Columns for HPLC. Columns for HPLC

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1 Analytical columns packed with NUCLEDUR NUCLEDUR reversed phase packings In reversed phase 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. NUCLEDUR silicas are synthesized in a unique and carefully controlled manufacturing process which provides silica particles, which are totally spherical. The important advantages of NUCLEDUR are: ideal particle shape and surface symmetry high purity minimized metal residues high pressure stability For a description of the properties of the NUCLEDUR base silica please see page. NUCLEDUR Sphinx RP the bifunctional RP phase distinct selectivity based on bifunctional surface coverage widens the scope in method development high density of covalently bonded silanes guarantees sharp peaks without tailing ph stabilility can be used from ph 0 low bleeding characteristics in LC/MS applications high reproducibility tight QC procedures provide consistant quality (CH ) hydrophobic interaction H π π (CH ) (CH ) H NEW NUCLEDUR Sphinx RP, µm 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. NUCLEDUR Sphinx RP is particularly suited for the separation of molecules containing aromatic and multiple bonds. For the separation of polar compounds NUCLEDUR Sphinx RP can be especially recommended and can also outperform many customary C8 phases. In addition, exhaustive endcapping steps minimize unwanted surface silanol activity and guarantee excellent peak shapes even for strong basic analytes. Different from standard phenyl phases, NUCLEDUR Sphinx RP is far more stable towards hydrolysis and is also suggested for LC/MS applications. Due to the additional intermolecular interactions NUCLE- DUR Sphinx RP is an interesting replenishment to the high density bonded phases NUCLEDUR C 8 /C 8 Gravity and the polar endcapped NUCLEDUR C 8 Pyramid.

2 Analytical columns packed with NUCLEDUR Columns: 0 x. mm Eluent: water methanol (0:0, v/v) Flow rate: ml/min Temperature: 0 C Inj. volume: µl Detection: 70 nm Separation of flavonoids on different NUCLEDUR phases H H H R H R H R H H. Catechin. Rutin R = R = H, R = -rutinose. Fisetin R = R = H, R = H. Quercetin R = R = R = H. Kaempferol R = H, R = R = H. Isorhamnetin R = CH, R = R = H NUCLEDUR C 8 Gravity, µm NUCLEDUR C 8 Gravity, µm NUCLEDUR Sphinx RP, µm 0,0,,0 7, 0,0 min rdering information Analytical columns with NUCLEDUR Sphinx RP Length 0 mm 70 mm 00 mm mm 0 mm 0 mm Guard columns NUCLEDUR Sphinx RP, µm Particle size µm, pore size 0 Å; special bifunctional RP phase, % C; eluent in column acetonitrile / water mm ID mm ID mm ID , mm ID Microbore columns ) mm ID EC columns ) mm ID mm ID mm ID , mm ID ChromCart guard column cartridges (8 mm) in packs of, all other columns in packs of. ) n request, Microbore columns are also available in lengths of 0, 0, 00 and 00 mm and with 0., 0., 0., 0., 0.7 and. mm ID. Guard columns for Microbore columns on request. ) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 79). 7

3 Analytical columns packed with NUCLEDUR NUCLEDUR C 8 Gravity and C 8 Gravity high-performance silicas for RP-HPLC for method development for HPLC at ph extremes for overall sophisticated analytical separations Base deactivation NUCLEDUR C 8 Gravity and C 8 Gravity are based on the new ultra-pure NUCLEDUR silica. A unique derivatization process generates a homogeneous surface with a high density of bonded silanes (carbon content ~ 8% for the C 8 phase, ~ % for the C 8 phase). The following thorough end-capping suppresses any unwanted polar interactions between the silica surface and the sample, which makes Gravity phases 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 C8 phases under isocratic conditions. Enhanced ph stability ne major disadvantage of using silica stationary phases is the limited stability at strong 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. Comparison of different base deactivated phases Columns: 0 x mm Eluent: methanol/0 mm KH P, ph 7.0 (7 :, v/v) Flow rate:.0 ml/min; temperature: 0 C. Dibutyl phthalat. Acenaphthene. Amitriptyline The special surface bonding technology and the low content of trace elements of NUCLEDUR Gravity allow for use at an expanded ph range from ph to. Surface silanols at different ph values H H H H H H + NH Me H + H C MeHN HC H + H + H H + H H 0 0 min Phase S (C8, µm) Phase P (C8, µm) Phase L (C8, µm) and overlap Phase I (C8, µm) NUCLEDUR C 8 Gravity, µm 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 C8 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 is the rule at ph 9 0. For acidic analytes it is exactly in inverse proportion, maximum retention can be attained at low ph. 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. 8

4 Analytical columns packed with NUCLEDUR k Correlation between retention and ph for basic and acidic compounds HA BH + A 0 8 ph The figure below shows the excellent column stability of NUCLEDUR C 8 Gravity in acidic conditions. Retention times of all compounds in the column performance test remain consistent and virtually unchanged, even after the column is run with 000 ml eluent. Due to the extremely stable surface modification, no cleavage of -- bonding occurs, column deterioration is therefore successfully prevented. Stability of NUCLEDUR C 8 Gravity at ph. Column: x mm NUCLEDUR 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, inj.volume: µl, peaks:. pyridine,. toluene,. ethylbenzene B Influence of the ph value on selectivity Column: x mm NUCLEDUR C 8 Gravity, µm eluents: A) acetonitrile/0 mm A) NH HC, ph.0 (0:0, v/v) B) acetonitrile/0 mm NH HC, ph 0.0 (0:0, v/v) flow rate: ml/min temperature: 0 C detection: UV, 0 nm inj. volume: µl. Lidocaine CH 0 min B) 0 min Steroids. Benzamide. Ketoprofen H C H C NH N(C H ) NH Column: x mm NUCLEDUR 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. α-methyl-β-hydroxyprogesterone. α-methyl-7α-hydroxyprogesterone. α-methyl-7α-hydroxyprogesterone acetate H 80 after 000 ml eluent st injection 0 0 min Another example for the effect of ph on selectivity is the separation of the acid ketoprofen, the base lidocaine and of 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, whereas the formally neutral ketoprofen is eluted after about min. Contrary, at ph 0 a reversal of elution order is observed, with a notably longer retention time for the basic lidocaine. 0 0 min 9

5 Analytical columns packed with NUCLEDUR The figure demonstrates the stability of NUCLEDUR C 8 Gravity under alkaline conditions in comparison with commercially available modern RP 8 phases. Again, the ultrapure Gravity with its unique high density surface bonding technology withstands strongly alkaline mobile phase conditions. Even after 00 injections no loss of column efficiency, identified e.g. by peak broadening or decrease in retention times, is observed. Phase X 0 min NUCLEDUR C 8 Gravity Phase L 0 min Stability of NUCLEDUR C 8 Gravity under alkaline conditions compared with different C 8 phases first injection after 00 injections and 0 l eluent 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 Inj. volume:.0 µl. Theophylline. Caffeine Phase K 0 min Phase I 0 min 0 min As mentioned above, enhanced ph stability of the stationary phase can be helpful for improving selectivity in method development. The two chromatograms on the right show the separation of basic drugs under acid and basic conditions. 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. The ph stability of sillica 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 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. For a detailed discussion of the different retention characteristics of NUCLEDUR octyl phases compared to the octadecyl phases see page. Separation of basic alkaloids Column: x mm NUCLEDUR C 8 Gravity, µm Eluent A: acetonitrile Eluent B: 0 mm (NH ) HP, ph. / 0.0 Gradient: 0% A ( min) 7% A in 0 min Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl ph. 0 ph min 0 0 min. Lidocaine. Papaverine. Noscapine. Diphenhydramine 0

6 Analytical columns packed with NUCLEDUR rdering information Analytical columns with NUCLEDUR Gravity Length 0 mm ) 0 mm 70 mm 00 mm mm 0 mm 0 mm NUCLEDUR C 8 Gravity, µm Particle size µm, pore size 0 Å; octadecyl phase, endcapped, 8% C; eluent in column acetonitrile / water Guard columns mm ID mm ID mm ID mm ID Microbore columns mm ID EC columns ) mm ID mm ID mm ID mm ID NUCLEDUR C 8 Gravity, µm Particle size µm, pore size 0 Å; octadecyl phase, endcapped, 8 % C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns mm ID EC columns ) mm ID mm ID mm ID mm ID NUCLEDUR C 8 Gravity, µm Particle size µm, pore size 0 Å; octyl phase, endcapped, % C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID EC columns ) mm ID mm ID mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of, all other columns in packs of. ) 0 mm require CC column holder 0 mm (Cat. No. 78). ) n request, Microbore columns are also available in lengths of 0, 0, 00 and 00 mm and with 0., 0., 0., 0., 0.7 and. mm ID. Guard columns for Microbore columns on request. ) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 79).

7 Analytical columns packed with NUCLEDUR NUCLEDUR C 8 Pyramid RP-HPLC with highly aqueous mobile phases stable in 00% aqueous mobile phase systems interesting polar selectivity features excellent base deactivation 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 selectivity. 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. 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. NUCLESIL NAUTILUS 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 ) (also see page 0). Stability features of NUCLEDUR C 8 Pyramid NUCLEDUR 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 behaviour of tartaric, acetic and maleic acid under purely aqueous conditions on NUCLEDUR C 8 Pyramid in comparison with a conventionally bonded RP phase. It can be shown that the retention times for NUCLEDUR C 8 Pyramid remain nearly unchanged between initial injection and NUCLEDUR C 8 Pyramid initial injection conventional RP column restart after the flow has been stopped for hours, whilst the performance of the conventional RP column collapsed totally after the same period. Retention characteristics of NUCLEDUR C 8 Pyramid Based on the ultrapure NUCLEDUR silica ) (also see page ) the polar surface derivatisation exhibits retention characteristics, which differentiate the Pyramid from conventional C 8 stationary phases. The following chromatogram shows the improved retention behaviour of very polar compounds such as short chain organic acids, which are insufficiently retained on RP columns with predominantly hydrophobic surface properties. 0 t 0 Separation of very polar compounds Column: NUCLEDUR C 8 Pyramid, µm, x mm ID Eluent: 0.% H P Flow rate: ml/min Temperature: C Detection: UV, 0 nm (injection volume µl). Formic acid. Acetic acid The figure below shows the separation of various organic acids at acidic ph under 00% aqueous eluent conditions. t 0 min Separation of organic acids Column: NUCLEDUR C 8 Pyramid, µm, 0 x mm ID Eluent: 0.% H P Flow rate: 0.7 ml/min Temperature: C Detection: UV, 0 nm (injection volume µl). Tartaric acid. Malic acid. Lactic acid. Succinic acid 0 0 min 0 min pump stopped! restart after h 0 min 0 min 0 min both columns x mm ID, injection volume µl 0 mm KH P ph., 0.7 ml/min; C; UV, 0 nm. tartaric acid,. acetic acid, maleic acid In addition to the exceptional polar selectivity NUCLEDUR C 8 Pyramid also provides adequate hydrophobic retention, which can be illustrated in the selectivity test according to Engelhardt ) (see below). The capacity factors of the nonpolar, alkyl-substituted benzenes toluene and ethylbenzene do not go too far in comparison with standard C 8 phases. Base deactivation of NUCLEDUR C 8 Pyramid The perceptible increase in polarity has no impact on the retention behaviour of ionizable analytes. Even with the strongly basic compounds of the tricyclic antidepressant test mixture, no unwanted interactions or a so-called lack in base deactivation are observed.

8 Analytical columns packed with NUCLEDUR Selectivity test Column: NUCLEDUR C 8 Pyramid, µm, 0 x mm ID Eluent: MeH H (:, v/v) Flow rate: ml/min. Uracil Temperature: 0 C. Aniline Detection: UV, nm. Phenol Inj. volume: µl. Dimethylaniline. p-ethylaniline. Toluene 7. Ethylbenzene 7 Tricyclic antidepressants Column: NUCLEDUR C 8 Pyramid, µm, x mm ID Eluent: MeH 0 mm NH H P ph.9 (70:0, v/v) Flow rate: ml/min Temperature: 0 C Detection: UV, nm (injection volume µl). Protriptyline. Nortriptyline. Doxepin. Imipramine. Amitriptyline min References ) D. Rieger and H. Riering, Int. Laboratory Aug. 000, Vol. 0 (A), 0 0 min ) D. Rieger, J. Pfeiffer, LaborPraxis () (00) 0 ) H. Engelhardt, M. Arangio, T. Lobert, LCGC Int. 0 (), 80 8 (997) rdering information Analytical columns with NUCLEDUR C 8 Pyramid Length 0 mm 70 mm 00 mm mm 0 mm 0 mm Guard columns NUCLEDUR C 8 Pyramid, µm Particle size µm, pore size 0 Å; octadecyl phase with hydrophic endcapping, % C; eluent in column acetonitrile / water mm ID mm ID mm ID mm ID Microbore columns ) mm ID EC columns ) mm ID mm ID mm ID mm ID NUCLEDUR C 8 Pyramid, µm as above, however, particle size µm mm ID mm ID mm ID mm ID Microbore columns ) mm ID EC columns ) mm ID mm ID mm ID mm ID ChromCart guard column cartridges (8 mm) in packs of, all other columns in packs of. ) n request Microbore columns are also available in lengths of 0, 0, 00 and 00 mm and with 0., 0., 0., 0., 0.7 and. mm ID. Guard columns for Microbore columns on request. ) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 79).

9 Analytical columns packed with NUCLEDUR NUCLEDUR C 8 ec and C 8 ec for daily routine analyses 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. NUCLEDUR 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. NUCLEDUR C 8 ec is also an ideal tool for scale-up purposes. NUCLEDUR Gravity and NUCLEDUR ec also available as octyl phases! In addition to the program of NUCLEDUR C 8 phases MACHEREY-NAGEL introduces the corresponding octyl modified NUCLEDUR C 8 Gravity and C 8 ec columns to expand the reversed phase tool box effectively. The C 8 phases exhibit the same excellent chemical and mechanical stability features as the C 8 counterparts. 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). NUCLEDUR C 8 ec and NUCLEDUR C 8 Gravity (for ordering information see page ) are most suitable for the development of new methods but also for robust routine analysis. different selectivity patterns of NUCLEDUR C 8 ec and NUCLEDUR C 8 ec. The separation of various nonsteroidal anti-inflammatory drugs illustrates the differences in polarity between C 8 and C 8 and the resulting impact on efficiency. NUCLEDUR 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 NUCLEDUR 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. Anti-inflammatory drugs Column: 0 x mm NUCLEDUR 00- C 8 ec / C 8 ec Eluent: acetonitrile water, % acetic acid (8 :, v/v) Flow rate: ml/min Temperature: C + Detection: UV, 0 nm (injection volume 0 µl). Piroxicam. Suprofen. Ketoprofen. Carprofen. Fenoprofen. Diclofenac C min + 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. The following comparative study reveals the C min 0 rdering information Analytical columns with NUCLEDUR C 8 and C 8 Length 0 mm ) 0 mm 70 mm 00 mm mm 0 mm 0 mm NUCLEDUR 00- C 8 ec Particle size µm, pore size 0 Å; octadecyl phase, endcapped, 7.% C; eluent in column acetonitrile / water Guard columns mm ID mm ID mm ID mm ID Microbore columns mm ID

10 Analytical columns packed with NUCLEDUR rdering information Analytical columns with NUCLEDUR C 8 and C 8 Length 0 mm ) 0 mm 70 mm 00 mm mm 0 mm 0 mm EC columns ) mm ID mm ID mm ID mm ID NUCLEDUR 00- C 8 ec as above, however, particle size µm mm ID mm ID mm ID mm ID Microbore columns mm ID Guard columns EC columns ) mm ID mm ID mm ID mm ID NUCLEDUR 00- C 8 ec Particle size µm, pore size 0 Å; octyl phase, endcapped, 0. % C; eluent in column acetonitrile / water mm ID mm ID mm ID , mm ID EC columns ) mm ID mm ID mm ID , mm ID NUCLEDUR 00- C 8 ec as above, however, particle size µm mm ID mm ID mm ID mm ID EC columns ) mm ID mm ID mm ID mm ID mm and CC guard column cartridges (8 mm) in packs of, all other columns in packs of. ) 0 mm require CC column holder 0 mm (Cat. No. 78). ) n request, Microbore columns are also available in lengths of 0, 0, 00 and 00 mm and with 0., 0., 0., 0., 0.7 and. mm ID. Guard columns for Microbore columns on request. ) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 79).

11 Analytical columns packed with NUCLEDUR NUCLEDUR CN cyano modified high-purity silica Widen your scope in selectivity! multi-mode column (RP and NP) different retention characteristics in comparison to C 8 / C 8 stable against hydrolysis at low ph high reproducibility from lot to lot 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 characterised by a hydrophobic layer of monomeric or polymeric bonded alkylsilanes. Reproducibility of NUCLEDUR CN-RP Column: NUCLEDUR 00- CN-RP, 0 x mm ID Eluent: acetonitrile water (0:0, v/v) Flow rate: ml/min Temperature: 0 C Detection: UV, nm Inj. volume: µl. Benzamide. Dimethyl phthalate. Phenetol. o-xylene. Biphenyl lot D lot C ne 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 NUCLEDUR 00 CN-RP phases have cyanopropyl groups on the surface able to generate a clearly recognizable different retention behaviour compared to purely alkyl-functionalized surface modifications. Comparison of NUCLEDUR C 8 ec and CN-RP for a separation of coldmedicine ingredients Columns: A) NUCLEDUR 00- CN-RP, 0 x mm ID B) NUCLEDUR 00- C 8 ec, 0 x mm ID Eluent: acetonitrile 00 mm sodium citrate ph. (:8, v/v) Flow rate: ml/min Temperature: C Detection: UV, 70 nm Inj. volume: 0 µl. Maleic acid. Norephedrine. Ephedrine. Acetaminophen. Chlorpheniramine. Brompheniramine lot B lot A 0 8 min 0 min The polarity of the NUCLEDUR 00 CN-RP phases can be classified as intermediate based on multiple retention mechanisms such as dipole-dipole, π-π, and also hydrophobic interactions ). 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) ). Short-chain bonded phases are sometimes suspected of revealing shortcomings in stability towards hydrolysis at low ph ). The chromatogram on the right shows that even after 00 sample injections and four weeks storage at ph (curve ), neither a considerable shift in retention, nor a visible change in peak symmetry could be noticed (curve = new column). 8 Due to the exceptional polarity features the cyano phase can also be run in the normal phase mode. NUCLEDUR 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 the chromatogram below. Moreover the high coverage combined with a thorough endcapping makes NUCLEDUR 00 CN-RP suitable for the separation of ionizable compounds such as basic drugs. References ) C. S. Young and R. J. Weigand, LCGC 0 (), 7 (00) ) V. R. Meyer, Practical High Performance Liquid Chromatography (John Wiley & Sons, New York, rd. ed., 999) ) J. J. Kirkland, LCGC (), 8 00 (99).

12 Analytical columns packed with NUCLEDUR Separation of steroids in normal phase and reversed phase mode Reversed phase mode Normal phase mode Column: NUCLEDUR 00- CN-RP, 0 x mm ID Column: NUCLEDUR 00- CN, 0 x mm ID Eluent: acetonitrile water (:7, v/v) Eluent: n-heptane -propanol (90:0, v/v) Flow rate: ml/min other conditions as for reversed phase mode Temperature: C Detection: UV, nm Peaks (injection volume 0 µl): +7. Methyltestosterone. Testosterone. Norgestrel 7. Medrysone. Cortisone. Hydrocortisone 7. Prednisolone min min rdering information Analytical columns with NUCLEDUR CN Length 0 mm 0 mm mm 0 mm 0 mm Guard columns NUCLEDUR 00- CN-RP Particle size µm, pore size 0 Å; cyano phase (nitrile), 7% C; eluent in column acetonitrile / water mm ID 70.0 mm ID mm ID EC columns ) mm ID mm ID mm ID mm ID NUCLEDUR 00- CN-RP as above, however, particle size µm mm ID mm ID EC columns ) mm ID mm ID NUCLEDUR 00- CN Particle size µm, pore size 0 Å; cyano phase (nitrile), 7% C; eluent in column n-heptane mm ID mm ID EC columns ) mm ID mm ID ChromCart guard column cartridges (8 mm) in packs of, all other columns in packs of. ) As guard columns for EC columns use ChromCart guard column cartridges with guard column adaptor EC (Cat. No. 79). 7

13 Analytical columns packed with NUCLEDUR Applications Selectivity test Column: 0 x mm NUCLEDUR C 8 Gravity, µm Eluent: methanol / 0 mm KH P, ph 7.0 (7:, v/v) Flow rate:.0 ml/min Detection: UV, nm Temperature: 0 C Inj. volume: µl. Uracil.,7-Dihydroxynaphthalene.,-Dihydroxynaphthalene. Lidocaine. Toluene. Naphthalene 7. Ethylbenzene 8. Dibutyl phthalate 9. Acenaphthene 0. Amitriptyline Separation of anti-inflammatory drugs Column: 0 x mm NUCLEDUR C 8 Gravity, µm Eluent: acetonitrile / 0 mm KH P, ph. (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Inj. volume: µl. Acetylsalicylic acid. Sulindac. Piroxicam. Suprofen. Tolmetin. Naproxen 7. Diflunisal 8. Flurbiprofen 9. Indomethazin 7 0. Ibuprofen min 0 0 min Separation of antidepressants Column: x mm NUCLEDUR C 8 Gravity, µm Eluent A: methanol / acetonitrile (0:0, v/v) Eluent B: 0 mm KH P, ph 7.0 Gradient: 0% A % A in min, then 0 min % A Flow rate:. ml/min Temperature: 0 C Detection: UV, 0 nm. Nordoxepin Inj. volume: µl. Protriptyline. Maprotiline. Nortriptyline. Norclomipramine. Doxepin 7. Imipramine 8. Amitriptyline 9. Clomipramine 0. Trimipramine Separation of a coldmedicine Column: x mm NUCLEDUR C 8 Gravity, µm Eluent A: 0 mm KH P + mm Na pentansulfate, ph. Eluent B: methanol Gradient: % A % A in min Flow rate:.0 ml/min Temperature: 0 C. Maleic acid Detection: UV, nm. Acetaminophen Inj. volume: µl. Pseudoephedrine. Benzoic acid. Chlorpheniramine. Dextromethorphan min 0 min 780 8

14 Analytical columns packed with NUCLEDUR Applications Separation of antibacterials Column: 0 x mm NUCLEDUR 00- C 8 ec Eluent: acetonitrile water (0:0, v/v) 0.0% TFA Flow rate:,0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl. floxacin. Ciprofloxacin. Cinoxacin. Penicillin G. Penicillin V. Cloxacillin 0 8 min 7870 Separation of pesticides Column: 0 x mm NUCLEDUR 00- C 8 ec Eluents: A) water (% acetic acid), B) acetonitrile Gradient: 0 % B in 0 min, 0% B in 0 min, min at 0% B, 0 0 % B in 0 min 0 0% B in 0 min, 0 min at 0% B Flow rate ml/min, temperature C, detection UV, 0 nm. Desisopropylatrazine. Metamitron. Desethylatrazine. Bromoxynil. mazine. Cyanazine 7. Metabenzthiazuron 8. Atrazine 9. Monolinuron Isoproturon 7. Diuron 8. Metobromuron. Metazachlor 0. Sebutylazine 9. Terbuthylazine. Linuron 7. Chloroxuron 8. Metolachlor min 80 Steroids Column: x mm NUCLEDUR 00- C 8 ec Cat. No Eluents: A) water, B) methanol Gradient: min at 0% B, 0 % B in 0 min, min at % B, 0% B in min, min at 0% B Flow rate: ml/min Temperature: 0 C Detection: UV, 0 nm 8 Peaks (each env. 0 0 µg/ml, inj. volume 0 µl):. Estriol. Prednisolone. Cortisone. Testosterone 0. α-methyl-β-hydroxyprogesterone 9. α-methyl-7α-hydroxyprogesterone 7. α-methyl-7α-hydroxyprogesterone acetate 7 8. Estradiol 9. Estrone 0. Progesterone 80 Analgesic and antiinflammatory drugs Column: 0 x mm NUCLEDUR 00- C 8 ec Cat. No Eluent: acetonitrile % acetic acid (8:, v/v) Flow rate: ml/min Temperature: C Detection: UV, 0 nm Inj. 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 9 ( µg/ml) 8. Flurbiprofen ( µg/ml) Indomethazin ( µg/ml) 0 0. Ibuprofen ( µg/ml) min 0 0 min 9