Columns for HPLC. Columns for HPLC

Transcription

1 In this chapter we describe analytical HPLC columns with our NUCLEOSIL silica phases. Silica as well as polymerbased phases for special separation problems are described in the chapter Columns for special applications from p. 73. NUCLEOSIL silica the standard in HPLC NUCLEOSIL is a family of totally porous spherical silicas. They feature a very pure and uniform SiO structure and have gained wide acceptance as outstanding chromatographic packing for very different fields of modern chromatography. Due to its particle sizes NUCLEOSIL finds application in analytical as well as in preparative columns. It allows high bed stability due to spherical particles high efficiency due to narrow particle size distribution high separation performance due to optimized binding techniques high chemical and mechanical stability high load capacity and recovery rates high reproducibility from lot to lot SEM photograph of NUCLEOSIL (magnification see scale, cm 0 µm) For our silica bulk packing materials, spherical NUCLEOSIL and NUCLEODUR materials and the irregular POLY- GOSIL and POLYGOPREP silicas, please see the chapter Packings for HPLC from p. 3. As can be seen in the figure, NUCLEOSIL is a spherically shaped totally porous silica. We manufacture this packing material with different pore diameters (50, 00, 0, 300, 500, 000 and 000 Å) and particle sizes from 3 µm (only NUCLEOSIL 50, 00 and 0) to 0 µm with very narrow fractionation. The different groups of NUCLEOSIL packing materials and columns packed with these phases are described in detail in the following chapters. All narrow-pore NUCLEOSIL packings are stable up to 600 bar (8 500 psi), for NUCLEOSIL 0 even pressures of up to 800 bar ( 500 psi) can be applied. The wide-pore NU- CLEOSIL silicas are stable up to 300 or 00 bar ( 00 or psi). Thus all NUCLEOSIL packings can be used in every field of HPLC without limitations. Modified packings NUCLEOSIL packings are available as unmodified silica or with numerous chemically bonded phases. Silica packings with chemically bonded phases can be classified in several groups, namely those intended mainly for reversed phase chromatography (the base deactivated RP phases C 8 AB, C 8 HD, C 8 HD, NAUTILUS and PROTECT I and the standard RP phases C 8, C 8 ec, C 8, C, C and Phenyl) as well as packings with chemically bonded polar groups with selective separation performance (such as CN, NO, NH, N(CH 3 ), OH types). Furthermore, for NUCLEOSIL 00 we produce ion exchangers (NUCLEOSIL SA and SB), which are stable from ph to 8,5 and do not swell. Compared to resin-based ion exchangers they offer the advantage of constant permeability, even when the ionic strength and/or ph of the eluent are changed. NUCLEOSIL silica summary of available modifications Type of modification base deactivated RP phases C 8 HD C 8 HD see p. 8 see p. 8 C 8 NAUTILUS PROTECT I see p. 30 functional group organic Si-C group Octadecyl, base deactivated monomer modification -(CH ) 7 -CH 3 Octyl, base deactivated monomer modification -(CH ) 7 -CH 3 Octadecyl, base deactivated embedded polar group for up to 00% aqueous eluents special RP phase base deactivated, monomer modification see p. 3 = as packed column in our standard programme; = available as bulk material Pore diameter [Å] Particles 3 µm 5 µm 7 µm 3 µm 5 µm 3 µm 5 µm 3 µm 5 µm 0

2 NUCLEOSIL silica summary of available modifications Type of modification C 8 AB see p. 3 Standard RP phases C 8 see p. 39 C 8 ec see p. 3 C 8 see p. 3 C 6 H 5 ec see p. 6 C 6 H 5 see p. 6 C see p. 7 C see p. 8 functional group Pore diameter [Å] Particles organic Si-C group Octadecyl, base deactivated polymer modification -(CH ) 7 -CH 3 5 µm Octadecyl, endcapped Octyl, endcapped Octyl, not endcapped Phenyl, endcapped Phenyl Butyl Dimethyl -(CH ) 7 -CH 3 3 µm 5 µm 7 µm 0 µm -(CH ) 7 -CH 3 5 µm -(CH ) 7 -CH 3 3 µm 5 µm 7 µm 0 µm -(CH ) 3 5 µm 5 µm -(CH ) 3 7 µm 5 µm 7 µm -(CH ) 3 -CH 3 0 µm -(CH 3 ) 7 µm Polar NUCLEOSIL phases and NUCLEOSIL ion exchangers Cyano (Nitrile) 5 µm CN 7 µm see p. 5 -(CH ) 3 -CN 0 µm Nitro 5 µm -(CH ) 3 NO 0 µm NO see p. 5 OH see p. 53 NH see p. 55 N(CH 3 ) see p. 56 SA 5 µm Diol 5 µm -(CH ) 3 -O-CH -CH-CH 7 µm OH OH Amino 3 µm 5 µm -(CH ) 3 -NH 7 µm 0 µm Dimethylamino -(CH ) 3 -N(CH 3 ) 0 µm Sulphonic acid 5 µm -(CH ) 3 SO 3 Na 0 µm see p. 57 quaternary ammonium groups 5 µm SB see p. 58 -(CH ) 3 CH -N + (CH 3 ) 3 Cl 0 µm = as packed column in our standard programme; = available as bulk material

3 Base deactivated phases for RP chromatography Si OH conventional NUCLEOSIL 00-5 C 8 Si O Si(CH 3 ) 3 It is a known fact that the surface of silica contains active groups that can cause unwanted interactions with the analytes, even after derivatization and endcapping. For NUCLEOSIL, MACHEREY-NAGEL use four different approaches to reduce silanol interactions. This results in four types of base deactivated RP phases, which cover a very wide range of selectivities solving almost all reversed phase separation problems. NUCLEOSIL 00-5 C 8 AB the classical Acid and Base deactivated octadecyl-phase with polymeric coating This phase exhibits a high steric selectivity. NUCLEOSIL 00-5 C 8 HD NUCLEOSIL 00-3 C 8 HD NUCLEOSIL 00-5 C 8 HD a family of base deactivated phases with a High Density monomeric coating available as octyl phase or octadecyl phase with 3 or 5 µm particles The dense coverage with alkyl groups results in phases with very low bleeding, which are ideal for trace analyses and especially recommended for LC/MS. NUCLEOSIL 00-3 C 8 NAUTILUS, NUCLEOSIL 00-5 C 8 NAUTILUS Si OH Pol Pol base deaktivated octadecyl phases for up to 00% aqueous eluents An embedded polar functional group allows chromatography under purely aqueous elution conditions. Si O Si(CH 3 ) 3 NUCLEOSIL 00-3 PROTECT I, NUCLEOSIL 00-5 PROTECT I Si Si OH O pro pro Si(CH 3 ) 3 a base deactivated silica RP phase with special monomeric coating A polar protective group in the carbon skeleton strongly interacts with the silanol groups, effectively shielding them from the analytes. While featuring an outstanding base deactivation, the phase NUCLEOSIL PROTECT I shows higher hydrophilicity than the above phases.

4 Base deactivated phases for RP chromatography Why is a good base deactivation of the silica so important? Due to interactions with residual silanol groups the RP chromatography of basic components on standard silica materials is subject to severe problems concerning peak symmetry and detection sensitivity. A normal surface modification reaction leaves many unreacted silanol groups on the surface Our adsorbents are manufactured according to very stringent quality criteria. All base deactivated NUCLEOSIL phases feature: versatile selectivity optimised endcapping high purity silica outstanding batch-to-batch reproducibility excellent peak shape for basic compounds of the silica, which can severely disturb the chromatography of ionizable compounds, especially if one works without buffers or PIC reagents. Even simple endcapping does not completely solve this problem. for every separation the optimised RP phase NUCLEOSIL C 8 NAUTILUS monomer-coated octadecyl phase with embedded polar group monomer-coated octadecyl phase NUCLEOSIL C 8 HD NUCLEOSIL C 8 AB polymer-coated octadecyl phase increasing carbon content relatively more hydrophobic base deactivated silicas for RPLC relatively more hydrophilic NUCLEOSIL C 8 HD NUCLEOSIL PROTECT I monomer-coated octyl phase monomer-coated octyl phase with embedded polar group 3

5 Fast LC of anilines Column: 30 x mm NUCLEOSIL 00-5 PROTECT I Eluent: methanol water (5:55, v/v) Flow rate: ml/min Detection: UV, 5 nm. Aniline. N,N-Dimethylaniline 3. N,N-Diethylaniline 3 RP method development kits For selection of the RP phase best suited for your separation we offer two RP method development kits with 30 mm : each kit consists of a CC column holder 30 mm and one column each 30 x or 30 x mm, respectively, packed with: NUCLEOSIL PROTECT I, NUCLEOSIL 00-5 C 8 HD, NUCLEOSIL 00-5 C 8 HD, NUCLEOSIL 00-5 C 8 AB and NUCLEOSIL 00-5 C 8 Ordering information Designation Cat. No. Method development kit ChromCart mm 75.0 Method development kit ChromCart mm 75.0 Method development kit ChromCart.6 mm min Comparison of selectivities for base deactivated RP phases Separation of a test mixture on four base deactivated NUCLEOSIL phases These chromatograms show the differences in retention times and selectivities of the four phases. Columns: A) 50 x mm NUCLEOSIL 00-5 C 8 HD; B) 50 x mm NUCLEOSIL 00-5 C 8 AB; C) 50 x mm NUCLEOSIL 00-5 C 8 HD; D) 50 x mm NUCLEOSIL 00-5 PROTECT I Eluent: methanol water (60:0); flow rate 0.8 ml/min; temperature: 35 C Detection: UV, 30 nm. p-ethylaniline. Diethyl phthalate 3. N,N-Dimethylaniline. Ethyl benzoate 5. Toluene 6. Naphthalene 7. Ethylbenzene C 8 HD C 8 AB C 8 HD PROTECT,3, min min min 0 0 min

6 Characterization of different NUCLEOSIL RP phases The separation of a substance mixture depends on many factors. Based on suggestions of Tanaka ) and Johnson ) we compare our RP adsorbents with respect to the following parameters: A capacity k (pentylbenzene) number of alkyl chains, C content B hydrophobicity α (pentylbenzene, butylbenzene) CH group selectivity C steric selectivity α (triphenylene, o-terphenyl) steric differences of the analytes D silanol capacity α (caffeine, phenol) residual silanol groups E ion exchange capacity at ph 7.6 α (benzylamine, phenol) residual silanol groups F ion exchange capacity at ph.8 α (benzylamine, phenol) residual silanol groups The capacity factor of pentylbenzene (axis A) and the selectivity between pentyl- and butylbenzene (axis B) are a measure for the hydrophobic interactions of a stationary phase. Naturally, octadecyl modified silica phases show very large values for these parameters. The capacity factor of NUCLEOSIL 00-5 C 8 HD proves the high density of the alkyl groups of this phase compared to NUCLEOSIL 00-5 C 8 AB and the conventional NUCLEOSIL 00-5 C 8 phase. Shortening of the alkyl chain from C 8 to C 8 results in a lower carbon content and a reduction of the hydrophobic interactions. The steric selectivity (axis C), i.e. the ability of an adsorbent to differentiate between molecules of equal hydrophobicity, but different shape (planar, non-planar) is especially large for the phase NUCLEOSIL 00-5 C 8 AB. Axes D F describe the quality of the base deactivation under different conditions.. N. Tanaka et al., J. Chromatogr. Sci. 7, 7 (989). C. M. Johnson et al., Chromatographia, 5 (997) Characteristics of some of our base deactivated NUCLEOSIL phases Phase Tanaka plot Hydrophobicity Base deactivation A 6.5 for comparison: NUCLEOSIL F 3.5 B C 0. 8 Steric selectivity NUCLEOSIL 00-5 C 8 HD E D. C F 0 0. A B.5 medium very good low E C 0. D please note: this phase is not endcapped A 6.5 NUCLEOSIL 00-5 C 8 HD F B.5 for comparison: NUCLEOSIL 00-5 C 8 very high very good medium E C A D A 6.5 F B.5 NUCLEOSIL 00-5 C 8 AB F 0 E D B.5. C E C D this phase is endcapped high good high 5

7 Base deactivated phases particle size and separation efficiency Due to the basic principles of HPLC ) short diffusion paths in the pores of the stationary phase are required for achieving high column efficiency. According to the van Deemter equation h A ν 0.33 B = C ν ν A: band broadening caused by eddy diffusion and stream splitting of the mobile phase B: band broadening caused by longitudinal diffusion C: band broadening caused by equilibrium distribution between stationary and mobile phase ν: velocity of the mobile phase the C-term describes the influence of band broadening, which is caused by the equilibrium distribution of the molecules between stationary and mobile phase. In the pores of smaller particles the diffusion paths are shorter. As a result the C-term is reduced, which means a favorable effect for gaining low HETP values (height equivalent to one theoretical plate). Provided that the velocity of the mobile phase is in its optimum range, smaller particles exhibit a considerably increased column efficiency. It is a rule of thumb, that a well-packed 3 µm HPLC column has about twice the separation efficiency of a 5 µm column. The choice of smaller particle size packings allows the use of shorter columns for rapid separations without loss of resolution. This is shown in the separation of vanillin compounds on the right. Analysis time can be reduced to a fifth by the replacement of a conventional 50 mm/5 µm NUCLEOSIL NAUTILUS column with a 70 mm high-speed column packed with the 3 µm counterparts. The drastic shortening of the analysis time is not only due to reducing column length, but also to increasing the flow rate by 50%. Problems with band broadening do not occur, because of the increased flow rate for smaller-sized packings. An additional benefit in the use of rapid resolution columns is the remarkable reduction of solvent consumption. The following 3 µm NUCLEOSIL base deactivated silicas are available: NUCLEOSIL 00-3 C8 NAUTILUS NUCLEOSIL 00-3 PROTECT I NUCLEOSIL 00-3 C8 HD NUCLEOSIL 00-3 C8 HD Besides the range of our well established standard materials NUCLEOSIL 00-3 C 8, NUCLEOSIL 0-3 C 8 and NU- CLEOSIL 0-3 C 8 are still available. All 3 µm packings are available in a variety of column configurations and can be used for standard and high-throughput applications. The currently available high performance NUCLEOSIL 3 µm RP packings are characterised by the following features: high purity NUCLEOSIL silica derivatised with unique monofunctional silanes optimised endcapping outstanding peak symmetry for basic compounds excellent reproducibility from batch to batch and column to column Separation of isovanillin, vanillin and ethoxyvanillin Column: a) 70 x mm NUCLEOSIL 00-3 C 8 NAUTILUS b) 50 x mm NUCLEOSIL 00-5 C 8 NAUTILUS Eluent: ACN water H 3 PO (0:80:0., v/v/v) ambient temperature Flow rate: a).5 ml/min, b) ml/min Detection: UV, 80 nm ) Isovanillin a) ) Vanillin 3) Ethoxyvanillin b) min ) V. R. Meyer, Practical High Performance Liquid Chromatography (John Wiley & Sons, New York, 3rd. ed. 999)

8 Base deactivated phases for RP chromatography: NUCLEOSIL HD NUCLEOSIL HD is a family of RP phases with optimised base deactivation, which is available with octyl or octadecyl modification and 3 or 5 µm particles. NUCLEOSIL 00-5 C 8 HD is an octyl modified RP phase with 5 µm particles. As can be seen in the figure below, the basic compound lidocaine is eluted without tailing. Compared to NUCLEOSIL 00-5 C 8 HD introduction of the shorter carbon chain results in a reduction of hydrophobic interactions. The chromatograms show the separation of compounds with different functional groups on both phases. The less polar aromatics toluene and naphthalene are eluted earlier from the C 8 HD phase resulting in a reversed elution sequence of toluene and lidocaine. Comparison of selectivities between NUCLEOSIL HD octadecyl and octyl phases Separation of a test mixture on NUCLEOSIL C 8 HD Column: 50 x mm NUCLEOSIL 00-5 C 8 HD Eluent: MeOH 5 mm NaH PO ph 7.0 (65:35) Flow rate: 0.8 ml/min Temperature: 5 C Detection: UV, 5 nm 3. Phenol. 5 -(p-methylphenyl)- 5-phenylhydantoin 3. Diethyl phthalate. Lidocaine 5 5. Toluene 6. Naphthalene Separation of a test mixture on NUCLEOSIL C 8 HD Column: 50 x mm NUCLEOSIL 00-5 C 8 HD Eluent: MeOH 5 mm NaH PO ph 7.0 (65:35) Flow rate: 0.8 ml/min Temperature: 5 C Detection: UV, 5 nm. Phenol (p-methylphenyl)- 5-phenylhydantoin 3. Diethyl phthalate. Lidocaine 5 5. Toluene 6. Naphthalene min min Highest quality! Batch-to-batch reproducibility of NUCLEOSIL HD Separation of a test mixture on three different batches of NUCLEOSIL 00-5 C 8 HD Column 50 x mm NUCLEOSIL 00-5 C 8 HD Eluent methanol water (55:5, v/v); flow rate ml/min temperature 0 C; detection: UV, 5 nm. Thiourea. Aniline 3. Phenol 5. o,m,p-toluidine N,N-Dimethylaniline 6. Methyl benzoate 7. Toluene 8 8. Ethylbenzene min Lot A Capacity factor k (toluene) k (ethyl benzoate) k (MPPH) B C α (MPPH/toluene) α (ethyl benzoate/toluene) D E F Selectivity NUCLEOSIL 00-5 C 8 HD, acetonitrile water 50:50 (v/v), ml/min, 5 C, UV 5 nm 7

9 Base deactivated NUCLEOSIL phases for RP chromatography NUCLEOSIL 00-3 C 8 HD Octyl 3 µm spherical 3 µm silica particles with monomeric octyl modification NUCLEOSIL 00-5 C 8 HD Octyl 5 µm spherical 5 µm silica particles with monomeric octyl modification NUCLEOSIL 00-3 C 8 HD Octadecyl 3 µm spherical 3 µm silica particles with monomeric octadecyl coating NUCLEOSIL 00-5 C 8 HD Octadecyl 5 µm spherical 5 µm silica particles with monomeric octadecyl coating These packings are manufactured from NUCLEOSIL 00 with 3 or 5 µm particles, respectively. Thus, surface shielding, capacity factors and selectivities are the same for both materials. However, the number of theoretical plates is higher when using the 3 µm starting silica. The 3 µm particle allows application of shorter columns (= reduction of time required for the analysis) and improves the detection sensitivity due to smaller peak widths. The following chromatogram exhibits the higher efficiency of the 3 µm NUCLEOSIL C 8 HD column compared to the 5µm equivalent for identical column lengths (curves a and b). The improved sensitivity of the 3 µm packing becomes evident by the increased peak heights in the chromatogram. However, if the column length is reduced from 50 mm to 50 mm, baseline separation, under the given chromatographic conditions, cannot be achieved (c). In this case the use of the 50 mm NUCLEOSIL 00-3 C 8 HD column is strongly recommended to obtain optimal results. Separation of paracetamol, caffeine and acetylsalicylic acid Sample preparation: dissolve tablet in 0 ml ACN/water (50:50), dilute :0. Eluent: ACN water H 3 PO (0:80:0., v/v/v) Columns: a) 50 x mm NUCLEOSIL 00-5 C 8 HD Temperature: 30 C b) 50 x mm NUCLEOSIL 00-3 C 8 HD Flow rate: ml/min c) 50 x mm NUCLEOSIL 00-3 C 8 HD Detection: UV, 5 nm. Paracetamol. Caffeine 3. Acetylsalicylic acid min Ordering information Analytical columns with NUCLEOSIL HD Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns NUCLEOSIL 00-3 C 8 HD Particle size 3 µm, pore size 00 Å; octyl phase, endcapped, monomer coating, 3% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID Each column is individually tested and supplied with test chromatogram and test conditions 8

10 Base deactivated NUCLEOSIL phases for RP chromatography Ordering information Analytical columns with NUCLEOSIL HD Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns NUCLEOSIL 00-5 C 8 HD Particle size 5 µm, pore size 00 Å; octyl phase, endcapped, monomeric coating, 3% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID NUCLEOSIL 00-3 C 8 HD Particle size 3 µm, pore size 00 Å; octadecyl phase, endcapped, monomeric coating, 0% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID NUCLEOSIL 00-5 C 8 HD Particle size 5 µm, pore size 00 Å; octadecyl phase, endcapped, monomeric coating, 0% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of. ) 30 mm require the CC column holder 30 mm (Cat. No. 783). ) On request, microbore columns are also available in lengths of 0, 60, 00 and 300 mm and with 0.5, 0.3, 0., 0.5, 0.75 and.5 mm ID. Guard columns for microbore columns on request. 3) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7359). Custom-packed columns with other lengths are available on request 9

11 H O H H H O Base deactivated NUCLEOSIL phases for RP chromatography NUCLEOSIL 00 C 8 Nautilus Pol Si Si the formula for RP chromatography in aqueous media O H O H With highly aqueous mobile phases (> 95 %) conventional reversed phase columns very often exhibit a plainly visible deterioration of column performance after a certain period of time. The non-polar alkyl chains lose their "brush-type structure" for maintaining the hydrophobic interactions between stationary phase and analyte. The result of this phase collapse is a drastic decrease of retention time and poor resolution. This phenomenon may occur very fast or after a while. H O H O A A H O H O A H O H O H O A H O H O H O A H O H O A H O H O A = analyte A H O A H O A Even after days or weeks of operation in purely aqueous eluents the C 8 chains of NUCLEOSIL 00-5 C 8 Nautilus neither are folded nor show any collapsing. A significant reduction of retention time cannot be observed as is shown in the following diagrams. % of initial retention time Phase collapse in aqueous media change of retention time with 00% aqueous eluents NUCLEOSIL 00-5 C 8 Nautilus conventional RP phase day Start 6 8 Phase collapse and decrease in retention times for a conventional RP phase with 00 % aqueous eluents NUCLEOSIL 00-5 C 8 Nautilus is a reversed phase C 8 column based on highly pure silica with thorough endcapping, which is totally stable if the mobile phase is 00% aqueous without any organic modifiers. This property is achieved by a covalently bonded C 8 silane (synthesised in MACHEREY-NAGEL s R&D laboratories) with an embedded functional group, which is the driving force for the excellent water stability of the RP surface. Compared to standard C 8 phases where hydrophobic interactions between analyte and stationary phase predominate, NUCLEOSIL 00-5 C 8 Nautilus also shows polar interaction via hydrogen bonding activities and dipole-dipole forces. This can influence retention time (t R ) and improve selectivity (α) particularly for the separation of polar compounds. Start min Separation of nucleobases in a purely aqueous eluent Column: 5 x mm NUCLEOSIL 00-5 C 8 Nautilus Eluent: 0 mm ammonium formate, ph. Flow rate: ml/min Temperature: 0 C Detection: UV, 5 nm. Cytosine. Uracil 3. Hypoxanthine. Xanthine 5. Adenine In spite of the polar character of the embedded functional group NUCLEOSIL C 8 Nautilus exhibits sufficient hydrophobic properties and is very well suited for analysing basic compounds just like NUCLEOSIL HD or NUCLEOSIL Protect I. It may be worthwhile to try this chemistry for difficult separations Each column is individually tested and supplied with test chromatogram and test conditions

12 Base deactivated NUCLEOSIL phases for RP chromatography In the selectivity test on the right the strong base p-ethylaniline is eluted as a sharp signal with remarkably good peak shape. Another example for the excellent base deactivation of NUCLEOSIL Nautilus is the tailing-free elution of pyridine. Chromatographic conditions: Column 50 x mm NUCLEOSIL 00-5 C 8 Nautilus; eluent methanol water (left 55:5, right 30:70, v/v); flow rate left 0.8 ml/min, right ml/min; temperature 0 C; detection UV, 5 nm Peaks (left):. uracil,. aniline, 3. phenol,. p-ethylaniline, 5. N,N-dimethylaniline, 6. toluene, 7. ethylbenzene References D. Rieger, H. Riering, Int. Laboratory, Vol. 30, p. Selectivity test N Pyridine/ phenol min 8 6 OH Ordering information Analytical columns with NUCLEOSIL 00-5 C 8 Nautilus Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns NUCLEOSIL 00-3 C 8 Nautilus Particle size 3 µm, pore size 00 Å; octadecyl phase, endcapped, embedded polar group, 6% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID NUCLEOSIL 00-5 C 8 Nautilus as above, however, particle size 5 µm mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of. ) 30 mm require the CC column holder 30 mm (Cat. No. 783). ) On request, Microbore columns are also available in lengths of 0, 60, 00 and 300 mm and with 0.5, 0.3, 0., 0.5, 0.75 and.5 mm ID. Guard columns for Microbore columns on request. 3) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7359). Custom-packed columns with other lengths are available on request 3

13 Base deactivated NUCLEOSIL phases for RP chromatography NUCLEOSIL 00-3 Protect I Special RP phase 3 µm spherical 3 µm silica particles with monomeric special RP modification NUCLEOSIL 00-5 Protect I Special RP phase 5 µm spherical 5 µm silica particles with monomeric special RP modification Although hydrophobic interactions of NUCLEOSIL 00-5 Protect I are comparatively low and closer to the standard octyl phase NUCLEOSIL 00-5 C 8, the special surface modification combined with a rigorous endcapping of the unreacted silanol groups reduces interaction successfully and ensures excellent peak shapes with basic compounds. In comparison with the well-established NUCLEOSIL 00-5 C 8 AB, NUCLEOSIL 00-5 C 8 HD and NUCLEOSIL 00-5 C 8 HD a key characteristic of the NUCLEOSIL 00-5 protect I is its higher polarity. Nevertheless, critical compounds like amines show outstanding peak symmetries. This stationary phase is well suited for the separation of low molecular-weight compounds (up to 5000 dalton), e. g. drugs or pesticides. For example, the tricyclic amines protriptyline and amitriptyline show especially strong interactions with residual silanol groups on a conventional C 8 column, while our NUCLEOSIL 00-5 Protect I features early elution and excellent peak symmetry for this critical pair of compounds (see chromatogram). The batch test underlines the outstanding base deactivation and unique selectivity of NUCLEOSIL 00-3 Protect I for the successful separation of critical bases such as amitriptyline and protriptyline, which are eluted with excellent peak shapes as for the aromatic hydrocarbons. Separation of a batch test mixture Column: 50 x mm NUCLEOSIL 00-3 Protect I, Cat. No Eluent: acetonitrile 5 mm KH PO, ph 7.5 (50:50) Flow rate: ml/min Detection: UV, 5 nm Temperature: 5 C 3. Uracil. Protriptyline 3. Amitriptyline. Ethyl benzoate 5. Toluene 6. Ethylbenzene min 6 3 Each column is individually tested and supplied with test chromatogram and test conditions

14 Base deactivated NUCLEOSIL phases for RP chromatography Ordering information Analytical columns with NUCLEOSIL 00-5 Protect I Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns NUCLEOSIL 00-3 Protect I Particle size 3 µm, pore size 00 Å; special RP phase, endcapped, monomer coating, % C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID NUCLEOSIL 00-5 Protect I Particle size 5 µm, pore size 00 Å; special RP phase, endcapped, monomer coating, % C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of. ) 30 mm require the CC column holder 30 mm (Cat. No. 783). ) On request, Microbore columns are also available in lengths of 0, 60, 00 and 300 mm and with 0.5, 0.3, 0., 0.5, 0.75 and.5 mm ID. Guard columns for Microbore columns on request. 3) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7359). Custom-packed columns with other lengths are available on request 33

15 Base deactivated NUCLEOSIL phases for RP chromatography NUCLEOSIL 00-5 C 8 AB Octadecyl 5 µm spherical 5 µm silica with polymeric octadecyl coating Tailing, changes of elution sequence and decreased sensitivity are common problems encountered in the separation of acid as well as basic substances on conventional RP phases. The polymer-coated octadecyl packing material NUCLEOSIL 00-5 C 8 AB features an outstanding shielding of the silica matrix and is inert towards acidic and basic substances, which have a high affinity for silica. The carbon content of 5% is considerably higher than for monomerically coated standard octadecyl phases. NUCLEOSIL 00-5 C 8 AB is a nonpolar, hydrophobic RP silica for HPLC with an increased stability to hydrolysis in alkaline media compared to standard RP phases. Moreover, the distinct steric selectivity of this phase can be emphasized. Steric selectivity of NUCLEOSIL C 8 AB Columns: a) 50 x mm NUCLEOSIL 00-5 C 8, b) 50 x mm NUCLEOSIL 00-5 C 8 AB, 50 x mm ID; eluent MeOH H O (80:0, v/v), flow rate ml/min, temperature 0 C, detection UV, 5 nm. Phenol 6. o-terphenyl. Diethyl phthalate 7. Anthracene 3. Naphthalene 8. Pentylbenzene. Dibutyl phthalate 9. Triphenylene 5. Butyl benzene a) b) min Ordering information Analytical columns with NUCLEOSIL 00-5 C 8 AB packing: particle size 5 µm, pore size 00 Å; octadecyl phase, endcapped, polymeric coating, 5% C; eluent in column acetonitrile / water Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of. ) 30 mm require the CC column holder 30 mm (Cat. No. 783). ) On request, Microbore columns are also available in lengths of 0, 60, 00 and 300 mm and with 0.3, 0., 0.5, 0.75 and.5 mm ID. Guard columns for Microbore columns on request. 3) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7359). Each column is individually tested and supplied with test chromatogram and test conditions 3

16 Base deactivated NUCLEOSIL phases for RP chromatography Applications Separation of phenols Column: 50 x mm NUCLEOSIL 00-5 Protect I Eluent: acetonitrile 5 mm KH PO ph 7 (50:50, v/v) Flow rate: ml/min Temperature: 0 C Detection: UV, 35 nm. p-benzoquinone. Phenol 3. o-nitrophenol. p-nitrophenol 5.,3-Dinitrophenol m-nitrophenol ,5-Dinitrophenol 8. 3,-Dinitrophenol 9.,6-Dinitrophenol 0.,-Dinitrophenol ,,6-Trinitrophenol 50 Separation of sunscreen agents V. Vanquerp et al. J. Chromatogr. 83 (999) Column: 50 x mm NUCLEOSIL 00-5 C 8 HD Eluents: A) % acetic acid in water, B) methanol Gradient: 80 00% B in 0 min Flow rate: ml/min Detection: UV, 300 nm Peaks (Injection volume 0 µl): A) Benzophenone-3 A B) Methylbenzylidene camphor C) Octyl methyl PABA D) Butyl methoxydibenzoylmethane E) PEG-5 PABA B C D E min Analysis of customary heroin, stretched with paracetamol/caffeine Column 50 x mm NUCLEOSIL 00-5 C 8 AB, with guard column 8 x 3 mm NUCLEOSIL 00-5 C 8 AB Injection volume 30 µl Eluents: A) mmol NaH PO, g/l -hexanesulphonic acid adjusted to ph.5 with H 3 PO, B) acetonitrile Gradient: 8 33% B in 30 min, then 5 min 90% B Flow rate: 0.3 ml/min, temperature: 0 C Detection: DAD, 0 nm. Paracetamol. Caffeine 3. 6-Monoacetylmorphine. Heroin 5. Papaverine 6 6. Noscapine courtesy of Mr. Kothe, Laboratory Dr. Krone, Prof. Hagedorn, Medizinal-Untersuchungsstelle Herford 3 5 Analysis of customary ecstasy Column: 50 x mm NUCLEOSIL 00-5 C 8 HD Eluents: A) mmol NaH PO adjusted to ph.5 with H 3 PO B) acetonitrile mau Gradient: 8 33% B in 30 min 30 Flow rate: 0.8 ml/min Detection: UV, 35 nm Peak:. Caffeine?. S(+)-3,-Methylenedioxymethamphetamine hydrochloride (Ecstasy) courtesy of Mr. Kothe, 0 Laboratory Dr. Krone, Prof. Hagedorn, Medizinal-Untersuchungsstelle Herford min min 35

17 Base deactivated NUCLEOSIL phases for RP chromatography Applications Separation of tricyclic antidepressants Column: 50 x mm NUCLEOSIL 00-5 Protect I Eluent: acetonitrile 5 mm KH PO, ph 7.0 (35 : 65, v/v) Flow rate: ml/min Temperature: 5 C Detection: UV, 5 nm. Protriptyline 3. Doxepin O 5. Amitriptyline NHCH 3 N(CH 3 ) N(CH 3 ). Nortriptyline. Imipramine 6. Trimipramine N N NHCH 3 N(CH 3 ) N(CH 3 ) min Separation of tricyclic antidepressants Column: 50 x mm NUCLEOSIL 00-5 C 8 HD 3 Eluent: MeOH 5 mm NaH PO ph 7.0 (70:30) Flow rate ml/min Temperature 30 C Detection UV, 5 nm. Protriptyline. Nortriptyline 3. Doxepin. Amitriptyline 5. Trimipramine Separation of tricyclic antidepressants Column: 50 x NUCLEOSIL 00-5 C 8 HD Injection volume 5 µl Eluent acetonitrile 0.% TEAPO ph 6.9 (5:55) Flow rate ml/min Temperature 5 C Detection UV, 5 nm. Protriptyline. Nortriptyline 3. Doxepin. Imipramine 5. Amitriptyline 6. Trimipramine min min 36

18 Standard phases for RP chromatography Reversed phase silica phases are by far the most widely used packings. Our base deactivated high performance RP materials are described in the preceding chapter. In this chapter you will find columns packed with standard silica phases for RPLC. For ordering information of the corresponding bulk packings please see the chapter starting on page 3. Octadecyl, Octyl, Butyl, Dimethyl and Phenyl phases differ in retention time for a given solute. Under identical conditions (mobile phase, flow rate, temperature etc.), e. g. the retention times on NUCLEOSIL C 8 are, for quite a number of compounds, by a factor of up to larger than on NUCLEOSIL C 8. The mechanism of separation with all NUCLEOSIL and NUCLEODUR RP phases is the attraction between hydrophobic groups of the test molecule and the bonded alkyl groups of the stationary phase by van der Waals forces. the less polar the sample molecules, the more they are attracted. Differences in the chemical structure of the sample molecules result in a stronger or weaker adsorption to the stationary phase and hence in a separation. The more polar the sample, the shorter are the retention times and the weaker are the van der Waals forces. The following is roughly the order of increasing polarity (reverse order of elution): aliphatic hydrocarbons, induced dipoles (CCI ), permanent dipoles (CHCI 3 ), weak Lewis bases (ethers, aldehydes, ketones), strong Lewis bases (amines), weak Lewis acids (alcohols, phenols) and strong Lewis acids (carboxylic acids). Also of considerable importance for retention times is the chain length of the molecules which are to be separated. The mobile phase in RP chromatography η [cp] at 0 C 3 viscosity Viscosity of solvent mixtures as a function of composition 3 value. Higher concentrations of organic solvent will cause shorter retention times. RP separations can be optimised by utilising the specific selectivity of organic solvents. The viscosity of the eluent should not be neglected. It is often not realised in practice, that eluents have very different viscosities (η) and that these differences in viscosity (with the same flow rate) cause changes in back pressure. The back pressure changes according to the following equation: η p = p η water n-propanol ethanol methanol acetonitrile % [w/w] organic The mobile phase for reversed phase chromatography is a mixture of water and an organic solvent which is miscible with water. The time of elution of the substances to be separated depends to a large degree on the water content of the organic solvent and is therefore easily adjusted to a suitable The viscosity of a solvent mixture is often higher than that of the single components, i.e. a higher pressure is needed to achieve the same flow rate. The graph on the left illustrates how strongly the viscosity of the eluent is influenced by its composition. For example the viscosity (and consequently the back pressure of the column) increases more than double when the eluent is changed from acetonitrile water (70:30) to methanol water (70:30). When water is used as a solvent or as a component in gradient mixtures, it is essential that it is completely degassed. Apparent instability can be caused by air bubbles (from not properly degassed solvents) which accumulate at the detector. The best way to remove such air is to first pump degassed solvent through the column and then follow up with degassed water. One can degas solvents with a vacuum pump, by passing helium through or by ultrasonic treatment. 37

19 Test mixture for reversed phase columns For reversed phase HPLC columns we supply a test mixture in acetonitrile to determine the quality of the packing. The figure on the right shows a typical test chromatogram of a column with the stationary phase NUCLEOSIL 00-5 C 8. Ordering information Designation Pack of Cat. No. Test mixture for reversed phase columns in acetonitrile ml 739 Test mixture for reversed phase HPLC columns (Cat. No. 739) Column: 50 x mm NUCLEOSIL 00-5 C 8 Sample volume: 0 µl Eluent: acetonitrile water (70:30) Flow rate: ml/min Pressure: 05 bar Temperature: C 3 Detection: UV, 5 nm (concentration in µg/ml eluent). Phenol (00). Naphthalene (80) 3. Anthracene () 8 min Individual test certificate enclosed with each NUCLEODUR and NUCLEOSIL column, respectively. 38

20 Octadecyl phases -(CH ) 7 CH 3 Analysis of nitroaromatic compounds (explosives) Column 50 x 3 mm NUCLEOSIL 0-3 C 8 ; eluents: A) methanol, B) water, gradient 35 55% A in min, then 0 min isocratic, finally 00% A, flow rate 0.35 ml/min; temperature 30 C, detection DAD, 30 nm; injection volume 0 µl ),6-Diamino--nitrotoluene ),-Diamino--nitrotoluene 3) Hexogene (Hexahydro-,3,5-trinitro-,3,5-triazine) ),3,5-Trinitrobenzene 5) -Methyl-3-nitroaniline 6) -Methyl-5-nitroaniline 7) -Methyl-3-nitroaniline mau ),3-Dinitrobenzene 9),,6-Trinitrotoluene 0)-Amino-,6-dinitrotoluene )-Amino-,6-dinitrotoluene ),6-Dinitrotoluene 3),-Dinitrotoluene )-Nitrotoluene 5)-Nitrotoluene 6)3-Nitrotoluene C 8 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) fat-soluble vitamins steroids prostaglandins PTH amino acids etc. Separation of triazines Column: 50 x mm NUCLEOSIL 50-5 C 8 ec Eluent: methanol acetonitrile water (0:0:0, v/v/v) Flow rate: ml/min, 30 C, 0 bar Detection: UV, 0 nm Peaks (injection volume 5 µl, 0 µg/ml each):. Simazine. Atrazine 3. Prometon. Ametryne 5. Propazine 3 6. Prometryne 5 7. Terbutryne min courtesy of Dr. Steinbach, Inst. f. Org. Chemistry, Univ. Marburg min Ordering information Analytical columns with NUCLEOSIL C 8 Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns NUCLEOSIL 50-5 C 8 ec Particle size 5 µm, pore size 50 Å; octadecyl phase, endcapped,.5% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of. ) 30 mm require the CC column holder 30 mm (Cat. No. 783). ) On request, Microbore columns are also available in lengths of 0, 60, 00 and 300 mm and with 0.5, 0.3, 0., 0.5, 0.75 and.5 mm ID. Guard columns for Microbore columns on request. 3) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7359). Custom-packed columns with other phases and other lengths are available on request 39

21 Ordering information Analytical columns with NUCLEOSIL C 8 Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns NUCLEOSIL 00-3 C 8 Particle size 3 µm, pore size 00 Å; octadecyl phase, endcapped, 5% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID NUCLEOSIL 00-5 C 8 Particle size 5 µm, pore size 00 Å; octadecyl phase, endcapped, 5% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID NUCLEOSIL 00-7 C 8 Particle size 7 µm, pore size 00 Å; octadecyl phase, endcapped, 5% C; eluent in column acetonitrile / water 3 mm ID mm ID mm ID mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of. ) 30 mm require the CC column holder 30 mm (Cat. No. 783). ) On request, Microbore columns are also available in lengths of 0, 60, 00 and 300 mm and with 0.5, 0.3, 0., 0.5, 0.75 and.5 mm ID. Guard columns for Microbore columns on request. 3) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7359). 0 Each column is individually tested and supplied with test chromatogram and test conditions

22 Ordering information Analytical columns with NUCLEOSIL C 8 Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns NUCLEOSIL 00-0 C 8 Particle size 0 µm, pore size 00 Å; octadecyl phase, endcapped, 5% C; eluent in column acetonitrile / water 3 mm ID mm ID mm ID mm ID mm ID NUCLEOSIL 0-3 C 8 Particle size 3 µm, pore size 0 Å; octadecyl phase, endcapped, % C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID NUCLEOSIL 0-5 C 8 Particle size 5 µm, pore size 0 Å; octadecyl phase, endcapped, % C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of. ) 30 mm require the CC column holder 30 mm (Cat. No. 783). ) On request, Microbore columns are also available in lengths of 0, 60, 00 and 300 mm and with 0.5, 0.3, 0., 0.5, 0.75 and.5 mm ID. Guard columns for Microbore columns on request. 3) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7359). Custom-packed columns with other phases and other lengths are available on request

23 Ordering information Analytical columns with NUCLEOSIL C 8 Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns NUCLEOSIL 0-7 C 8 Particle size 7 µm, pore size 0 Å; octadecyl phase, endcapped, % C; eluent in column acetonitrile / water mm ID mm ID NUCLEOSIL 0-0 C 8 Particle size 0 µm, pore size 0 Å; octadecyl phase, endcapped, % C; eluent in column acetonitrile / water mm ID mm ID NUCLEOSIL C 8 Particle size 5 µm, pore size 300 Å; octadecyl phase, endcapped, 6.5% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID mm ID NUCLEOSIL C 8 Particle size 7 µm, pore size 500 Å; octadecyl phase, endcapped, % C; eluent in column acetonitrile / water mm ID mm ID NUCLEOSIL C 8 Particle size 7 µm, pore size 000 Å; octadecyl phase, endcapped, ~% C; eluent in column acetonitrile / water mm ID mm ID NUCLEOSIL C 8 Particle size 7 µm, pore size 000 Å; octadecyl phase, endcapped, <% C; eluent in column acetonitrile / water mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of. ) 30 mm require the CC column holder 30 mm (Cat. No. 783). ) On request, Microbore columns are also available in lengths of 0, 60, 00 and 300 mm and with 0.5, 0.3, 0., 0.5, 0.75 and.5 mm ID. Guard columns for Microbore columns on request. 3) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7359). Each column is individually tested and supplied with test chromatogram and test conditions

24 Wide pore silica packings Silica as HPLC packing material has several advantages: pore size and surface are defined it is mechanically stable numerous methods for surface modification are available Many biologically interesting molecules can not be separated using conventional narrow pore silicas with pore sizes of about 00 Å. This is why we offer a complete line of wide pore packings with pore sizes of 300, 500, 000 and 000 Å. These materials can also be used for size exclusion chromatography (SEC). Octyl phases -(CH ) 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 Anthranoids in senna based drugs L. Kabelitz, K. Reif, Dtsch. Apotheker Ztg. 3 (99) 37 0 Column: 50 x mm NUCLEOSIL 00-5 C 8 Eluents: A) 0.0 M KH PO, adjusted to ph with H 3 PO (85%), B) acetonitrile Gradient: flow rate ml/min: 0 min % B, in 5 min to 0% B, 0 min at 0% B, flow rate 0.8 ml/min: in min to % B, in 9 min to 80% B, flow rate ml/min: in min to % B, then 3 min at % B Photodiode array: dianthrones at 70 nm, anthraquinones at 35 nm Detection:. Aloe emodin-8-glucoside. Rhein-8-glucoside 3: Sennoside D : Sennoside D 5: Sennoside B 6: Sennoside A 7: Sennoside C 8: Sennoside A 9: Sennidin-B-monoglucoside 0: Sennidin-A-monoglucoside : Aloe emodin : Rhein 3: Sennidin A : Emodin 5: Sennidin B min Ordering information Analytical columns with NUCLEOSIL C 8 Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns NUCLEOSIL 50-5 C 8 ec Particle size 5 µm, pore size 50 Å; octyl phase, endcapped, 9% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of. ) 30 mm require the CC column holder 30 mm (Cat. No. 783). ) On request, Microbore columns are also available in lengths of 0, 60, 00 and 300 mm and with 0.5, 0.3, 0., 0.5, 0.75 and.5 mm ID. Guard columns for Microbore columns on request. 3) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7359). Custom-packed columns with other phases and other lengths are available on request 3

25 Ordering information Analytical columns with NUCLEOSIL C 8 Length 30 mm ) 70 mm 00 mm 5 mm 50 mm 50 mm Guard columns mm ID mm ID NUCLEOSIL 00-5 C 8 ec Particle size 5 µm, pore size 00 Å; octyl phase, endcapped, 9% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID NUCLEOSIL 00-5 C 8 Particle size 5 µm, pore size 00 Å; octyl phase, not endcapped, 8.5% C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID mm ID mm ID mm ID NUCLEOSIL 00-7 C 8 Particle size 7 µm, pore size 00 Å; octyl phase, not endcapped, 8.5% C; eluent in column acetonitrile / water mm ID mm ID NUCLEOSIL 00-0 C 8 Particle size 0 µm, pore size 00 Å; octyl phase, not endcapped, 8.5% C; eluent in column acetonitrile / water mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of 3, all other columns in packs of. ) 30 mm require the CC column holder 30 mm (Cat. No. 783). ) On request, Microbore columns are also available in lengths of 0, 60, 00 and 300 mm and with 0.5, 0.3, 0., 0.5, 0.75 and.5 mm ID. Guard columns for Microbore columns on request. 3) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 7359). Each column is individually tested and supplied with test chromatogram and test conditions