ACE CN-ES. Extra Stability. Extended Spacer. Enhanced Selectivity ACE. Combining CN polar selectivity with enhanced hydrophobicity

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1 ACE CN-ES Combining CN polar selectivity with enhanced hydrophobicity Extended Spacer Extra Stability New stationary phase technology for UHPLC and HPLC separations Alternative polar selectivity for method development High efficiency µm, µm, µm and 0µm particles Ultra-inert for maximum performance and reproducibility Enhanced Selectivity ACE ACE CN-ES Combining CN polar selectivity with enhanced UHPLC and HPLC hydrophobicity s

2 ACE CN-ES Mechanism of Separation Strength of Interaction Fig. - ACE CN-ES Provides Alternative Selectivity Application # 70 Dipole-dipole interactions Hydrophobic binding interactions Target Analytes Strong Strong Polar and non-polar analytes Analytes with double and triple bonds Analytes differing in hydrophobicity Polar molecules having insufficient retention on traditional CN bonded phases ACE Excel CN-ES ACE CN-ES phase provides complete separation due to hydrophobic AND dipole-dipole interactions 0 Recommended Separations Mixtures of very polar, polar and non-polar analytes Suitable for NP and RP separations Separations where traditional CN bonded phases show insufficient stability/lifetime Applications where a typical C8 column does not provide adequate separation As an orthogonal phase in method development ACE Excel CN,, CN phase shows insufficient resolution (greatly reduced hydrophobic interaction) 0 ACE Excel C8 C8 phase provides insufficient resolution (no dipole-dipole interaction), If ACE does not outperform the column you are currently using, simply contact us for a full refund and keep the ACE column FREE F CHARGE. 0 Sample: ) metronidazole ) benzyl alcohol ) hydrochlorothiazide ) vanillin ) methyl paraben ),-dinitrobenzene Mobile Phase: A = 0.% formic acid in H B = 0.% formic acid in 0:0 MeH/H Gradient: - 00% B in minutes Dimensions: 0 x.mm Flow Rate: 0.0ml/min Temperature: 0 C Wavelength: nm

3 Why does ACE CN-ES provide alternative selectivity? Combines a polar CN group with an extended spacer that provides retention similar to a C8 column Extended spacer technology additionally provides increased column lifetime compared to traditional CN bonded phases with short alkyl spacer (typically C /propyl) CN Phase ACE CN-ES C8 Phase R Si R CN group for polar interaction Short alkyl spacer (typically C /propyl) results in low hydrophobicity and reduced stability R Si R Extended spacer technology provides enhanced hydrophobic interaction Extended spacer also increases column lifetime and stability Ultra inert, ultra pure, ultra reproducible ACE silica R Si R C8 group for hydrophobic interaction Dipole-dipole interaction ACE CN-ES provides multiple modes of interaction (dipole-dipole AND hydrophobic interactions) Hydrophobic interaction ACE CN-ES provides retention similar to a C8 column Fig. - Separation of Neutral Compounds under RP conditions Application # 70 ACE Excel CN-ES ACE CN-ES phase offers increased retention due to contribution from the extended spacer ACE Excel C8 C8 group provides hydrophobic interaction ACE Excel CN CN phases with short alkyl spacer group show minimal hydrophobic retention Sample: ) uracil ) dimethyl phthalate ) toluene ) biphenyl ) phenanthrene Mobile Phase: 0:0 MeCN/H Dimensions: 0 x.mm Flow Rate:.00ml/min Temperature: C Wavelength: nm

4 Alternative Selectivity with MeH or MeCN Fig. - Advantages of Multi-Mode Interactions on Separations Application # 70 0 ACE Excel CN-ES 7 8 Multi-Mode interaction with hydrophobic AND dipole-dipole interactions ACE Excel C8 7 8 Hydrophobic interaction (no dipole-dipole interaction) 0 8 0,,, ACE Excel CN 7, 8 0 Dipole-dipole interaction (reduced hydrophobic interaction) Sample: ),-dihydroxybenzene ) catechol ) hydrochlorothiazide ) oxyprenolol ) salicylic acid ) myricetin 7) piroxicam 8),-dinitrobenzene ) tolmetin 0) -naphthol ) piperine ) diflunisal ) propylbenzene ),,-trichlorobenzene Mobile Phase: A = 0.% formic acid in H B = 0.% formic acid in MeCN Gradient: - 00% B in 0 minutes Dimensions: 00 x.mm Flow Rate: 0.0ml/min Temperature: 0 C Wavelength: 0nm Fig. - Fast Gradient with NSAIDs Application # 70 -Acetamidophenol NH H CH ACE Excel CN-ES 0 Acetanilide H C NH NH Salicylamide H ACE Excel C8 H 0 Aspirin CH, ACE Excel CN Phenacetin H C NH CH 0 H Sample: ) -acetamidophenol ) acetanilide ) salicylamide ) aspirin ) phenacetin ) salicylic acid Mobile Phase: A = 0.% formic acid in H B = 0.% formic acid in MeH Gradient: - 8% B in.7 minutes, hold at 8% B until minutes Dimensions: 0 x.mm Flow Rate: 0.0ml/min Temperature: 0 C Wavelength: 0nm Salicylic acid H

5 Exploit Enhanced Selectivity for UHPLC Fig. - Steroids by UHPLC Application # 70 ACE Excel CN-ES 7 8 ACE CN-ES provides complete separation due to hydrophobic AND dipole-dipole interactions 0 ACE Excel C8, C8 phase provides insufficient resolution (no dipole-dipole interaction) ACE Excel CN 7 8 CN phase shows insufficient resolution (reduced hydrophobic interaction) 0 Sample: ) cortisone ) corticosterone ) α-hydroxyprogesterone ) cortisone--acetate ) -ketoprogesterone ) β-estradiol 7) 7α-estradiol 8) 7α-ethynylestradiol ) estrone Mobile Phase: A = 0.% formic acid in H B = 0.% formic acid in MeCN Gradient:.% B in minutes Dimensions: 0 x.mm Flow Rate: 0.0ml/min Temperature: 0 C Wavelength: 0nm ACE CN-ES is additionally compatible with Normal Phase conditions Fig. - Retention Comparison with Substituted Aromatic Compounds under NP Conditions Application # 70 ACE Excel CN-ES ACE CN-ES provides separation similar to a CN column ACE Excel CN Dipole-dipole interaction provides separation ,, ACE Excel C8 No dipole-dipole interaction to enable separation Sample: ) butylbenzene ) methyl benzoate ) nitrobenzene Mobile Phase: 0:0 heptane/ethyl acetate Dimensions: 0 x.mm Flow Rate:.00ml/min Temperature: C Wavelength: nm

6 Product Availability and Specifications Phase Functional Group Endcapped ACE CN-ES Cyano with proprietary extended alkyl spacer Particle Size (µm) Pore Size (Å) Surface Area (m /g) Carbon Load (%) Maximum ph Range 00% Aqueous Compatible USP Listing Yes,,, a Yes L0 a For optimum column lifetime, a ph range of -8 is recommended. To increase column lifetime at higher ph, organic buffers, low buffer concentrations, high % organic solvent and low temperatures must be considered. Further information is contained within A Guide to HPLC and LC/MS Buffer Selection by John Dolan please contact your distributor to request your FREE copy or visit. ACE Excel µm CN-ES UHPLC/HPLC s (supplied in dual compatible UHPLC/HPLC Excel hardware format with 000bar/000psi pressure limit) 0mm 0mm mm 0mm 7mm 00mm mm 0mm.mm EXL-0-00U EXL-0-00U EXL-0-0U EXL-0-00U EXL-0-70U EXL-0-00U EXL-0-0U EXL-0-0U.0mm EXL-0-00U EXL-0-00U EXL-0-0U EXL-0-00U EXL-0-70U EXL-0-00U EXL-0-0U EXL-0-0U.mm EXL-0-0U EXL-0-0U EXL-0-U EXL-0-0U EXL-0-7U EXL-0-0U EXL-0-U EXL-0-U ACE Excel µm CN-ES UHPLC/HPLC s (supplied in dual compatible UHPLC/HPLC Excel hardware format with 000bar/000psi pressure limit) 0mm 0mm mm 0mm 7mm 00mm mm 0mm 0mm.mm EXL--00U EXL--00U EXL--0U EXL--00U EXL--70U EXL--00U EXL--0U EXL--0U EXL--0U.0mm EXL--00U EXL--00U EXL--0U EXL--00U EXL--70U EXL--00U EXL--0U EXL--0U EXL--0U.mm EXL--0U EXL--0U EXL--U EXL--0U EXL--7U EXL--0U EXL--U EXL--U EXL--U ACE Excel µm CN-ES UHPLC/HPLC s (supplied in dual compatible UHPLC/HPLC Excel hardware format with 000bar/000psi pressure limit) 0mm 0mm mm 0mm 7mm 00mm mm 0mm 0mm.mm EXL--00U EXL--00U EXL--0U EXL--00U EXL--70U EXL--00U EXL--0U EXL--0U EXL--0U.0mm EXL--00U EXL--00U EXL--0U EXL--00U EXL--70U EXL--00U EXL--0U EXL--0U EXL--0U.mm EXL--0U EXL--0U EXL--U EXL--0U EXL--7U EXL--0U EXL--U EXL--U EXL--U ACE µm CN-ES Semi-Prep and Preparative HPLC s (supplied in HPLC hardware format with 7bar/000psi pressure limit) 0mm 7mm 00mm mm 0mm 0mm 7.7mm ACE--008 ACE--708 ACE--008 ACE--08 ACE--08 ACE--08 0mm ACE--00 ACE--70 ACE--00 ACE--0 ACE--0 ACE--0.mm ACE--00 ACE--70 ACE--00 ACE--0 ACE--0 ACE--0 ACE 0µm CN-ES Analytical, Semi-Prep and Preparative HPLC s (supplied in HPLC hardware format with 7bar/000psi pressure limit) 0mm 7mm 00mm mm 0mm 0mm.mm ACE--0 ACE--7 ACE--0 ACE-- ACE-- ACE-- 7.7mm ACE--008 ACE--708 ACE--008 ACE--08 ACE--08 ACE--08 0mm ACE--00 ACE--70 ACE--00 ACE--0 ACE--0 ACE--0.mm ACE--00 ACE--70 ACE--00 ACE--0 ACE--0 ACE--0 0mm ACE--00 ACE--70 ACE ACE--0 ACE--0 ACE CN-ES Capillary and Nano HPLC s ACE CN-ES capillary and nano columns are available in mm, 0.mm (00µm), 00µm, 00µm and 7µm internal diameters with µm, µm and 0µm particle sizes. Please enquire for further information.

7 UHPLC and HPLC Method Development Kits Method development kits enable the optimum bonded phase for an application to be identified. ACE columns are available with a unique range of highly selective phases in µm, µm, µm and 0µm particle sizes, specially developed for challenging UHPLC and HPLC applications. The availability of a range of phases offering complementary selectivity enables resolution to be maximised, improves the chances of impurity detection and increases the speed at which methods can be systematically developed. Please enquire for further information. UHPLC and HPLC Accessories UHPLC Connectors Pressure rating >700 bar (>000 psi) Compatible with all UHPLC systems Compatible with all UHPLC column brands Eliminates poor connections Innovative reusable design Wrench Flat ¼ (0.cm) Proprietary PEEK cone Reuseable column connector with 0- threads for / D tubing ACE Excel UHPLC Connector (p/n EXL-CC0, 0 pack) All UHPLC column brands require correct installation in order to realise maximum column efficiency. To avoid connection problems, permanently swaged fittings are not recommended as they do not allow free movement between the tubing, fitting and column inlet on installation. This can result in a poorly connected column that shows unexpected peak tailing due to the introduction of extra column volume (dead volume) to the system. Alternatively, a leak at the inlet fitting connection may be observed. ACE Excel UHPLC Connectors (p/n EXL-CC0, 0 pack) enable the inlet end of UHPLC columns to be correctly installed every time. Their unique reuseable design ensures that they maintain a 700 bar (000 psi) pressure rating with repeated use, yet do not permanently swage onto the inlet tubing. To maximise the lifetime of the fitting, the use of torque wrench (p/n EXL-TW) is required. At the outlet end of the UHPLC column (where pressure demands are lower but a correct connection remains important), ACE Fingertight HPLC Connectors (p/n ACE-CC0, 0 pack, see overleaf) may alternatively be used. Note: For inlet connections onto a Waters Acquity system (containing a Waters Acquity / fitting and ferrule on the inlet tubing) the use of a pre-column filter incorporating the unique Waters Acquity column port profile is alternatively recommended (p/n EXL-PCF0/ACQ - 0 pack) to ensure maximum compatibility with the Waters Acquity system fittings. UHPLC Pre-column Filters To further extend column lifetime under UHPLC conditions up to 000 psi (000 bar), ACE Excel UHPLC Pre-column filters are recommended. By one estimate, over 70% of the failures of UHPLC and HPLC columns are caused by inlet frit plugging. Due to their ultra low dispersion design, column performance and retention remain unaffected, whilst the column inlet is protected from blockage. ACE Excel UHPLC Pre-column filters can be installed on any brand of UHPLC column in seconds. Simply finger tighten initially, then wrench tighten a further ¼ turn. 000 bar (000 psi) pressure rating Eliminates poor connections Compatible with all UHPLC column brands Compatible with all UHPLC systems including Waters Acquity Two UHPLC pre-column filter options are available, for compatibility with all UHPLC systems: ACE Excel UHPLC Pre-column Filters (p/n EXL-PCF0, 0 pack) are compatible with almost all UHPLC system configurations. For inlet connections onto a Waters Acquity system (containing a Waters Acquity / fitting and ferrule on the inlet tubing) the use of a pre-column filter incorporating the Waters Acquity column port profile is alternatively recommended (p/n EXL-PCF0/ACQ - 0 pack) to ensure maximum compatibility. ACE Excel UHPLC Pre- Filter (p/n EXL-PCF0, 0 pack) ACE Excel UHPLC Pre- Filter (Waters Acquity System Compatible) (p/n EXL-PCF0/ACQ, 0 pack) 7

8 UHPLC and HPLC Accessories (continued) HPLC Connectors Fingertight to 0 bar (000 psi) Reuseable and simple to install Eliminates poor connections Compatible with all HPLC column brands and instruments ACE Fingertight HPLC Connectors (p/n ACE-CC0, 0 pack) are recommended for the connection of both the inlet and outlet ends of HPLC columns. Manufactured from premium quality PEEK, the fittings simply hand tighten to provide a perfect column connection, and are pressure rated to 0 bar/000 psi. ACE Fingertight HPLC Connectors may additionally be used at the outlet end of UHPLC columns, where pressure demands are lower but a correct connection remains important. ACE Fingertight HPLC Connector (p/n ACE-CC0, 0 pack) HPLC Guard Cartridges Pressure rated to 7 bar (000 psi) Simple to install and exchange Increase HPLC column lifetime Guard cartridges are recommended to prevent both inlet frit blockage and irreversible sample adsorption onto the top of the column. Guard cartridges are available for all ACE HPLC columns from nano and capillary dimensions through to preparative scale. For assistance identifying the appropriate guard for the column selected, please contact our Technical Support Department at info@ace-hplc.com HPLC Pre-column Filters Pressure rated to 0 bar (000 psi) Simple to install and replace Effective low cost column protection Compatible with all HPLC column brands ACE Analytical HPLC Pre-column Filter (p/n ACE-CS0, 0 pack) ACE Microbore HPLC Pre-column Filter (p/n ACE-HP0, 0 pack) As an alternative to guard cartridges, pre-column filters may be used to protect the column inlet frit from blockage. Due to their ultra low dispersion design, column performance and retention remain unaffected. ACE HPLC pre-column filters utilise a PEEK fingertight design that connects directly into any / 0- internal thread column inlet, and are thus compatible with all HPLC column brands. For.0 -.mm id HPLC columns, the use of ACE Analytical HPLC Pre-column Filters (p/n ACE-CS0, 0 pack) are recommended. For.mm id HPLC columns, the use of ACE Microbore HPLC Pre-column Filters (p/n ACE-HP0, 0 pack) are recommended. ACE HPLC pre-column filters are simply hand tightened into the column inlet to achieve a pressure rating of 0 bar (000 psi). If higher pressure rating is required, ACE Excel UHPLC Pre-column Filters (suitable for use up to 000 bar/000 psi) are alternatively recommended. ACE and Excel are registered trademarks of Advanced Chromatography Technologies Limited. Comparative data may not be representative of all applications. Advanced Chromatography Technologies Limited acknowledges the registered and unregistered trademarks of Victrex Plc and Waters Corporation and has no association with either company. ACE UHPLC and HPLC columns are available through our international distributor network Available from: ACE UHPLC and HPLC s Advanced Chromatography Technologies Limited, Berry Street, Aberdeen, AB HF, Scotland Tel: + (0) 70 Fax: + (0) info@ace-hplc.com 00000Q/07/00-A

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