 7 HILIC mmonium-sulphonic acid No 1.8, 3, 5 110 340 7 NH 2 / NH 2 -RP mino Yes 3, 5 110 340 2.5 PFP Pentafluorophenyl propyl modification Yes 1.8, 3, 5 110 340 8 PolarTec C18 modification with embedded polar group Yes 1.8, 3, 5 110 340 16 Sphinx RP ctadecyl/phenyl No 1.8, 3, 5 110 340 15 NUCLEDUR Gravity NUCLEDUR C18 Gravity and C8 Gravity are based on the ultrapure NUCLEDUR silica. The high density bonding of these phases leads to an extended operation ph range (1-11). This can be helpful for improving selectivity in method development and for more challenging applications. NUCLEDUR C18 ec and C8 ec The NUCLEDUR C18 ec and C8 ec phases are recommended for routing analyses and for scaling up to preparative LC. NUCLEDUR C18 Isis NUCLEDUR C18 Isis is a C18 bonded phase with specially cross-linked surface modification. This polymeric bonding leads to high steric selectivity and is therefore suited for separation of positional and structural isomers and planar/non-planar molecules. The phase has an enhanced ph stability of 1-10. 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 (see Figure 1). NUCLEDUR C18 Pyramid NUCLEDUR C18 Pyramid is a C18 bonded phase with hydrophilic endcapping designed especially for use with eluents containing up to 100% water. Figure 2 shows the retention behaviour of tartaric, acetic and maleic acids under purely aqueous conditions on NUCLEDUR C18 Pyramid in comparison with a conventionally bonded RP phase. Figure 2. Stability test Figure 1. Steric selectivity of NUCLEDUR C18 Isis NUCLEDUR NH 2 The aminopropyl modification of the NUCLEDUR NH 2 phase enables the column to be used in reversed and normal-phase modes. The polarity of the phase can be classified as intermediate, based on multiple retention mechanisms such as dipoledipole and hydrophobic. NUCLEDUR NH 2 shows excellent selectivity for polar organic compounds, such as sugar, anilines and esters. Toll Free: 1-888-226-2775 E-mail: info@lifescience.ca Web: www.lifescience.ca 187
NUCLEDUR (Macherey-Nagel) verview of NUCLEDUR Phases Phase Specification Characteristics* Stability Structure octadecyl phase, high density coating multi-endcapping C 18 Gravity: 18 % C USP L1 Gravity: 11 % C USP L7 C 18 C 18 C C 18 ph stability 1-11, octadecyl phase with specially crosslinked surface modification endcapping 20 % C USP L1 C ph stability 1-10, C 18 modification with polar endcapping 14 % C USP L1 C stable against 100 % aqueous eluents, ph stability 1-9, bifunctional RP phase, propylphenyl and C 18 ligands; endcapping 15 % C; USP L1 and L11 C ph stability 1-10, octadecyl / octyl phase, medium density coating endcapping C 18 ec: 17.5 % C USP L1 ec: 10.5 % C USP L7 C 18 C 18 C C 18 ph stability 1 9 (CH3) 3 (CH3) 3 zwitterionic ammonium sulfonic acid modification 7 % C ph stability 2 8.5, N S 3 + N S 3 + cyano (nitrile) phase for NP and RP separations 7 % C USP L10 ph stability 1 8, stable towards highly aqueous mobile phases C N C N (CH3) 3 amino phase for NP and RP separations 2.5 % C USP L8 ph stability 1 8, stable towards highly aqueous mobile phases NH 2 NH 2 unmodified high purity silica USP L3 n.a. ph stability 2 8 * = hydrophobic selectivity, = polar/ionic selectivity, C = steric selectivity 188 Toll Free: 1-888-226-2775 E-mail: info@lifescience.ca Web: www.lifescience.ca
verview of NUCLEDUR Phases NUCLEDUR (Macherey-Nagel) pplication milar phases** Separation principle Retention mechanism in general compounds with ionizable functional groups such as basic pharmaceuticals and pesticides for Gravity generally shorter retention times for nonpolar compounds NUCLESIL C 18 HD Waters Xterra RP 18 / MS C 18 ; Phenomenex Luna C18 (2), Synergi and Max RP; Zorbax Extend C18; Purospher RP-18; Star RP-18 NUCLESIL HD; Waters Xterra RP 8 / MS ; Phenomenex Luna C8; Zorbax Eclipse; XD-C8 only hydrophobic (van der Waals ) (CH3)3 H3C CH3 N high steric selectivity, thus suited for separation of positional and structural isomers, planar/nonplanar molecules NUCLESIL C 18 YMC Pro C18RS steric and hydrophobic basic pharmaceutical ingredients, very polar compounds, organic acids Phenomenex qua; YMC Q; Waters tlantis dc18 hydrophobic CH3 and polar N H3C (H bonds) compounds with aromatic and multiple bond systems no similar phases π-π and hydrophobic N2 robust C 18 / phase for routine analyses hydrophilic compounds such as organic polar acids and bases, polar natural compounds NUCLESIL C 18 Spherisorb DS II; Hypersil DS; Waters Symmetry C18; Kromasil C18 LiChrospher RP 18 NUCLESIL ec / Spherisorb C8; Hypersil MS; Waters Symmetry C8; Kromasil C8; LiChrospher RP 8 Merck Sequant ZIC -HILIC; elc belisc only hydrophobic (van der Waals ) some residual silanol ionic / hydrophilic, electrostatic CH3 (CH3)3 N H3C H 3C N + S 3 CH3 H N NH H 3C NH 2 N + S 3 polar organic compounds (basic drugs), molecules containing π electron systems NUCLESIL CN / CN-RP π-π, polar (H bonds), hydrophobic C N C N H sugars, sugar alcohols and other hydroxy compounds, DN bases, polar compounds in general NUCLESIL NH 2 / NH 2 -RP polar /ionic, hydrophobic + NH3 polar compounds in general unmodified NUCLESIL polar /ionic 2N ** phases which provide a similar selectivity based on chemical and physical properties Toll Free: 1-888-226-2775 E-mail: info@lifescience.ca Web: www.lifescience.ca 189
NUCLEDUR (Macherey-Nagel) NUCLEDUR HILIC 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 NUCLEDUR HILIC is a special zwitterionic modified stationary phase based on ultra spherical NUCLEDUR 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. Retention characteristics Commonly HILIC is described as partition chromatography or liquid/liquid extraction system between the mobile and stationary phase. water-poor layer of mobile phase is formed on the surface of the polar stationary phase versus a water-rich layer. Therefore, a distribution of the analytes between these two layers will occur. HILIC includes weak electrostatic mechanisms as well as hydrogen donor between neutral polar molecules under high organic elution conditions. This distinguishes HILIC from ion exchange chromatography. More polar compounds will have stronger interaction with the stationary aqueous layer than less polar compounds resulting in a stronger retention. Non-polar compounds exhibit faster elution profiles due to minor hydrophobic. s seen in Figure 1, the elution order is quite often inverse on HILIC columns compared to RP columns. Figure 1. Separation of uracil and naphthalene Figure 2. Stability and equilibration Fields of application Due to its high loadability, NUCLEDUR HILIC is suitable for preparative and semi-preparative applications. ased on their special interaction and retention features, HILIC columns are ideal for analysis of medium-polar and polar compounds. Determinations of polar compounds in foods, such as melamine in milk or acrylamide in bakery products, are as necessary as separations of vitamins and organic acids in food and pharmaceutical products. Stable and effective application notes were developed on NUCLEDUR HILIC for pharmaceutical and physiological substances (ie. the chemotherapeutic agent 5-fluorouracil, the energy carrier of muscle metabolism creatine), catecholamines and amino acids. In bioanalysis, nucleotides as well as pyrimidine and purine bases can be determined efficiently. ecause well-biodegradable and polar pesticides have to be analyzed, the determination of chlormequat, mepiquat, paraquat and diquat gains increasing importance (see Figure 3). Figure 3. Separation of growth regulators Stability features Due to an advanced and unique surface modification procedure (pat. pend.) NUCLEDUR HILIC columns provide short equilibration times (see Figure 2). fter just 5 min. equilibration the 2 nd injection shows stable and reproducible results. NUCLEDUR HILIC columns are characterized by an outstanding column lifetime. fter nearly 800 runs, the columns show no loss of pristine performance. Peak shape and retention are still immaculate. Determinations of hydrocarbons, peptides and glycolized or phosphorylized compounds are other successful HILIC applications. 190 Toll Free: 1-888-226-2775 E-mail: info@lifescience.ca Web: www.lifescience.ca
NUCLEDUR (Macherey-Nagel) NUCLEDUR 1.8µm Phases Ultra fast LC Shorter columns with high separation efficiency gnificant improvement in resolution Increased detection sensitivity Suitable for LC-MS due to low bleeding characteristics ll NUCLEDUR premium phases are now available in 1.8µm particle sizes. Columns packed with these microspherical particles show improvement in terms of plate numbers, column efficiencies and resolution compared with their 3µm counterparts. Increase in separation efficiency n increase in plate number of approximately 67% in transferring from 3µm to 1.8µm particles offers the possibility of using shorter columns with equal plate numbers resulting in a decrease in analysis time. 50mm x 4.6mm NUCLEDUR C18 Gravity 3µm: N 100,000 plates/m (h value 10) 1.8µm: N 166,667 plates/m (h value 6) Improvement in resolution The use of 1.8µm instead of 3µm particles can lead to an increase in resolution of approximately 30%. This is illustrated in Figure 1 for the separation of naphthalene and ethylbenzene. Column back pressure The smaller particle size results in an increased back pressure. However, the high sphericity of the NUCLEDUR particles and their very narrow particle size distribution means that back pressure is kept at a moderate level. In addition, the particles are fractionated in order to limit the increase in back pressure. Nevertheless, the use of columns packed with sub 2µm particles generally makes special demands on the LC equipment. Pumps should be designed for pressures of 250-1000 psi and the entire system should feature the lowest possible dead volume for optimum column performance. Higher flow rates and shorter run times The optimal flow rate for 1.8µm particles is higher than for 3 and 5µm particles. Figure 2 shows the considerable decrease in analysis time on converting from a 5µm to a 1.8µm column and increasing the flow rate. See pg. 195 for all NUCLEDUR part numbers and pricing Figure 1. Resolution as a function of particle size Macherey-Nagel literature available, please enquire Figure 2. Reduction of analysis time Toll Free: 1-888-226-2775 E-mail: info@lifescience.ca Web: www.lifescience.ca 191
NUCLEDUR (Macherey-Nagel) NUCLEDUR PolarTec NUCLEDUR PolarTec shows outstanding stability in highly aqueous media (>95%) due to a covalently bonded C18 silane with an embedded polar functional group. In addition to the hydrophobic between analyte and stationary phase shown by standard C18 RP phases, Nucleodur PolarTec also shows polar interaction via hydrogen bonding and dipole-dipole forces. This can have an influence on retention time and improve selectivity, particularly for the separation of polar compounds. Figure 1 shows the separation of organic acids with Nucleodur PolarTec compared with a standard RP18 phase. Figure 1. rganic acids comparison Eluents: : cetonitrile +0.1%TF Sample : Water +0.1%TF 1. Dihydroxymandelic cid 6. Vanillic cid Gradient: 20% > 60% in 12 min 2. Gallic cid 7. 3-Hydroxybenzoic cid Flow: 0.73 ml/min 3. Dihydroxyphenylacetic cid 8. 2.5-Dihydroxybenzoic cid Temperature: 20 C 4. 3,4-Dihydroxybenzoic cid 9. 2.4-Dihydroxybenzoic cid Detection: 254nM 5. Syringic cid NUCLEDUR PolarTec 5µm, EC 150/3 RP18 5µm, EC 150/3 0,0 1,0 2,0 3,0 4,0 5,0 7,0 8,0 9,0 10,0 0,0 1,0 2,0 3,0 4,0 5,0 7,0 8,0 9,0 10,0 NUCLEDUR PFP The functional group of Nucleodur PFP is a covalently bonded, Pentafluorophenyl propyl ligand that allows the separation of small, water soluble molecules, as well as peptides, polar or basic compounds and positional isomers. Nucleodur PFP shows a variety of between the analyte and the solid phase. esides the hydrophobic and dipole-dipole, the perfluorinated aromatic ring system (which is electron poor due to I effect of the fluor atoms), additional p-p and hydrogen bonding allow the base line separation of positional isomeric dichlorophenols. Figure 2. 80,0 40,0 20,0 0,0 2,0 4,0 8,0 10,0 12,0 14,0 Column: Eluents: Flow: Temperature: 35 C Detection: UV @ 280nM Sample 1. o-cresol 2. m-cresol 3. 3,4-Dimethylphenol 4. 3,5-Dimethylphenol 5. 2,5-Dimethylphenol 6. 2,6-Dichlorophenol 7. 2,2-Dichlorophenol 8. 2,4-Dichlorophenol 9. 3,4-Dichlorophenol 10. 2,4-Dibromophenol 11. 3,5-Dichlorophenol NUCLEDUR PFP 5µm, 125mm x 4mm : cetonitrile +0.1%F/ Water +0.1%F (35:65, v/v) 1 ml/min See pg. 195 for part numbers and pricing 192 Toll Free: 1-888-226-2775 E-mail: info@lifescience.ca Web: www.lifescience.ca