Packings for HPLC. Packings for HPLC
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1 Summary of packings for HPLC In analytical HPLC, packings with particle sizes of 3 to 10 µm are preferred. For preparative separation tasks, also particles with diameters larger than 10 µm are applied. The high standard of quality, the broad range of available packing materials, and ready-to-use columns, combined with efficient instrumentation, allow short analysis times optimum selectivity high sensitivity of detection These parameters can be optimised by variation of particle size and pore structure modification of the packing column dimension eluent and chromatographic conditions We offer a versatile programme of HPLC packings, briefly summarised in the following groups: silica, spherical particles totally porous available under the trade names and silica, irregular particles totally porous available under the trade names POLYGOSIL and POLYGOPREP All silica packing materials are available as unmodified silica, as well as with chemically bonded phases with different organic groups and thus specific selective behaviour. At this point it should be mentioned, that in practice most separation problems can be solved using only a few stationary phases, if the different selectivities of eluents are taken into account. However, in order to optimize certain separations, advantage can be taken of the specific selectivities of chemically bonded phases, which can save time and effort needed for determining the best separation conditions. The actual particle size of the packing plays an important role for the efficiency of a column. In general, higher efficiencies are achieved with smaller particles. Depending on the separation problem, however, somewhat larger particles (e. g. 7 µm instead of 5 µm) can still give good efficiency with shorter analysis times and longer column life. Consistent use of very narrow particle size distributions is another means of optimising separations. We supply packings with very narrow particle size distribution for optimum analytical performance. Furthermore, the permeability and the pressure required for a given eluent velocity depend strongly on the particle size and distribution. silica packings As silica packings for HPLC MACHEREY-NAGEL supply spherical and as well as irregular POLYGOSIL and ( 12 µm) POLYGOPREP. These types are available as unmodified silica as well as with chemically bonded phases. Columns packed with the high purity silica are described from page 6. For a detailed description of the high performance silica and columns packed with these phases please see pages All available modifications are listed on page 20. The high performance sillica is described on page 141. For POLYGOSIL and POLYGOPREP packings see page 153 and following. 140
2 spherical silicas Requirements for a high performance silica Purity For achieving an optimum resolution and symmetric peak shapes, a highly pure silica is required. It is well known, that metal ions, on the surface of silica largely are responsible for unwanted interactions with ionizable analytes, e. g. amines or phenolic compounds. The unique manufacturing process of provides a highly pure silica virtually free of metal impurities, and low acidic surface silanols. The following table shows the elementary analysis (metal ions), measured by AAS, of silica, 100 Å, 5 µm. Aluminium < 5 ppm Iron < 5 ppm Sodium < 5 ppm Calcium < 10 ppm Titanium < 1 ppm Zirconium < 1 ppm Arsenic < 0.5 ppm Mercury < 0.05 ppm Stability Physical stability: This totally spherical, 100% synthetic silica gel exhibits an outstanding mechanical stability, even at high pressures, and elevated flow rates of the mobile phase. Moreover, the material is re-usable. After several cycles of repeated packing, no significant changes in pressure drop can be observed. This is an important prerequisite for process-scale applications. Chemical stability: The utmost purity of the base silica and the exceptional silane bonding chemistry minimizes the risk of dissolution, or hydrolysis at ph extremes. The following chromatograms show the retention behaviour at ph values of 1.5 and 10.0 for C 18 endcapped Separation of uracil, veratrol, toluene and ethylbenzene at ph 1.5 Column: CC 30/ C 18 ec Mobile phase: ACN/H 2 O (65:35, v/v), TFA, ph 1.5 Flow rate: 1.0 ml/min Temperature: 25 C Detection: UV, 254 nm min Stability of at ph 1.5 over 1000 cycles inj back pressure [bar] Packing # (conditions see chromatogram) For ordering information of packings see pages 143 and
3 spherical silicas Separation of theophylline and caffeine at ph 10 Column: CC 30/ C 18 ec Mobile phase: mau MeOH/aq. NH 3 (20:80, v/v), ph Flow rate: 0.5 ml/min Temperature: 25 C Detection: UV, 254 nm inj. 750 inj. 500 inj. 250 inj. Start 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 C 18 ec column describes the correlation between the increase of column loading and the decrease of separation efficiency. Surface bonding chemistry The efficiency of a separation is controlled by particle size and selectivity of the stationary phase. is available in 8 different particle sizes (3, 5, 7, 10, 12, 16, 20, 30 and 50 µm) as silica and with RP derivatization (C 8 and C 18 ). The exceptional surface coverage of monomerically bonded alkylsilanes combined with an exhaustive endcapping, results in a surface with lowest silanol activity. The elution of critical compounds such as basic drugs is possible without adsorption or peak deformation. The wide range of different particle sizes allow the use in analytical, preparative HPLC and for scale-up applications. Separation of phenols Column: EC 250/ C18 ec Mobile phase: Methanol/H 2 O, 60:40 (v/v) Flow: 1.0 ml/min Injection volume: 4 µl Temperature: 30 C Detection: UV, 254nm Peaks: 1) Pyrocatechol 2) Resorcinol 1 3 3) Phenol 4 2 4) Guaiacol 5) Veratrole min Loading curve Column: EC 250/ C 18 ec Mobile phase: acetonitrile/h 2 O 80:20 (v/v) Flow: 1.0 ml/min Temperature: 25 C acetophenone Detection: UV, nm butyrophenone theoret. plates / m µg / compound 142
4 and spherical silicas Octadecyl phases -(CH 2 ) 17 CH 3 C 18 phases are suited for reversed phase chromatography and ion-pairing chromatography for separation of nonpolar to moderately polar compounds such as fatty acids glycerides polycyclic aromatics esters (phthalates) vitamins steroids prostaglandins PTH amino acids etc C 18 ec spherical silica, octadecyl phase, endcapped (HMDS), 14.5% C, pore size 50 Å, ph stability at 20 C: 2 8 particle size 5 µm pack of 10 g pack of 100 g C 18 AB spherical silica, octadecyl phase, endcapped, base deactivated, polymer coating, 24% C, pore size 100 Å, ph stability at 20 C: 1 9 particle size 5 µm pack of 10 g pack of 100 g C 18 ec spherical silica, octadecyl phase, endcapped (HMDS), 17.5% C, pore size 110 Å, ph stability at 20 C: C 18 ec C 18 ec C 18 ec C 18 ec C 18 ec C 18 ec particle size 10 µm 12 µm 16 µm 20 µm 30 µm 50 µm pack of 100 g pack of 1000 g C 18 spherical silica, octadecyl phase, endcapped (HMDS), 15% C, pore size 100 Å, ph stability at 20 C: C C C C 18 particle size 3 µm 5 µm 7 µm 10 µm pack of 10 g pack of 100 g C 18 spherical silica, octadecyl phase, endcapped (HMDS), 11% C, pore size 120 Å, ph stability at 20 C: C C C C 18 particle size 3 µm 5 µm 7 µm 10 µm pack of 10 g pack of 100 g
5 spherical silicas Wide pore silica packings Silica as HPLC packing has several advantages: defined pore size and surface mechanical stability of the silica structure numerous methods for surface modification are available Many biologically interesting molecules can not be separated using conventional narrow pore silicas with pore diameters of about 100 Å. This is why we offer a complete line of wide pore packings with pore sizes of 300, 500, 1000 and 4000 Å. These materials can also be used for size exclusion chromatography (SEC). 300 C 18 spherical silica, octadecyl phase, endcapped (HMDS), 6.5% C, pore size 300 Å, ph stability at 20 C: C C C 18 particle size 5 µm 7 µm 10 µm pack of 10 g pack of 100 g C 18 spherical silica, octadecyl phase, endcapped (HMDS), 2% C, pore size 500 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g C 18 spherical silica, octadecyl phase, endcapped (HMDS), ~ 1% C, pore size 1000 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g C 18 spherical silica, octadecyl phase, endcapped (HMDS), <1% C, pore size 4000 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g
6 spherical silicas Octyl phases -(CH 2 ) 7 CH 3 C 8 phases are suited for reversed phase chromatography and ion-pairing chromatography for separation of moderately to highly polar (water-soluble) compounds such as steroids nucleosides cyclodextrins pharmacological plant constituents 50-5 C 8 ec spherical silica, octyl phase, endcapped (HMDS), 9% C, pore size 50 Å, ph stability at 20 C: 2 8 particle size 5 µm pack of 10 g pack of 100 g C 8 ec spherical silica, octyl phase, endcapped (HMDS), 9% C, pore size 100 Å, ph stability at 20 C: 2 8 particle size 5 µm pack of 10 g pack of 100 g C 8 spherical silica, octyl phase, not endcapped, 8.5% C, pore size 100 Å, ph stability at 20 C: C C C 8 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g C 8 spherical silica, octyl phase, not endcapped, 6.5% C, pore size 120 Å, ph stability at 20 C: C C C C 8 particle size 3.5 µm particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g C 8 spherical silica, octyl phase, not endcapped, ~ 3% C, pore size 300 Å, ph stability at 20 C: C C C 8 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g C 8 spherical silica, octyl phase, not endcapped, <1% C, pore size 500 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g
7 spherical silicas Phenyl phases -(CH 2 ) 3 C 6 H 5 phases are suited for reversed phase chromatography and ion pairing chromatography for separation of moderately polar compounds. The retention characteristics are similar to the C 8 phase, but with different selectivity for polycyclic aromatic hydrocarbons, polar aromatics, fatty acids etc C 6 H 5 ec spherical silica, phenyl phase, endcapped (HMDS), 8% C, pore size 100 Å, ph stability at 20 C: 2 8 particle size 5 µm pack of 10 g pack of 100 g C 6 H 5 spherical silica, phenyl phase, not endcapped, 8% C, pore size 100 Å, ph stability at 20 C: C 6 H C 6 H 5 particle size 5 µm pack of 10 g pack of 100 g C 6 H 5 spherical silica, phenyl phase, not endcapped, 6,5% C, pore size 120 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g C 6 H 5 spherical silica, phenyl phase, not endcapped, ~ 3% C, pore size 300 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g C 6 H 5 spherical silica, phenyl phase, not endcapped, pore size 500 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g C 6 H 5 spherical silica, phenyl phase, not endcapped, pore size 1000 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g
8 spherical silicas Butyl phases -(CH 2 ) 3 CH 3 C 4 phases are suited for reversed phase chromatography and ion-pairing chromatography for separation of macromolecules and hydrophobic substances. Retention times on butyl phases are shorter than on C 8 and C 18 phases C 4 spherical silica, butyl phase, not endcapped, pore size 120 Å, ph stability at 20 C: 2 8 particle size 5 µm pack of 10 g pack of 100 g C 4 spherical silica, butyl phase, endcapped (HMDS), ~ 2% C, pore size 300 Å, ph stability at 20 C: C C C 4 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g C 4 spherical silica, butyl phase, endcapped (HMDS), <1% C, pore size 500 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g C 4 spherical silica, butyl phase, endcapped (HMDS), <1% C, pore size 1000 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g C 4 spherical silica, butyl phase, endcapped (HMDS), <1% C, pore size 4000 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g Dimethyl phases -(CH 3 ) 2 Dimethyl phases are suited for reversed phase chromatography and ion-pairing chromatography. Retention times on the dimethyl phase are much shorter than for the other RP phases C 2 spherical silica, dimethyl phase, not endcapped, 3.5% C, pore size 100 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g
9 spherical silicas Cyano phases (nitrile) CN phases are suited for reversed phase chromatography and for normal phase chromatography: Normal phase chromatography: with low-polarity solvents for many compounds, which can also be separated on unmodified silica, however, due to the -(CH 2 ) 3 CN rapid equilibration much more suitable for gradient separations Reversed phase chromatography: with different selectivity than C 18, C 8 or phenyl modified packings. 100 CN spherical silica, cyano phase (nitrile), 5% C, pore size 100 Å, ph stability at 20 C: CN CN particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g CN spherical silica, cyano phase (nitrile), pore size 120 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g CN spherical silica, cyano phase (nitrile), pore size 300 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g CN spherical silica, cyano phase (nitrile), pore size 500 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g Nitro phases -(CH 2 ) 3 NO 2 NO 2 phases are suited for the separation of compounds with double bonds or for aromatic compounds. 100 NO 2 spherical silica, nitro phase, pore size 100 Å, ph stability at 20 C: NO NO 2 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g
10 spherical silicas Diol phases OH phases are suited for reversed phase chromatography and normal phase chromatography; they are less polar than unmodified silica and very easily wettable with water. -(CH 2 ) 3 O CH 2 CH CH 2 OH OH OH (Diol) spherical silica, diol phase, 5% C, pore size 100 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g OH (Diol) spherical silica, diol phase, pore size 300 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g OH (Diol) spherical silica, diol phase, pore size 500 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g OH (Diol) spherical silica, diol phase, pore size 1000 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g OH (Diol) spherical silica, diol phase, pore size 4000 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g Unmodified silica Due to its narrow pore structure, 50 is recommended for adsorption chromatography, while SiOH and 100 as well as 120 can be used for adsorption as well as for partition chromatography. 50 spherical silica, unmodified, pore size 50 Å, pore volume 0.8 ml/g, surface (BET) 420 m 2 /g, density 0.45 g/ml, pressure stability 600 bar particle size 3 µm 5 µm 7 µm 10 µm pack of 10 g pack of 100 g
11 and spherical silicas 100 spherical silica, unmodified, pore size 100 Å, pore volume 1 ml/g, surface (BET) 350 m 2 /g, density 0.36 g/ml, pressure stability 600 bar particle size 3 µm 5 µm 7 µm 10 µm pack of 10 g pack of 100 g spherical silica, unmodified, pore size ~110 Å, pore volume 0.9 ml/g, surface (BET) 340 m 2 /g, density 0.47 g/ml, pressure stability 800 bar particle size 10 µm 12 µm 16 µm 20 µm 30 µm 50 µm pack of 100 g pack of 1000 g spherical silica, unmodified, pore size 120 Å, pore volume 0,65 ml/g, surface (BET) 200 m 2 /g, density 0.55 g/ml, pressure stability 800 bar particle size 3 µm 5 µm 7 µm 10 µm pack of 10 g pack of 100 g spherical silica, unmodified, pore size 300 Å, pore volume 0.8 ml/g, surface (BET) 100 m 2 /g, density 0.45 g/ml, pressure stability 400 bar particle size 5 µm 7 µm 10 µm pack of 10 g pack of 100 g spherical silica, unmodified, pore size 500 Å, pore volume 0.8 ml/g, surface (BET) 35 m 2 /g, density 0.45 g/ml, pressure stability 400 bar particle size 5 µm 7 µm 10 µm pack of 10 g pack of 100 g
12 spherical silicas 1000 spherical silica, unmodified, pore size 1000 Å, pore volume 0.8 ml/g, surface (BET) 25 m 2 /g, density 0.45 g/ml, pressure stability 300 bar particle size 5 µm 7 µm 10 µm pack of 10 g pack of 100 g spherical silica, unmodified, pore size 4000 Å, pore volume 0.7 ml/g, surface (BET) 10 m 2 /g, density 0.48 g/ml, pressure stability 300 bar µm 7 µm 10 µm pack of 10 g pack of 100 g Amino phases -(CH 2 ) 3 NH 2 NH 2 phases feature versatile applicability: in normal phase chromatography with hexane, CH 2 CI 2 or isopropanol as mobile phase for polar compounds such as substituted anilines, esters, chlorinated pesticides etc. in aqueous-organic eluent systems for reversed phase chromatography of polar compounds like carbohydrates as weak anion exchanger for anions and organic acids using common buffers (e. g. acetate or phosphate) in conjunction with organic modifiers (e. g. acetonitrile). 100 NH 2 spherical silica, amino phase, 3.5% C, pore size 100 Å, ph stability at 20 C: NH NH 2 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g NH 2 spherical silica, amino phase, pore size 120 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g NH 2 spherical silica, amino phase, pore size 300 Å, ph stability at 20 C: 2 8 pack of 10 g pack of 100 g
13 spherical silicas Dimethylamino phases -(CH 2 ) 3 N(CH 3 ) 2 The DMA phase is a weakly basic anion exchanger for the separation of many anions; it can also be used in a similar way as the NH 2 phase. 100 N(CH 3 ) 2 spherical silica, dimethylamino phase, 4% C, pore size 100 Å, ph stability at 20 C: N(CH 3 ) N(CH 3 ) 2 particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g Cation exchanger, strongly acidic -(CH 2 ) 3 SO 3 Na The cation exchanger SA is used for ion exchange chromatography. It is a strongly acidic cation exchanger with sulphonic acid modification and a capacity of about 1 meq/g 100 SA spherical silica, strongly acidic cation exchanger, sulphonic acid modification, 6.5% C, pore size 100 Å, ph stability at 20 C: SA SA particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g Anion exchanger, strongly basic -(CH 2 ) 3 CH 2 N + (CH 3 ) 3 Cl The anion exchanger SB is used for ion exchange chromatography. It is a strongly basic anion exchanger with a quaternary ammonium modification and a capacity of about 1 meq/g. 100 SB spherical silica, strongly basic anion exchanger, quaternary ammonium modification, 10% C, pore size 100 Å, ph stability at 20 C: SB SB particle size 5 µm particle size 10 µm pack of 10 g pack of 100 g
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