High performance HPLC packings and Applications

Transcription

1 High performance HPLC packings and Applications MACHEREY-NAGEL MN

2 NUCLEODUR Reversed Phase Packings In RP liquid chromatography the efficiency of the packing is strongly affected by the quality of the base silica itself. Shortcomings in the surface geometry of the particles or metal contaminants are the main reasons for inadequate coverage with the covalently bonded alkylsilanes in the subsequent derivatization steps. It is well known, that poor surface coverage and, in consequence, high activity of residual free silanols often results in peak tailing or adsorption, particularly with basic compounds. NUCLEODUR Particle Shape and Surface Symmetry Billiard Balls in HPLC NUCLEODUR silicas are synthesized in a unique and carefully controlled manufacturing process which provides silica particles, which are totally spherical. The pictures show the uniform particle size distribution and the outstanding smoothness of the NUCLEODUR surface is emphasized. Fig. : High performance totally spherical NUCLEODUR silica Fig. : Surface of a NUCLEODUR particle (magnified) NUCLEODUR Purity Intolerant Towards Metals As already mentioned above, a highly pure silica is required for achieving symmetric peak shapes and maximum resolution. Inclusions of e.g. iron or alkaline earth metal ions on the silica surface are largely responsible for the unwanted interactions with ionizable analytes, e.g. amines or phenolic compounds. NUCLEODUR is virtually free of metal impurities and low acidic surface silanols. Elemental analysis data of NUCLEODUR µm measured by AAS are listed beside. Aluminium < ppm Iron < ppm Sodium < ppm Calcium < 0 ppm Titanium < ppm Zirconium < ppm Arsenic < 0. ppm Mercury < 0.0 ppm Elementary analysis (metal ions) of NUCLEODUR 00-

3 NUCLEODUR Reversed Phase Packings NUCLEODUR Pressure Stability Hard as a Rock The totally spherical and 00% synthetic silica gel exhibits an outstanding mechanical stability, even at high pressures and elevated eluent flow rates. In addition, after several cycles of repeated packing, no significant drop in pressure can be observed. The latter is of prime importance for preparative and process-scale applications min Column: x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile water (80:0, v/v) Temperature: C Detection: UV, nm 0 inj. 00 inj. 0 inj. 00 inj. 0 inj. start. Phenol. Naphthalene. Anthracene back pressure [bar] Packing # Fig. : Column stability of NUCLEODUR 00- C 8 ec Fig. : Repeated packing NUCLEODUR C 8 ec the Workhorse for Daily Routine Analysis and Up-Scaling for Preparative HPLC The efficiency of a separation is controlled by particle size and selectivity of the stationary phase. The exceptional surface coverage of monomeric bonded alkylsilanes, combined with an exhaustive endcapping, results in a surface with lowest silanol activity. This allows the tailing-free elution of polar compounds such as basic drugs. NUCLEODUR C 8 ec is available in 9 different particle sizes (,, 7, 0,,, 0, 0 and 0 µm) which cover the whole range from high speed analytical HPLC up to medium and low pressure prep LC. NUCLEODUR C 8 ec is also an ideal tool for scale-up purposes. Surface area (BET) 0 m /g Pore size 0 Å Pore volume 0.9 ml/g Carbon load 7. % Physical data of NUCLEODUR C 8 ec

4 NUCLEODUR C 8 Gravity The column for method development HPLC at ph extremes overall sophisticated analytical separations Base deactivation NUCLEODUR C 8 Gravity is based on the ultrapure NUCLEODUR silica, which is described above. An unique derivatization process generates a homogeneous surface with a high density of bonded silanes (carbon content ~ 8%). The following thorough endcapping suppresses any unwanted polar interactions between the silica surface and the sample, which makes Gravity particularly suitable for the separation of basic and other ionizable analytes. Figure shows a comparison study, where the strongly basic amitriptyline is eluted on various highly base deactivated C 8 phases under isocratic conditions. More examples to emphasize the outstanding suitability of the NUCLEODUR C 8 Gravity phase are shown below (figures and 7) and in the application section of this brochure. Phase S (C 8, µm) Phase P (C 8, µm) Phase L (C 8, µm) and overlap Phase I (C 8, µm) NUCLEODUR C 8 Gravity, µm 0 0 min Columns: 0 x mm Eluent: methanol 0 mm KH PO, ph 7.0 (7 :, v/v). Dibutyl phthalate Flow rate:.0 ml/min. Acenaphthene Temperature: 0 C. Amitriptyline Detection: UV, nm Fig. : Comparison of different base deactivated phases 080. Pyridine. Phenol min Column: 0 x mm NUCLEODUR C 8 Gravity, µm Eluent: methanol 0 mm KH PO, ph 7.0 (7 :, v/v) Flow rate:.0 ml/min Detection: UV, nm Temperature: 0 C Inj. volume: µl 0 0 min Column: 0 x mm NUCLEODUR C 8 Gravity, µm Eluent: methanol water (0 : 70, v/v) Flow rate:.0 ml/min Detection: UV, nm Temperature: 0 C Inj. volume: µl Fig. : Separation of pyridine and phenol. Uracil.,7-Dihydroxynaphthalene.,-Dihydroxynaphthalene. Lidocaine. Toluene Fig. 7: Selectivity test. Naphthalene 7. Ethylbenzene 8. Dibutyl phthalate 9. Acenaphthene 0. Amitriptyline

5 NUCLEODUR C 8 Gravity Enhanced ph Stability One major disadvantage of using silica stationary phases is the limited stability at strongly acidic or basic ph ranges. Cleavage of the siloxane bonding by hydrolysis, or dissolution of the silica will rapidly lead to a considerable loss in column performance. Therefore conventional RP phases are usually not recommended to be run with mobile phases at ph > 8 or ph < for extended periods of time. The special surface bonding technology and the low concentration of trace elements of NUCLEODUR C 8 Gravity allow for use at an expanded ph range from ph to. When is enhanced ph stability beneficial? The option to work at an expanded ph range is often required in method development. Many nitrogen containing compounds like basic drugs are protonated at acidic or neutral ph and exhibit poor retention on a standard C 8 phase. The retention behavior can be improved by working at a higher ph, where the analyte is no longer protonated, but formally neutrally charged, as a rule between ph 9 0. For acidic analytes it is exactly in inverse proportion, maximum retention can be attained at low ph. Figure 8 shows the extent of protonation of surface silanols and of two exemplary analytes at acidic and alkaline ph. The graph in figure 9 explains the general correlation between retention and ph. k HA BH + A 0 8 ph Fig. 9: Correlation between retention and ph for basic and acidic compounds In figure 0 the excellent column stability of NUCLEODUR C 8 Gravity in acidic conditions is shown. The retention time of all three compounds in the column performance test remains consistent and virtually unchanged, even after the column is run with 000 ml eluent. Due to the extremely stable surface modification, no cleavage of the Si-O-Si bonding occurs, column deterioration is therefore successfully prevented. B 080 after 000 ml eluent st injection 0 0 min Column: x mm NUCLEODUR C 8 Gravity, µm eluent: acetonitrile % TFA in water (0:0, v/v), ph. flow rate:.0 ml/min, temperature: 0 C, detection: UV, 0 nm, inj.volume: µl. pyridine,. toluene,. ethylbenzene Fig. 8: Surface silanols at different ph values Fig. 0: Stability of NUCLEODUR C 8 Gravity at ph.

6 NUCLEODUR C 8 Gravity Enhanced ph Stability Fig. : Stability of NUCLEODUR C 8 Gravity under alkaline conditions compared with different C 8 phases first injection after 00 injections Phase X 0 min NUCLEODUR C 8 Gravity Phase L 0 min 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 0 min Phase K Phase I 0 min 0 min 080 Fig. demonstrates the stability of NUCLEODUR 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 strong alkaline mobile phase conditions. Even after 00 injections no loss of column efficiency, identified e.g. by peak broadening or decrease in retention times, could be observed. As it was previously mentioned, ph stability of the stationary phase can be helpful for improving selectivity in method development. Figure shows the separation of basic drugs under acidic and basic conditions.

7 NUCLEODUR C 8 Gravity Enhanced ph Stability ph. 800 The ph stability of silica under alkaline conditions is mainly a kinetic effect and based on the velocity of the dissolution of the silica support. It is worth mentioning, that this phenomenon also depends on type and concentration of buffers, as well as on the temperature. It is well known, that the use of phosphate buffers, particularly at elevated temperatures, can reduce column lifetime even at moderate ph. If possible, phosphate buffers should be replaced by less harmful alternatives. 0 0 min A) 080 ph min ph.0 Column: x mm NUCLEODUR C 8 Gravity, µm Eluent A: acetonitrile Eluent B: 0 mm (NH ) HPO, ph. / 0.0 Gradient: 0% A ( min) 7% A in 0 min Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl. Lidocaine. Papaverine. Noscapine. Diphenhydramine Fig. : Separation of basic alkaloids 0 min B) ph 0.0 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. A further example how selectivity can be controlled by the ph value is demonstrated in figure. The sample mixture consists of an acid (ketoprofen), a base (lidocaine) and benzamide. Under acidic conditions the protonated lidocaine is eluted very fast due to lack of sufficiently strong hydrophobic interactions between analyte and C 8 chains, in contrary to the formally neutral ketoprofen, which is eluted after about minutes. However at ph 0 a reversal of the elution order, with a visibly longer retention time for the basic lidocaine, can be achieved. Column: x mm NUCLEODUR C 8 Gravity, µm Eluents: A) acetonitrile 0 mm ammonium formate, ph.0 (0:0, v/v) B) acetonitrile 0 mm ammonium bicarbonate, ph 0.0 (0:0, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, 0 nm Inj. volume: µl. Lidocaine. Benzamide. Ketoprofen H C 0 min CH NH N(C H ) O O NH H C Fig. : Influence of the ph value on selectivity O O OH 7

8 NUCLEODUR C 8 Pyramid RP-HPLC with Highly Aqueous Mobile Phases stable in 00% aqueous mobile phase systems interesting polar selectivity features excellent base deactivation ph stability 8 The efforts to neutralize unwanted activity of unreacted surface silanols often results in well basedeactivated 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 caused by the mobile phase expelled from the pores due to the fact, that hydrophobic RP phases are incompletely wetted with the mobile phase ). 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. NUCLEOSIL 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 ). Stability features NUCLEODUR C 8 Pyramid is a silica phase with hydrophilic endcapping, designed especially for use in eluent systems of up to 00% water. In figure we studied the retention behaviour of tartaric, acetic and maleic acid under purely aqueous conditions on NUCLEODUR C 8 Pyramid in comparison with a conventionally bonded RP phase. It can be shown that the retention times for NUCLEODUR C 8 Pyramid remain nearly unchanged between initial injection and restart after the flow has been stopped for hours (see figure ), whilst the performance of the conventional RP column collapsed totally after the same period. ) U. D. Neue et al., Chromatographia (00) 9 77 ) D. Rieger, H. Riering, Int. Laboratory Aug. 000, Vol. 0 (A), ) D. Rieger, J. Pfeiffer, LaborPraxis () (00) 0 8

9 NUCLEODUR C 8 Pyramid RP-HPLC with Highly Aqueous Mobile Phases Retention characteristics Based on the ultrapure NUCLEODUR silica ) the polar surface derivatization exhibits retention characteristics, which differentiate the Pyramid from conventional C 8 stationary phases. Figure shows the improved retention behavior of very polar compounds such as short chain organic acids, which are insufficiently retained on RP columns with predominantly hydrophobic surface properties. Figure shows the separation of various organic acids at acidic ph under 00% aqueous eluent conditions. 970 Column: NUCLEODUR C 8 Pyramid, µm, x mm Eluent: 0.% H PO Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm (injection volume µl). Formic acid. Acetic acid 0 min Fig. : Separation of very polar compounds t min Column: NUCLEODUR C 8 Pyramid, µm, 0 x mm Eluent: 0.% H PO Flow rate: 0.7 ml/min Temperature: C Detection: UV, 0 nm Fig. : Separation of organic acids (injection volume µl). Tartaric acid. Malic acid. Lactic acid. Succinic acid 980 NUCLEODUR C 8 Pyramid conventional RP column 0870 initial injection 0 min 0 min pump stopped! restart after h 0 min 0 min Both columns: x mm Inj. volume: µl Eluent: 0 mm KH PO ph. Flow rate: 0.7 ml/min Temperature: C Detection: UV, 0 nm. Tartaric acid. Acetic acid. Maleic acid Fig. : Stability test comparison 9

10 NUCLEODUR C 8 Pyramid RP-HPLC with Highly Aqueous Mobile Phases In addition to the exceptional polar selectivity NUCLEODUR C 8 Pyramid also provides adequate hydrophobic retention, which can be illustrated in the selectivity test according to Engelhardt ) (see figure 7). The capacity factors of the non-polar, alkyl-substituted benzenes toluene and ethylbenzene do not go too far in comparison with standard C 8 phases. Base deactivation The perceptible increase in polarity has no impact on the retention behavior of ionizable analytes. Even with the strongly basic compounds of the tricyclic antidepressant drug test mixture, no unwanted interactions or a so-called lack in base deactivation are observed (see figure 8).. Uracil. Aniline. Phenol. Dimethylaniline. p-ethylaniline. Toluene 7. Ethylbenzene. Protriptyline. Nortriptyline. Doxepin. Imipramine. Amitriptyline min Column: 0 x mm NUCLEODUR C 8 Pyramid, µm Eluent: methanol water (:, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm Inj. volume: µl Fig. 7: Selectivity test min Column: x mm NUCLEODUR C 8 Pyramid, µm Eluent: MeOH 0 mm NH H PO ph.9 (70:0, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm Inj. volume: µl Fig. 8: Tricyclic antidepressants 900 NUCLEODUR C 8 Pyramid NUCLEODUR C 8 Gravity NUCLEODUR C 8 Gravity t R hydrophobic compounds t R hydrophobic compounds t R polar compounds t R polar compounds min min 0 0 min Columns: 0 x mm, µm particles Eluent: methanol mm NH H PO, ph 7 (:, v/v) Flow rate: 0.8 ml/min Temperature: 0 C Detection: UV, nm Inj. volume: µl. Chlorpheniramine. Dimethyl phthalate. Benzamide. Ethyl benzoate. Benzophenone. Lidocaine 7. Naphthalene 8. Biphenyl 9. Acenaphthene Fig. 9: Retention behavior of polar and non-polar compounds on different NUCLEODUR RP columns 0

11 NUCLEODUR C 8 Phases NUCLEODUR C 8 ec and NUCLEODUR C 8 Gravity In addition to the program of NUCLEODUR C 8 phases MACHEREY-NAGEL offers the corresponding octyl modified NUCLEODUR C 8 ec and NUCLEODUR C 8 Gravity columns to expand the reversed phase tool box effectively. Based on the same totally spherical and highly pure silica the C 8 phases exhibit the same excellent chemical and mechanical stability features as the C 8 counterparts. Indeed NUCLEODUR C 8 Gravity can also be run at ph extremes (ph ) by choosing appropriate elution parameters. Due to the shorter chain and less hydrophobic properties of the stationary phase the retention of non-polar compounds is decreased, and in consequence a reduction in time of analysis can be achieved. Moreover a stronger polar selectivity, particularly with the separation of ionizable analytes is frequently observed (as distinct from the C 8 phases). NUCLEODUR C 8 ec and NUCLEODUR C 8 Gravity are most suitable for the development of new methods but also for robust routine analysis. C 8 or C 8 the best of both worlds Chromatographers now might wonder about the differences between C 8 and C 8 phases and the preferred range of application.. Piroxicam. Suprofen. Ketoprofen. Carprofen. Fenoprofen. Diclofenac C 8 C min 0 Column: 0 x mm NUCLEODUR 00- C 8 ec / C 8 ec Eluent: acetonitrile water, % acetic acid (8 :, v/v) Flow rate:.0 ml/min Detection: UV, 0 nm Temperature: C Inj. volume: 0 µl Fig. 0: Anti-inflammatory drugs NUCLEODUR 00- C 8 ec NUCLEODUR 00- C 8 ec Columns: 0 x mm Flow rate:.0 ml/min Temperature: C Detection: UV, 7 nm Inj. volume: 0 µl Fig. : Phenols. Resorcinol. Pyrocatechol. Phenol. -Methoxyphenol. -Methoxyphenol. -Ethoxyphenol 7. Veratrol 8. Biphenyl--ol 9. Phenetole min Eluents: A) water, B) methanol Gradient for C 8 : min 0% B, then to 0% B in min Gradient for C 8 : min % B, then to % B in min 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 two comparative studies reveal some different selectivity patterns of NUCLEODUR C 8 ec and NUCLEODUR C 8 ec. In figure baseline separation for -ethoxyphenol and dimethoxybenzene (veratrol) and in addition a reversal of the elution order of phenol and -methoxyphenol can be shown on the octyl phase. The separation of various nonsteroidal anti-inflammatory drugs (figure 0) illustrates the differences in polarity between C 8 and C 8 and the resulting impact on efficiency. NUCLEODUR C 8 ec exhibits enhanced selectivity and excellent resolution for the polar compounds piroxicam and suprofen which co-elute on the C 8 column. However due to the longer alkyl chain NUCLEODUR C 8 ec shows a distinct hydrophobic selectivity that leads to baseline separation of the more non-polar analytes carprofen and fenoprofen with superior peak shapes

12 NUCLEODUR 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 NUCLEODUR Sphinx RP is characterized by exceptional selectivity features generated by a wellbalanced 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. NU- CLEODUR Sphinx RP is particularly suited for the separation of molecules containing aromatic and multiple bonds. For the separation of polar compounds NUCLEODUR Sphinx RP can be especially recommended and can also outperform many customary C 8 phases. In addition, exhaustive endcapping steps minimize unwanted surface silanol activity and guarantee excellent peak shapes even for strong basic analytes. Different from standard phenyl phases, NUCLEODUR Sphinx RP is far more stable towards hydrolysis and is also suggested for LC/MS applications. after 00 injections (column run with l eluent). Theophyllline. Caffeine st injection 0 min Column: 0 x, mm NUCLEODUR Sphinx RP, µm Eluent: methanol dil. NH, ph 0 (0:80, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, 7 nm Inj. volume: µl Fig. : Stability at ph Due to the additional intermolecular interactions NUCLEODUR Sphinx RP is an interesting replenishment to the high density bonded phases NUCLEODUR C 8 /C 8 Gravity and the polar endcapped NUCLEODUR C 8 Pyramid.

13 NUCLEODUR Sphinx RP The Bifunctional RP Phase 0 min 0 min 0 min 0 min NUCLEODUR Sphinx-RP, µm Competitor (column XP) Competitor (column LP) Competitor (column SP) Columns: 0 x. mm Eluent: methanol water (0:70, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm Peaks (inj. volume µl):. Pyridine. Phenol Fig. : Comparison of surface deactivation of different phenyl modified RP phases 090 Columns: 0 x. mm Eluent: water methanol (0:0, v/v) Flow rate: ml/min Temperature: 0 C Inj. volume: µl Detection: 70 nm HO OH HO R O O O OH OH R R OH OH. Catechin. Rutin R = R = OH, R = O-rutinose. Fisetin R = R = OH, R = H. Quercetin R = R = R = OH. Kaempferol R = H, R = R = OH. Isorhamnetin R = OCH, R = R = OH NUCLEODUR C 8 Gravity, µm NUCLEODUR C 8 Gravity, µm NUCLEODUR Sphinx RP, µm min Fig. : Separation of flavonoids on different NUCLEODUR phases 980

14 NUCLEODUR CN and CN-RP 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, ph working range 8 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 characterized by a hydrophobic layer of monomeric or polymeric bonded alkylsilanes. One approach to improve the resolution of compounds poorly separated on nonpolar stationary phases, is to change bonded-phase functionality. The fully endcapped and highly reproducible (figure ) NUCLEODUR 00- CN-RP phase has cyanopropyl groups on the surface able to generate a clearly recognizable different retention behavior compared to purely alkyl-functionalized surface modifications (figure ).. Benzamide. Dimethyl phthalate. Phenetole. o-xylene. Biphenyl. Maleic acid. Norephedrine. Ephedrine. Acetaminophen. Chlorpheniramine. Brompheniramine 0 8 min Columns: 0 x mm NUCLEODUR 00- C 8 ec 0 x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile 00 mm sodium citrate ph. (:8, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 70 nm Inj. volume: 0 µl Fig. : Comparison of NUCLEODUR C 8 ec and CN-RP for a separation of cold-medicine ingredients 90 lot D lot C lot B lot A 0 min Column: 0 x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile water (0:0, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm Inj. volume: µl Fig. : Reproducibility of NUCLEODUR CN 8 080

15 NUCLEODUR CN and CN-RP Cyano-modified High-purity Silica The polarity of the NUCLEODUR 00- CN-RP phase 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 in figure 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). Due to the exceptional polarity features the cyano phase can also be run in the normal phase mode. NUCLEODUR CN columns for normal phase applications are shipped in n-heptane. The drastic change in selectivity and order of elution for a mixture of various steroids in normal and reversed phase mode is displayed in figure 7 below. Moreover the high coverage combined with a thorough endcapping makes NUCLEODUR 00- CN-RP suitable for the separation of ionizable compounds such as basic drugs.. Benzamide. Dimethyl phthalate. Phenetole. o-xylene. Biphenyl 0 min Column: x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile water, % TFA ph (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl Fig. : Stability of NUCLEODUR CN-RP at ph 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) Peaks :. Methyltestosterone. Testosterone. Norgestrel. Medrysone. Cortisone. Hydrocortisone 7. Prednisolone min Reversed phase mode Column: 0 x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile water (:7, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj.volume: 0 µl min Normal phase mode Column: 0 x mm NUCLEODUR 00- CN Eluent: n-heptane -propanol (90:0, v/v) other conditions as for reversed phase mode Fig. 7: Separation of steroids in normal phase and reversed phase mode

16 Applications Appl. : Analgesics Appl. : Analgesics. Paracetamol. Acetylsalicylic acid. Salicylic acid. Ketoprofen. Diclofenac. Ibuprofen. Paracetamol. Acetylsalicylic acid. -Hydroxymethyl benzoate. Ketoprofen. Flurbiprofen. Ibuprofen 0 0 min 0 8 min Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile 0 mm KH PO, ph. (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Inj. volume: µl MN Appl. No Column: 0 x mm NUCLEODUR C 8 Pyramid, µm Eluent: acetonitrile 0.% TFA (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No. 90 Appl. : Rapid separation of analgesics. Acetylsalicylic acid (about 0. µg). Salicylic acid (about 0. µg) A Appl. : Analgesics. Paracetamol. Caffeine. -Acetamidophen. Acetanilide. Acetylsalicylic acid. Phenactin min Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile 0 mm KH PO, ph. (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No B 0 Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: methanol 0.% phosphoric acid (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm A: Thomapyrin tablet; B: standard Thomapyrin is a trademark of Boehringer Ingelheim Pharma KG MN Appl. No. 800 min

17 Applications Appl. : Analgesic and antiinflammatory drugs µg/ml. Acetylsalicylic. acid. Tolmetin. Piroxicam. Suprofen. Naproxen 0.. Diflunisal. 7. Fenoprofen 8. Flurbiprofen 9. Indomethazin 0. Ibuprofen Appl. 7: Antiinflammatory drugs Acetylsalicylic acid. Sulindac. Piroxicam. Suprofen. Tolmetin. Naproxen 7. Diflunisal 8. Flurbiprofen 9. Indomethazin 0. Ibuprofen min Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile % acetic acid (8:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Inj. volume: 0 µl MN Appl. No min Column: 0 x mm NUCLEODUR C 8 Gravity, µm Eluent: acetonitrile 0 mm KH PO, ph. (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Inj. volume: µl MN Appl. No. 780 Appl. : Antiinflammatory drugs Appl. 8: Barbiturates. Acetylsalicylic acid. Sulindac. Tolmetin. Ketoprofen. Flurbiprofen. Diclofenac 7. Ibuprofen 8. Meclofenamic acid. Phenobarbital. Pentobarbital. Hexobarbital. Mephobarbital. Thiamylal min Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile 0 mm KH PO, ph. (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Inj. volume: µl MN Appl. No min Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile water (0:0, v/v) Flow rate: 0.7 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No

18 Applications Appl. 9: Antidepressants Nordoxepin. Protriptyline. Maprotiline. Nortriptyline. Norclomipramine. Doxepin 7. Imipramine 8. Amitriptyline 9. Clomipramine 0. Trimipramine Appl. : Tricyclic antidepressants. Protriptyline. Nortriptyline. Doxepin. Imipramine. Amitriptyline. Trimipramine 0 0 min Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: A) methanol acetonitrile (0:0, v/v) B) 0 mm KH PO, ph % B in min, then 0 min at % B Flow rate:. ml/min Temperature: 0 C Detection: UV, 0 nm Inj. volume: µl MN Appl. No min Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: methanol 0 mm KH PO, ph 7 (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm MN Appl. No. 80 Appl. 0: Tricyclic antidepressants Appl. : Cold medicine. Protriptyline. Nortriptyline. Doxepin. Imipramine. Amitriptyline. Trimipramine. Maleic acid. Acetaminophen. Pseudoephedrine. Benzoic acid. Chlorpheniramine. Dextromethorphan 0 min 0 min Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: acetonitrile 0 mm KH PO, ph 7.0 (:, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm Inj. volume: µl MN Appl. No Column: x mm NUCLEODUR C 8 Gravity, µm Eluents: A) 0 mm KH PO + mm pentanesulfate (Na salt), ph.; B) methanol Gradient: % B in min Flow rate:.0 ml/min Temperature: 0 C Detection: UV, 0 nm Inj. volume: µl MN Appl. No

19 Applications Appl. : Cold medicine ingredients. Ascorbic acid. Paracetamol. Caffeine. Chlorpheniramine. Dextromethorphan. Diphenhydramine Appl. : Basic drugs. Procainamide. Clonidin. Clenbuterol 0 8 min Column: 0 x mm NUCLEODUR C 8 Pyramid, µm Eluent: A) 0 mm NH H PO, ph.; B) acetonitrile Gradient: 0 0% B in min Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm (. min) UV, nm Inj. volume: µl MN Appl. No min Column: x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile 0 mm KH PO, ph. (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume:.0 µl MN Appl. No. 90 Appl. : Cold medicine (purchasable OTC drug). Ascorbic acid. Paracetamol. Caffeine. Chlorpheniramine. Ofloxacin. Ciprofloxacin. Cinoxacin. Penicillin G. Penicillin V. Cloxacillin Appl. : Antibacterial drugs min Column: 0 x mm NUCLEODUR C 8 Pyramid, µm Eluent: A) 0 mm NH H PO, ph.; B) acetonitrile 0 0% B in min Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm (. min) UV, nm Inj. volume: µl MN Appl. No min Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile water (0:0, v/v) 0.0% TFA Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No

20 Applications. Cinoxacin. Oxolinic acid. Nalidixic acid Appl. 7: Quinolone antibiotics Appl. 9: Penicillin antibiotics. Amoxicillin. Penicillin G. Penicillin V. Nafcillin. Dicloxacillin 0 0 min min Column: 0 x. mm NUCLEODUR Sphinx RP, µm Eluent: methanol 0 mm KH PO, ph, (0:0, v/v) Flow rate:.0 ml/min Temperature: ambient Detection: UV, nm Inj. volume: µl MN Appl. No Column: 0 x mm NUCLEODUR C 8 Pyramid, µm Eluent: acetonitrile 0.% TFA (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No. 90 Appl. 8: Penicillin antibiotics Appl. 0: Sulfonamides. Penicillin G. Penicillin V. Cloxacillin. Nafcillin. Dicloxacillin. Sulfanilamide. Sulfadiazine. Sulfathiazole. Sulfamerazine. Sulfadimidine. Succinylsulfathiazole 0 min Column: x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile 0 mm KH PO, ph.0 (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No min Column: x mm NUCLEODUR Sphinx RP, µm Eluent: methanol 0.% TFA (0:80, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Inj. volume: µl MN Appl. No

21 Applications Appl. : Sulfonamides Appl. : Sedative drugs. Sulfanilamide. Sulfadiazine. Sulfathiazole. Sulfamerazine. Sulfadimidine. Succinylsulfathiazole. Promethazine. Promazine. Chlorpromazine 0 0 min min Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: methanol 0.% TFA (0:80, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Inj. volume: µl MN Appl. No Column: x mm NUCLEODUR 00- CN-RP Eluent: A) methanol B) 0 mm ammonium acetate, ph % B in 0 min, then 0 min 0% B Flow rate:. ml/min Temperature: 0 C Detection: UV, nm Inj. volume: µl (70 µg/ml +, µg/ml ) MN Appl. No Bromazepam. Oxazepam. Lorazepam. Temazepam Appl. : Benzodiazepines Appl. : Coronary therapeutic drugs (Ca-antagonists). Nifedipine. Nitrendipine. Nimodipine. Nisoldipine. Nicardipine 0 min Column: x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile 0 mm KH PO, ph. (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No min Column: x mm NUCLEODUR 00- CN-RP Eluent: A) acetonitrile, B) 0 mm KH PO, ph. 0 0% B in 7. min, then 7. min 0% B Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume:. µl ( µg/compound) MN Appl. No. 90

22 Applications Appl. : Nucleic acid bases Appl. 7: Alkaloids. Cytosine. Adenine. Uracil. Thymine. Codeine. Quinine. Strychnine. Atropine. Papaverine. Noscapine 0 8 min 0 0 min Column: 0 x mm NUCLEODUR C 8 Pyramid, µm Eluent: A) 0 mm NH H PO, ph., B) acetonitrile. min 00% A, then to 90% A in 0 min Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No. 90 Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: A) acetonitrile, B) 0 mm KH PO, ph % B in min, then 70 90% B in min Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Inj. volume: µl MN Appl. No Chloroquine. Quinine. Mefloquine Appl. : Quinine alkaloids Appl. 8: Catecholamines. Norephedrine. Adrenaline. Dihydroxyphenylalanine. Hydroxytyramine. Tyrosine 0 0 min Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: A) methanol, B) 0 mm KH PO, ph % B in min, then 70 0% B in 7 min Flow rate:. ml/min Temperature: C Detection: UV, 0 nm Inj. volume: 0 µl MN Appl. No min Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: 00 mm NaH PO, ph.0 Flow rate: 0.8 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No. 790

23 Applications Appl. 9: Steroids. Cortisone. Hydrocortisone. Hydrocortisone -acetate. α-methyl-βhydroxyprogesterone. α-methyl-7αhydroxyprogesterone. α-methyl-7αhydroxyprogesterone acetate Peaks (each env. 0 0 µg/ml):.estriol.prednisolone.cortisone.testosterone.α-methyl-β-hydroxyprogesterone.α-methyl-7α-hydroxyprogesterone 7.α-Methyl-7α-hydroxyprogesterone acetate 8.Estradiol 9.Estrone 0.Progesterone Appl. : Steroids min Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: acetonitrile water (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm MN Appl. No min Column: x mm NUCLEODUR 00- C 8 ec Eluent: A) water, B) methanol min 0% B, 0 % B in 0 min, min % B, 0% B in min, min 0% B Flow rate:.0 ml/min Temperature: 0 C Detection: UV, 0 nm Inj. volume: 0 µl MN Appl. No. 80. Cortisone. Hydrocortisone. Corticosterone. Hydrocortisone acetate. Corticosterone acetate Appl. 0: Corticosteroids Appl. : Xylometazoline in nasal spray b). Xylometazoline.. Benzalkonium chlorides with different chain lengths (C 8, C 0, C, C ) a) standard b) nasal spray 0 0 min a) Column: x mm NUCLEODUR 00- C 8 ec Eluent: methanol water (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, 0 nm Inj. volume: µl MN Appl. No min Column: x mm NUCLEODUR 00- CN-RP Eluent: acetonitrile 0 mm Na citrate, ph.0 (0:0, v/v) Flow rate: 0.8 ml/min; temperature: 0 C Detection: UV, nm MN Appl. No. 090

24 Applications Appl. : Soft drink additives Appl. : Aromatic aldehydes. Ascorbic acid (acidic). Acesulfam K. Saccharin. Caffeine (basic). Aspartame. Quinine (strongly basic) 7. Vanillin 8. Sorbic acid (acidic) 9. Benzoic acid (acidic). p-carboxybenzaldehyde. p-hydroxybenzaldehyde. Vanillin. -Ethoxyvanillin. Benzaldehyde min 0 8 min Column: 0 x. mm NUCLEODUR 00- C 8 ec Eluent: 0 mm KH PO, ph acetonitrile (:, v/v) Flow rate:.9 ml/min Temperature: C Detection: UV, 0 nm Inj. volume: 0 µl MN Appl. No. 80 Column: x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile water, ph.0 (:78, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No Appl. : Fast separation of sweeteners. Acesulfam K. Saccharin. Aspartame 7 8 Appl. : Substituted aromatics 0 9. Uracil. Benzamide. Phenol. Benzaldehyde. Acetophenone. -Nitrophenol 7. Nitrobenzene A) C 8 Gravity Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile water (0:80, v/v) 0.% TFA Flow rate:. ml/min Temperature: C Detection: UV, nm Inj. volume: 0 µl MN Appl. No min 9 0 B) Sphinx RP min 8. Propyl -hydroxybenzoate 9. Toluene 0. Benzophenone. Xylene Columns: 0 x. mm NUCLEODUR Eluent: methanol water (:, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm Inj. volume: µl MN Appl. No. 980/980

25 Applications Appl. 7: Separation of a test mixture. Ethyl p-aminobenzoate. Propranolol. Lidocaine. Imipramine. Amitriptyline. Trimipramine Appl. 9: Fat-soluble vitamins 7 8. Vitamin K. Vitamin A. Vitamin A acetate. Vitamin D. Vitamin D. Vitamin E 7. Vitamin E acetate 8. Vitamin K min Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: methanol 0 mm KH PO, ph 7 (70:0, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm MN Appl. No min Column: 0 x mm NUCLEODUR 00- C 8 ec Eluent: acetonitrile Flow rate:. ml/min Temperature: 0 C Detection: UV, 80 nm Inj. volume: µl MN Appl. No Peaks (inj. volume µl):. Uracil.,7-Dihydroxynaphthalene.,-Dihydroxynaphthalene. Ethyl benzoate. Lidocaine. Biphenyl 7. Acenaphthene Appl. 8: Selectivity test. δ-tocopherol. γ-tocopherol. α-tocopherol. α-tocopherol acetate Appl. 0: Tocopherols 7 0 min 0 min 0 Column: x mm NUCLEODUR Sphinx RP, µm Eluent: methanol mm NH H PO, ph 7 (:, v/v) Flow rate:.0 ml/min Temperature: 0 C Detection: UV, nm MN Appl. No Column: x mm NUCLEODUR 00- C 8 ec Eluent: methanol (00%) Flow rate:.0 ml/min Temperature: C Detection: UV, 9 nm Inj. volume: 8 µl MN Appl. No. 790

26 Applications Appl. : Water-soluble vitamins (mg/ml). Nicotinic acid (0.). Nicotinamide (0.). -Aminobenzoic acid (0.0). Folic acid (0.). Pyridoxin (B ) [hydrochloride] (0.0). Riboflavin (B ) (0.0) 7. Thiamine hydrochloride (B ) (0.0) 8. Rutin (0.0) red curves: vitamin test mixture (in eluent A) blue curves: multivitamin juice (undiluted) Appl. : Complexing agents acc. to DIN 8-8. NTA (nitrilotriacetic acid). EDTA (ethylenediamintetraacetic acid). DTPA (diethylenetrinitrilopentaacetic acid) chromatogram courtesy of H. Albrich, C. Geis, Gewerbliches Institut für Umweltanalytik GmbH, Teningen, Germany 7 8 nm 0 8 min Column: 0 x mm NUCLEODUR C 8 Pyramid, µm Eluent: 0. mmol/l HNO, 7. mmol/l N(C H 9 ) HSO,. mmol/l N(C H 9 ) OH, 7 µmol/l Fe + Flow rate: 0. ml/min Temperature: 0 C Detection: UV, 0 nm Inj. volume: 0 µl MN Appl. No nm nm Appl. : Phenolic compounds. Resorcinol. Pyrocatechol. -Methoxyphenol. Phenol. -Methoxyphenol. -Ethoxyphenol min Column: x mm NUCLEODUR C 8 Pyramid, µm Eluent: A) water, mm heptanesulfonic acid (Na salt), mm NaH PO, 0.% CH COOH, 0.00% triethylamine (ph.), B: acetonitrile water (0:0, v/v), mm heptanesulfonic (Na salt), 0. % CH COOH, 0.00% triethylamine (ph ~.); multistep gradient: min 0% B, 0 0% B in. min, 0 % B in. min, 0% B in 8 min, 0 70% B in 7 min, 70 0%B in min Flow rate:.0 ml/min Temperature: C Detection: UV,, 7 and nm Inj. volume: 0 µl MN Appl. No min 0 Column: x mm NUCLEODUR 00- C 8 ec Eluent: methanol water, 0.% H PO (0:0, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm Inj. volume: µl MN Appl. No. 7970

27 Applications. Aspartic acid. Fumaric acid. Maleic acid Appl. : Organic acids. Desisopropylatrazine. Metamitron. Desethylatrazine. Bromoxynil. Simazine. Cyanazine 7. Metabenzthiazuron 8. Atrazine 9. Monolinuron Appl. : Pesticides 0.Isoproturon.Diuron.Metobromuron.Metazachlor.Sebutylazine.Terbuthylazine.Linuron 7.Chloroxuron 8.Metolachlor min Column: 0 x mm NUCLEODUR 00- CN-RP Eluent: mm KH PO, ph.0 Flow rate: 0. ml/min Temperature: 0 C Detection: UV, 0 nm Inj. volume: µl MN Appl. No min Appl. : Test for metal ions in silica adsorbent.,7-dihydroxynaphthalene.,-dihydroxynaphthalene Column: Eluent: Gradient: Flow rate Temperature: C Detection UV 0 nm 0 x mm NUCLEODUR 00- C 8 ec A) water (% acetic acid), B) acetonitrile 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.0 ml/min MN Appl. No min The ratio of the asymmetry factors of,-dihydroxynaphthalene () and,7-dihydroxynaphthalene () is a measure for the metal ion content of the silica phase, because () can form complexes with metal ions, resulting in broad peaks for this compound. Column: x mm NUCLEODUR C 8 Gravity, µm Eluent: methanol 0 mm KH PO, ph 7 (:, v/v) Flow rate:.0 ml/min Temperature: C Detection: UV, nm MN Appl. No. 80 7

28 Alphabetical Index of Analytes Analyte NUCLEODUR phase page Analyte NUCLEODUR phase page A Acenaphthene C 8 Gravity, µm Fig., 7 C 8 Pyramid, µm Fig. 9 0 Sphinx RP, µm Appl. 8 Acesulfam K 00- C 8 ec Appl. 00- C 8 ec Appl. -Acetamidophen C 8 Gravity, µm Appl. Acetaminophen 00- CN-RP Fig. C 8 Gravity, µm Appl. 8 Acetanilide C 8 Gravity, µm Appl. Acetic acid C 8 Pyramid, µm Fig., 9 Acetophenone Sphinx RP, µm Appl. Acetylsalicylic acid 00- C 8 ec Appl., Appl C 8 ec Appl. 7 C 8 Gravity, µm Appl. 7 7 C 8 Pyramid, µm Appl. C 8 Gravity, µm Appl. Adenine C 8 Pyramid, µm Appl. Adrenaline C 8 Gravity, µm Appl. 8 -Aminobenzoic acid C 8 Pyramid, µm Appl. Amitriptyline C 8 Gravity, µm Fig., 7 Appl. 9, 0 8 C 8 Pyramid, µm Fig. 8 0 C 8 Gravity, µm Appl. 8 Appl. 7 Amoxicillin C 8 Pyramid, µm Appl. 9 0 Aniline C 8 Pyramid, µm Fig. 7 0 Anthracene 00- C 8 ec Fig. Ascorbic acid 00- C 8 ec Appl. C 8 Pyramid, µm Appl., 9 Aspartame 00- C 8 ec Appl. 00- C 8 ec Appl. Aspartic acid 00- CN-RP Appl. 7 Atrazine 00- C 8 ec Appl. 7 Atropine C 8 Gravity, µm Appl. 7 B Benzaldehyde 00- C 8 ec Appl. Sphinx RP, µm Appl. Benzalkonium chlorides 00- CN-RP Appl. Benzamide 00- CN-RP Fig. Fig. C 8 Gravity, µm Fig. 7 C 8 Pyramid, µm Fig. 9 0 Sphinx RP, µm Appl. Benzoic acid 00- C 8 ec Appl. C 8 Gravity, µm Appl. 8 Benzophenone C 8 Pyramid, µm Fig. 9 0 Sphinx RP, µm Appl. Biphenyl 00- CN-RP Fig. Fig. C 8 Pyramid, µm Fig. 9 0 Sphinx RP, µm Appl. 8 Biphenyl--ol 00- C 8 ec Fig. Bromazepam 00- C 8 ec Appl. Bromoxynil 00- C 8 ec Appl. 7 Brompheniramine 00- CN-RP Fig. C Caffeine 00- C 8 ec Appl. C 8 Gravity, µm Fig. C 8 Pyramid, µm Appl., 9 C 8 Gravity, µm Appl. Sphinx RP, µm Fig. p-carboxybenzaldehyde 00- C 8 ec Appl. Carprofen 00- C 8 ec Fig. 0 Catechin Sphinx RP, µm Fig. Chloroquine C 8 Gravity, µm Appl. Chloroxuron 00- C 8 ec Appl. 7 Chlorpheniramine 00- CN-RP Fig. C 8 Gravity, µm Appl. 8 C 8 Pyramid, µm Fig. 9 0 Appl., 9 Chlorpromazine 00- CN-RP Appl. Cinoxacin 00- C 8 ec Appl. 9 Sphinx RP, µm Appl. 7 0 Ciprofloxacin 00- C 8 ec Appl. 9 Clenbuterol 00- CN-RP Appl. 9 Clomipramine C 8 Gravity, µm Appl. 9 8 Clonidin 00- CN-RP Appl. 9 Cloxacillin 00- C 8 ec Appl. 9 Appl. 8 0 Codeine C 8 Gravity, µm Appl. 7 Corticosterone acetate 00- C 8 ec Appl. 0 Corticosterone 00- C 8 ec Appl. 0 Cortisone 00- C 8 ec Appl C 8 ec Appl. 00- CN Fig. 7 C 8 Gravity, µm Appl. 9 Cyanazine 00- C 8 ec Appl. 7 Cytosine C 8 Pyramid, µm Appl. D Desethylatrazine 00- C 8 ec Appl. 7 Desisopropylatrazine 00- C 8 ec Appl. 7 Dextromethorphan C 8 Gravity, µm Appl. 8 C 8 Pyramid, µm Appl. 9 Dibutyl phthalate C 8 Gravity, µm Fig., 7 Diclofenac 00- C 8 ec Appl. Appl C 8 ec Fig. 0 Dicloxacillin 00- C 8 ec Appl. 8 0 C 8 Pyramid, µm Appl. 9 0 Diethylenetrinitrilopentaacetic acid C 8 Pyramid, µm Appl. Diflunisal 00- C 8 ec Appl. 7 C 8 Gravity, µm Appl. 7 7 Dihydroxynaphthalene isomers C 8 Gravity, µm Fig. 7 C 8 Gravity, µm Appl. 7 Sphinx RP, µm Appl. 8 Dihydroxyphenylalanine C 8 Gravity, µm Appl. 8 Dimethyl phthalate 00- CN-RP Fig. Fig. C 8 Pyramid, µm Fig. 9 0 Dimethylaniline C 8 Pyramid, µm Fig. 7 0 Diphenhydramine C 8 Gravity, µm Fig. 7 C 8 Pyramid, µm Appl. 9 Diuron 00- C 8 ec Appl. 7 Doxepin C 8 Gravity, µm Appl. 9, 0 8 C 8 Pyramid, µm Fig. 8 0 C 8 Gravity, µm Appl. 8 DTPA C 8 Pyramid, µm Appl. E EDTA C 8 Pyramid, µm Appl. Ephedrine 00- CN-RP Fig. Estradiol 00- C 8 ec Appl. Estriol 00- C 8 ec Appl. 8

29 Alphabetical Index of Analytes Analyte NUCLEODUR phase page Analyte NUCLEODUR phase page Estrone 00- C 8 ec Appl. -Ethoxyphenol 00- C 8 ec Appl. 00- C 8 ec Fig. -Ethoxyvanillin 00- C 8 ec Appl. Ethyl benzoate C 8 Pyramid, µm Fig. 9 0 Ethyl p-aminobenzoate C 8 Gravity, µm Appl. 7 p-ethylaniline C 8 Pyramid, µm Fig. 7 0 Ethylbenzene C 8 Gravity, µm Fig. 7 Fig. 0 C 8 Pyramid, µm Fig. 7 0 Ethyl benzoate Sphinx RP, µm Appl. 8 Ethylenediaminetetraacetic acid C 8 Pyramid, µm Appl. F Fenoprofen 00- C 8 ec Fig. 0 Appl. 7 Fisetin Sphinx RP, µm Fig. Flurbiprofen 00- C 8 ec Appl C 8 ec Appl. 7 C 8 Gravity, µm Appl. 7 7 C 8 Pyramid, µm Appl. Folic acid C 8 Pyramid, µm Appl. Formic acid C 8 Pyramid, µm Fig. 9 Fumaric acid 00- CN-RP Appl. 7 H Hexobarbital 00- C 8 ec Appl. 8 7 Hydrocortisone 00- C 8 ec Appl CN Fig. 7 C 8 Gravity, µm Appl. 9 Hydrocortisone acetate 00- C 8 ec Appl. 0 C 8 Gravity, µm Appl. 9 -Hydroxybenzaldehyde 00- C 8 ec Appl. -Hydroxymethyl benzoate C 8 Pyramid, µm Appl. Hydroxytyramine C 8 Gravity, µm Appl. 8 I Ibuprofen 00- C 8 ec Appl. Appl C 8 ec Appl. 7 C 8 Gravity, µm Appl. 7 7 C 8 Pyramid, µm Appl. Imipramine C 8 Gravity, µm Appl. 9, 0 8 C 8 Pyramid, µm Fig. 8 0 C 8 Gravity, µm Appl. 8 Appl. 7 Indomethazin 00- C 8 ec Appl. 7 C 8 Gravity, µm Appl. 7 7 Isoproturon 00- C 8 ec Appl. 7 Isorhamnetin Sphinx RP, µm Fig. K Kaempferol Sphinx RP, µm Fig. Ketoprofen 00- C 8 ec Appl. Appl C 8 ec Fig. 0 C 8 Gravity, µm Fig. 7 C 8 Pyramid, µm Appl. L Lactic acid C 8 Pyramid, µm Fig. 9 Lidocaine C 8 Gravity, µm Fig. 7, Fig. 7, Fig. 7 C 8 Pyramid, µm Fig. 9 0 C 8 Gravity, µm Appl. 7 Sphinx RP, µm Appl. 8 Linuron 00- C 8 ec Appl. 7 Lorazepam 00- C 8 ec Appl. M Maleic acid 00- CN-RP Fig., Appl. 7 C 8 Gravity, µm Appl. 8 C 8 Pyramid, µm Fig. 9 Malic acid C 8 Pyramid, µm Fig. 9 Maprotiline C 8 Gravity, µm Appl. 9 8 Meclofenamic acid 00- C 8 ec Appl. 7 Medrysone 00- CN Fig. 7 Mefloquine C 8 Gravity, µm Appl. Mephobarbital 00- C 8 ec Appl. 8 7 Metabenzthiazuron 00- C 8 ec Appl. 7 Metamitron 00- C 8 ec Appl. 7 Metazachlor 00- C 8 ec Appl. 7 Methoxyphenol isomers 00- C 8 ec Appl. 00- C 8 ec Fig. α-methyl-β-hydroxyprogesterone 00- C 8 ec Appl. C 8 Gravity, µm Appl. 9 α-methyl-7α-hydroxyprogesterone 00- C 8 ec Appl. C 8 Gravity, µm Appl. 9 α-methyl-7α-hydroxyprogesterone acetate 00- C 8 ec Appl. C 8 Gravity, µm Appl. 9 Methyltestosterone 00- CN Fig. 7 Metobromuron 00- C 8 ec Appl. 7 Metolachlor 00- C 8 ec Appl. 7 Monolinuron 00- C 8 ec Appl. 7 N Nafcillin 00- C 8 ec Appl. 8 0 C 8 Pyramid, µm Appl. 9 0 Nalidixic acid Sphinx RP, µm Appl. 7 0 Naphthalene 00- C 8 ec Fig. C 8 Gravity, µm Fig. 7 C 8 Pyramid, µm Fig. 9 0 Naproxen 00- C 8 ec Appl. 7 C 8 Gravity, µm Appl. 7 7 Nicardipine 00- CN-RP Appl. Nicotinamide C 8 Pyramid, µm Appl. Nicotinic acid C 8 Pyramid, µm Appl. Nifedipine 00- CN-RP Appl. Nimodipine 00- CN-RP Appl. Nisoldipine 00- CN-RP Appl. Nitrendipine 00- CN-RP Appl. Nitrilotriacetic acid C 8 Pyramid, µm Appl. Nitrobenzene Sphinx RP, µm Appl. -Nitrophenol Sphinx RP, µm Appl. Norclomipramine C 8 Gravity, µm Appl. 9 8 Nordoxepin C 8 Gravity, µm Appl. 9 8 Norephedrine 00- CN-RP Fig. C 8 Gravity, µm Appl. 8 Norgestrel 00- CN Fig. 7 9

30 Alphabetical index of analytes Analyte NUCLEODUR phase page Analyte NUCLEODUR phase page Nortriptyline C 8 Gravity, µm Appl. 9, 0 8 C 8 Pyramid, µm Fig. 8 0 C 8 Gravity, µm Appl. 8 Noscapine C 8 Gravity, µm Fig. 7 Appl. 7 NTA C 8 Pyramid, µm Appl. O Ofloxacin 00- C 8 ec Appl. 9 Oxazepam 00- C 8 ec Appl. Oxolinic acid Sphinx RP, µm Appl. 7 0 P Papaverine C 8 Gravity, µm Fig. 7 Appl. 7 Paracetamol 00- C 8 ec Appl. C 8 Pyramid, µm Appl. Appl., 9 C 8 Gravity, µm Appl. Penicillin G 00- C 8 ec Appl. 9 Appl. 8 0 C 8 Pyramid, µm Appl. 9 0 Penicillin V 00- C 8 ec Appl. 9 Appl. 8 0 C 8 Pyramid, µm Appl. 9 0 Pentobarbital 00- C 8 ec Appl. 8 7 Phenactin C 8 Gravity, µm Appl. Phenetole 00- C 8 ec Fig. 00- CN-RP Fig. Fig. Phenobarbital 00- C 8 ec Appl. 8 7 Phenol 00- C 8 ec Fig. Appl. 00- C 8 ec Fig. C 8 Gravity, µm Fig. C 8 Pyramid, µm Fig. 7 0 Sphinx RP, µm Fig. Phenol Sphinx RP, µm Appl. Piroxicam 00- C 8 ec Fig. 0 Appl. 7 C 8 Gravity, µm Appl. 7 7 Prednisolone 00- C 8 ec Appl. 00- CN Fig. 7 Procainamide 00- CN-RP Appl. 9 Progesterone 00- C 8 ec Appl. Promazine 00- CN-RP Appl. Promethazine 00- CN-RP Appl. Propranolol C 8 Gravity, µm Appl. 7 Propyl -hydroxy benzoate Sphinx RP, µm Appl. Protriptyline C 8 Gravity, µm Appl. 9, 0 8 C 8 Pyramid, µm Fig. 8 0 C 8 Gravity, µm Appl. 8 Pseudoephedrine C 8 Gravity, µm Appl. 8 Pyridine C 8 Gravity, µm Fig. Fig. 0 Sphinx RP, µm Fig. Pyridoxin C 8 Pyramid, µm Appl. Pyrocatechol 00- C 8 ec Appl. 00- C 8 ec Fig. Q Quercetin Sphinx RP, µm Fig. Quinine 00- C 8 ec Appl. C 8 Gravity, µm Appl., 7 R Resorcinol 00- C 8 ec Appl. 00- C 8 ec Fig. Riboflavin C 8 Pyramid, µm Appl. Rutin C 8 Pyramid, µm Appl. Sphinx RP, µm Fig. S Saccharin 00- C 8 ec Appl. 00- C 8 ec Appl. Salicylic acid 00- C 8 ec Appl., Sebutylazine 00- C 8 ec Appl. 7 Simazine 00- C 8 ec Appl. 7 Sorbic acid 00- C 8 ec Appl. Strychnine C 8 Gravity, µm Appl. 7 Succinic acid C 8 Pyramid, µm Fig. 9 Succinylsulfathiazole C 8 Gravity, µm Appl. Sphinx RP, µm Appl. 0 0 Sulfadiazine C 8 Gravity, µm Appl. Sphinx RP, µm Appl. 0 0 Sulfadimidine C 8 Gravity, µm Appl. Sphinx RP, µm Appl. 0 0 Sulfamerazine C 8 Gravity, µm Appl. Sphinx RP, µm Appl. 0 0 Sulfanilamide C 8 Gravity, µm Appl. Sphinx RP, µm Appl. 0 0 Sulfathiazole C 8 Gravity, µm Appl. Sphinx RP, µm Appl. 0 0 Sulindac 00- C 8 ec Appl. 7 C 8 Gravity, µm Appl. 7 7 Suprofen 00- C 8 ec Fig. 0 Appl. 7 C 8 Gravity, µm Appl. 7 7 T Tartaric acid C 8 Pyramid, µm Fig., 9 Temazepam 00- C 8 ec Appl. Terbuthylazine 00- C 8 ec Appl. 7 Testosterone 00- C 8 ec Appl. 00- CN Fig. 7 Theophylline C 8 Gravity, µm Fig. Sphinx RP, µm Fig. Thiamine C 8 Pyramid, µm Appl. Thiamylal 00- C 8 ec Appl. 8 7 Thymine C 8 Pyramid, µm Appl. Tocopherols 00- C 8 ec Appl. 0 Tolmetin 00- C 8 ec Appl C 8 ec Appl. 7 C 8 Gravity, µm Appl. 7 7 Toluene C 8 Gravity, µm Fig. 7 Fig. 0 C 8 Pyramid, µm Fig. 7 0 Sphinx RP, µm Appl. Trimipramine C 8 Gravity, µm Appl. 9, 0 8 C 8 Gravity, µm Appl. 8 Appl. 7 Tyrosine C 8 Gravity, µm Appl. 8 U Uracil C 8 Gravity, µm Fig. 7 C 8 Pyramid, µm Fig. 7 0 Appl. Sphinx RP, µm Appl. Appl. 8 0