ACE C18-PFP. Hydrophobic and pentafluorophenyl mixed mode interaction. High efficiency 2µm, 3µm, 5µm and 10µm particles for UHPLC and HPLC

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1 ACE C-PFP A C bonded phase with unique selectivity F F F F F Guaranteed reproducibility Exceptional bonded phase stability Hydrophobic and pentafluorophenyl mixed mode interaction High efficiency µm, µm, µm and 0µm particles for UHPLC and HPLC UHPLC and HPLC Columns F F F F F

2 ACE C-PFP a C phase with unique selectivity Explore the Advantages of ACE C-PFP a unique C bonded HPLC column with the extra selectivity of a pentafluorophenyl (PFP) phase Combines C and PFP mechanisms of separation to separate mixtures not readily possible with either phase alone Improved retention of polar basic compounds for better separations Ultra inert, ultra high purity silica, for excellent peak shape and reproducibility Exceptional bonded phase stability for elevated temperature applications Ultra low bleed phase ensures UV and LC/MS compatibility Available in high throughput column dimensions High efficiency µm, µm, µm and 0µm particles for UHPLC and HPLC Contents Page Alternative Selectivity to Standard C Columns Improving Resolution Resolution Equation Improving Resolution Selectivity or Efficiency? PFP Separation Mechanisms Applications # Substituted Methoxybenzene Isomers Leading C Columns # Substituted Methoxybenzene Isomers PFP Columns # Substituted Dinitrobenzene Isomers # Pharmaceutically Relevant Analytes # Structurally Diverse Analytes # Acidic Analytes Comparison of Column Inertness Temperature and ph Stability Reproducibility and Scalability Low Bleed for UV and LC/MS Compatibility ACE Excel UHPLC Columns Material Characteristics Ordering Information ACE Excel UHPLC Columns and Accessories ACE HPLC Columns and Accessories ACE Capillary and Nano Columns ACE Method Development Kits ACE Ultra Inert Base Deactivated HPLC Columns

3 ACE C-PFP a C phase with unique selectivity Why do I need another new C phase? The use of an ultra pure, ultra inert silica has many recognised benefits including improved reproducibility, lifetime and chromatographic performance (particularly with basic molecules). However, since the ultra inert silica surface effectively no longer contributes to the separation, C columns manufactured with high purity silicas show near identical selectivity. It is therefore highly likely that a problem separation on one leading brand will not be significantly improved by changing to an alternative manufacturer s equivalent product. For many years, experienced chromatographers have been seeking phases with the proven performance and reproducibility benefits shown by such leading C column brands, but which additionally provide the alternative selectivity required for their challenging applications. How is ACE C-PFP different? C bonded phases currently dominate the HPLC market, with recent surveys indicating that they are still responsible for 0-0% of all HPLC columns sold. In recent years the use of PentaFluoroPhenyl (PFP) bonded phases has grown significantly due to the alternative selectivity they provide. However, compared to C bonded phases, PFP phases have traditionally been compromised with reduced hydrophobicity, reduced stability and significant column bleed. ACE C-PFP shows alternative selectivity to C bonded columns ACE C, The ACE C-PFP phase utilises a specially developed ligand combining a C chain with integral PFP functionality, resulting in a phase that maintains the hydrophobic, stability and low bleed characteristics of leading C phases, yet provides the multiple retention mechanisms of a PFP phase that are responsible for the unique selectivity of ACE C-PFP (as further detailed on page ). ACE C-PFP ACE C-PFP is a valuable method development tool a column combining C retention and stability with PFP selectivity R&D Team Leader, Leading Pharmaceutical Company 0 Compounds: ),,-trimethoxybenzene ),,-trimethoxybenzene ),-dimethoxybenzene Column: 0 x. mm, µm Flow Rate:.00 ml/min Temperature: 0 C Detection: UV, nm Mobile Phase: 0:0 v/v MeOH/H O When should I use ACE C-PFP? Due to their similar hydrophobic characteristics, ACE C-PFP columns may be used for applications in which standard C columns would normally be considered. However, due to its integral pentafluorophenyl functionality, ACE C-PFP is additionally recommended for separations that involve halogenated aromatic compounds, regioisomers and those analytes with differing shape constraints. As the applications contained within this brochure demonstrate, ACE C-PFP can be used to improve separations that are proving problematic on C columns. The unique ACE C-PFP phase provides an alternative selectivity to C columns, but remains a valid selection for methods in which C bonded columns are specified. In many instances, the same evaluation conditions that prove unsuitable for the C column prove suitable for the C-PFP column, avoiding the need for lengthy method redevelopment. ACE Ultra Inert Base Deactivated HPLC Columns

4 ACE C-PFP a C phase with unique selectivity Improve Chromatographic Resolution The goal of chromatographic separation is to obtain adequate resolution (R s ) of the components of interest in the minimum time. Baseline separation is achieved with a resolution of., although for rugged, reproducible methods that can be readily transferred between laboratories, a resolution of.-.0 is desirable. The resolution equation tells us what variables affect resolution: R s = Resolution between peaks of interest N=Efficiency measuredbytheoreticalplates α = Selectivity the ratio of retention (k values) for two peaks k = Retention factor the number of column volumes required to elute a peak Efficiency Selectivity Retention Factor Resolution, R s, can be increased by increasing either N, α or k. However, increasing either N or k to improve R s suffers from quickly diminishing returns, as can be seen graphically demonstrated in Figure below. For example, R s increases only with the square root of the increase in N. N can be increased by either adding column length or decreasing the particle size of the column packing material, or some combination of the two. Either way, the system back pressure increases with increases in N, so the cost of achieving a satisfactory separation by increasing N can be extremely high pressure. Similarly, increasing retention (k values) will increase R s, but also with quickly diminishing returns. Increasing k beyond a value of 0 is usually a poor trade-off between R s and analysis time, as only marginal gains in R s are achieved with increasing retention times. A graphic representation of this effect can also be seen in Figure below. Figure. The Effect of N, α and k on Resolution (R s ) For a typical separation where: N = 0,000, k =, α =. Resolution (R s ) Increasing N, α or k increases resolution (R s ). However, as can be seen from these plots, increasing either N or k suffers from quickly diminishing returns. Increasing selectivity (α) on the other hand, does not have this problem and, therefore, becomes the most powerful of these three variables to optimise when developing a separation. Increasing α increases R s but, unlike N and k, without the constraint of diminishing returns. Changes in α also have no effect on pressure and only negligible effects on separation time (see Figure ). Therefore, α is the most powerful variable to change when developing a separation. Optimising α can allow you to achieve satisfactory resolution between all peaks of interest, while keeping system back pressure and separation times acceptable. ACE Ultra Inert Base Deactivated HPLC Columns

5 ACE C-PFP a C phase with unique selectivity Improve Chromatographic Resolution Selectivity or Efficiency? Selectivity (α) is controlled by the mobile phase, temperature and stationary phase chemistry. Most method development strategies will explore all of these chromatographic variables. When sufficient resolution is not achieved with a standard µm C phase, it is recommended to optimise the chromatographic selectivity of the separation rather than the separation efficiency, as highlighted in the following example. By simply changing the stationary phase chemistry (i.e. column) to one with an alternative chromatographic selectivity, the desired resolution can be readily obtained on a standard HPLC system without the need for expensive UHPLC instrumentation. Complex mobile phase compositions, elevated temperature and aggressive ph conditions may also be avoided. Figure. Leveraging Selectivity to Achieve Fast, High Resolution Separations ACE C (P max = 0 bar), 0 Reducing particle size to sub-µm does not materially improve separation Leading <µm C Column (P max = 0 bar) 0 Increase Efficiency (N) (reduce particle size) Improved Efficiency Selectivity Improved Increase Selectivity (α) (change bonded phase) Enhanced resolution Using the power of selectivity gives a better separation ACE C-PFP (P max = 0 bar), Sample: ) paracetamol ) hydrochlorothiazide ) methylphenylsulphoxide ) methylphenylsulphone ) aspirin ) phenacetin ),-dinitrobenzene ),,-trimethoxybenzene ) ethylbenzoate 0) nimesulide ) ibuprofen ) indomethacin ) mefenamic acid Column Dimensions: 0 x. mm Flow Rate: 0.0 ml/min Temperature: 0 C Detection: UV, nm Mobile Phase: A = mm formic acid in H O and B = mm formic acid in MeOH, Gradient = to 00% B in minutes Comparative data may not be representative of all applications. Phenomenex columns were not used in the above comparison. Reducing particle size from µm to sub-µm whilst retaining the C bonded phase does not materially improve the separation and additionally results in a significant pressure increase. The ACE C-PFP column provides better selectivity (α) for the three critical pairs and therefore provides a superior separation compared to the sub-µm C column, even though the sub-µm column provides higher efficiency. Leveraging the power of selectivity leads to a better separation than that obtained by trying to force peaks apart using a column with a high plate count and high pressure. ACE Ultra Inert Base Deactivated HPLC Columns

6 ACE C-PFP a C phase with unique selectivity PFP Separation Mechanisms The ACE C-PFP phase exhibits multiple retention mechanisms including hydrophobic, π-π interaction, dipole-dipole, hydrogen bonding and shape selectivity. Whilst approximations of relative strengths are provided below, the predominance of each retention mechanism is dictated by the solute s physico/chemical properties, its structure and the chromatographic conditions employed. Separation Mechanism Typical C Typical PFP ACE C-PFP Hydrophobicity / π-π Interaction Dipole-Dipole Hydrogen Bonding Shape Selectivity π-π Interaction The PFP rings add aromatic character to the surface of the phase. However, PFP phases are different from phenyl phases since the electronegative fluorine atoms produce an electron deficient phenyl ring, which makes the PFP phase act as a Lewis acid. This will interact with an analyte able to donate electrons (i.e. a Lewis base). This is the opposite of phenyl phases, which contain an electron rich aromatic ring (due to the absence of electron withdrawing groups) and which therefore act as Lewis bases. Analyte Stationary phase Dipole-dipole and hydrogen bonding The carbon-fluorine bonds in the PFP ring are extremely polar. Therefore, PFP phases can additionally retain analytes by dipole-dipole or hydrogen bonding interactions that occur between the analyte and the electronegative fluorine atoms. Any such interactions will result in increased retention. Analytes Stationary phase Analytes Shape selectivity The PFP has a rigid ring structure which, when combined with the other retention mechanisms that are possible, confers outstanding shape selectivity on the PFP phase. Stationary phase The ACE C-PFP phase exhibits the multiple retention mechanisms of a PFP phase, which chromatographers may exploit in order to resolve mixtures that are difficult, if not impossible, to separate on traditional C phases (which rely primarily on hydrophobic retention mechanisms only). ACE Ultra Inert Base Deactivated HPLC Columns

7 ACE C-PFP a C phase with unique selectivity Application # - Substituted Methoxybenzene Isomers - Leading C Columns, Hydrophobic reference ACE C,,, Waters Sunfire. C,, Zorbax Eclipse XDB. C, Enhanced resolution ACE C-PFP Sample: ),,-trimethoxybenzene ),,-trimethoxybenzene ),-dimethoxybenzene ),-dimethoxybenzene ) methoxybenzene ),-dimethoxybenzene ),,-trimethoxybenzene ) neutral molecule (reference). Column Dimensions: 0 x. mm Flow Rate:.00 ml/min Temperature: 0 C Detection: UV, nm Mobile Phase: 0:0 v/v MeOH/H O Comparative data may not be representative of all applications. Please see page for acknowledgement of trademarks. The above example highlights the fact that leading C brands provide similar selectivity and all fail to separate tri-, di- and monomethoxybenzene isomers. The differences in absolute retention (as illustrated by the neutral reference marker) are due to purely hydrophobic effects and related to parent silica characteristics (e.g. surface area). The ACE C-PFP exhibits excellent resolution of all components, resulting from the integral PFP functionality contained within the unique ACE C-PFP ligand. ACE Ultra Inert Base Deactivated HPLC Columns

8 ACE C-PFP a C phase with unique selectivity Application # - Substituted Methoxybenzene Isomers - PFP Columns, Hydrophobic reference, ACE C Loss of Hydrophobicity Alternative selectivity but significant decrease in hydrophobicity Typical µm PFP (propyl spacer) ACE C-PFP Alternative selectivity but hydrophobicity maintained Sample: ),,-trimethoxybenzene ),,-trimethoxybenzene ),-dimethoxybenzene ),-dimethoxybenzene ) methoxybenzene ),-dimethoxybenzene ),,-trimethoxybenzene ) neutral molecule (reference) Column Dimensions: 0 x. mm Flow Rate:.00 ml/min Temperature: 0 C Detection: UV, nm Mobile Phase: 0:0 v/v MeOH/H O Comparative data may not be representative of all applications. Phenomenex columns were not used in the above comparison. The above application highlights the usefulness of pentafluorophenyl phases in the separation of regioisomers. As previously shown on page, standard C phases fail to separate the tri-, di- and monomethoxybenzene isomers. Traditional PFP phases provide an alternative selectivity but at the expense of a significant decrease in hydrophobicity, which compromises the separation. The ACE C-PFP maintains the hydrophobic characteristics of a C phase and provides a superior separation that can be further optimised to reduce analysis time. ACE Ultra Inert Base Deactivated HPLC Columns

9 ACE C-PFP a C phase with unique selectivity Application # - Substituted Dinitrobenzene Isomers Hydrophobic reference ACE C ACE C-PFP Enhanced resolution C-PFP achieves baseline separation of all isomers Sample: ),-dinitrobenzene ),-dinitrobenzene ),-dinitrobenzene ) neutral molecule (reference). Column Dimensions: 0 x. mm Flow Rate:.00 ml/min Temperature: 0 C Detection: UV, nm Mobile Phase: 0:0 v/v MeOH/H O This test containing aromatic dinitrobenzene isomers, performed under simple isocratic conditions, highlights that while these phases possess similar hydrophobicities, the C-PFP phase exhibits differing chromatographic selectivity (via an enhanced dipole-dipole interaction) towards the dinitrobenzene isomers compared to C phases. The C phase fails to separate the isomers under these conditions while the C-PFP achieves baseline separation of all the isomers due to its integral PFP functionality providing additional retention mechanisms. ACE Ultra Inert Base Deactivated HPLC Columns

10 ACE C-PFP a C phase with unique selectivity Application # - Pharmaceutically Relevant Analytes,,, ACE C 0, A typical PFP column Typical µm PFP (propyl spacer), 0 changes selectivity - but critical pairs still result C-PFP achieves ACE C-PFP 0 resolution of all components 0 Sample: ) sulphanilamide ) nizatidine ) metronidazole ) amiloride ) hydrochlorothiazide ) caffeine ) pindolol ) metoprolol ) phenacetin 0),-dinitrobenzene ) hexobarbitol ) furosemide ) piroxicam ) carvedilol ) ketoprofen ) ibuprofen ) indomethacin Column Dimensions: 0 x. mm Flow Rate: 0.0 ml/min Temperature: 0 C Detection: UV, nm Mobile Phase: A = 0.% v/v formic acid in H O and B = 0.% v/v formic acid in MeOH Gradient = to 00% B in minutes Comparative data may not be representative of all applications. Phenomenex columns were not used in the above comparison. The above application shows the separation of a range of pharmaceutically active analytes on an ACE C column, with sets of co-eluting peaks being observed. This separation on the ACE C is also consistent with that expected with other leading C column brands, which exhibit very similar selectivity due to the same (predominantly hydrophobic) retention mechanism. Changing to a typical PFP phase results in a change of selectivity, but different critical pairs now result. The same evaluation conditions that proved unsuitable for the ACE C and a typical PFP phase, were found to be suitable for the ACE C-PFP column, enabling resolution of all components including all critical pairs previously identified. ACE Ultra Inert Base Deactivated HPLC Columns

11 ACE C-PFP a C phase with unique selectivity Application # - Structurally Diverse Analytes ACE C, 0 ACE C-PFP pulls peaks apart! ACE C-PFP 0 0 Sample: ) paracetamol ) hydrochlorothiazide ) methylphenylsulphoxide ) methylphenylsulphone ) aspirin ) phenacetin ),-dinitrobenzene ),,-trimethoxybenzene ) ethylbenzoate 0) nimesulide ) ibuprofen ) indomethacin ) mefenamic acid Column Dimensions: 0 x. mm Flow Rate: 0.0 ml/min Temperature: 0 C Detection: UV, nm Mobile Phase: A = mm formic acid in H O and B = mm formic acid in MeOH Gradient = to 00% B in minutes Based upon the same ultra inert, ultra high purity silica platform as ACE C, the unique ACE C-PFP phase again provides alternative selectivity, leading to superior resolution of all structurally diverse analytes, without the need for lengthy method redevelopment. ACE Ultra Inert Base Deactivated HPLC Columns

12 ACE C-PFP a C phase with unique selectivity Application # - Acidic Analytes ACE C, 0 ACE C-PFP Additional retention mechanisms result in dramatically different selectivity 0 Sample: ) benzene sulphonic acid ) -hydroxybenzoic acid ) -hydroxybenzoic acid ) phenol ) -hydroxybenzoic acid ) sorbic acid ) benzoic acid ) dimethylphthalate ) -phenylpropionic acid 0) cinnamic acid ) -hydroxybenzoic acid propyl ester ) neutral molecule (reference) Column Dimensions: 0 x. mm, µm Flow Rate:.00 ml/min Temperature: 0 C Detection: UV, nm Mobile Phase = : v/v mm KH PO (ph.) in H O/MeOH The additional retention mechanism provided by the ACE C-PFP phase results in dramatically different chromatographic selectivity compared to a typical C phase, including many reversals in peak elution order. 0 ACE Ultra Inert Base Deactivated HPLC Columns

13 HPLC COLUMNS Comparison Data on Commonly Used C Phases Stationary Phase Specifications Phases Compared According to Relative Hydrophobicity Comparison of Column Efficiency for a Neutral Compound Phases Compared According to Metal Activity Phases Grouped According to Silanol Activity Comparison of Column Efficiency for Basic Compounds Categorization of Phases According to Silanol Activity and Inertness ACE C-PFP a C phase with unique selectivity Comparison of Column Inertness Leading µm, small pore C column brands 0 x. mm i.d. LC/MS compatible dimensions Basic molecule inertness test Peak efficiency and asymmetry investigation Peak Efficiency Comparison ACE C-PFP ACE C-AR ACE C Sunfire. C HyPurity C Zorbax XDB. C Zorbax SB. C Luna C() XBridge. C Inertsil ODS- XTerra MS. C Ascentis Express. C Hypersil BDS C Spherisorb ODS Symmetry. C Hypersil ODS 00,00,00,00 0,000,000,000,000 0,000 N 0. (pyridine) (plates/m),00,00 0,00,00,00,00 0,00,00,00,00,00 0,00 Lower Tailing Higher Tailing ACE C-PFP N 0. (pyr) = 0,00pl/m Waters XTerra MS. C N 0. (pyr) =,00pl/m Waters Symmetry. C N 0. (pyr) =,00pl/m Column Dimensions: 0 x. mm Sample: ) uracil ) pyridine ) phenol Mobile Phase: 0:0 v/v MeOH/H O Flow Rate: 0.0 ml/min Temperature: C Wavelength: nm Please see page for acknowledgement of trademarks. Comparative data may not be representative of all applications. Conclusion Significant differences in efficiency, peak shape and selectivity are seen when analysing pyridine a small highly basic molecule. Increased tailing and retention are indicative of undesirable secondary interactions between pyridine and silanol groups on the stationary phase surface. These interactions can also result in poor column reproducibility. ACE C columns have been previously independently tested and found to be exceptionally efficient and extremely inert. The ACE C-PFP maintains this excellent performance. Si-OH ACE Stationary Phases Virtually Eliminate the Negative Effects of Silanols on UHPLC and HPLC Separations Comparison Guide Guide TO C REVERSED P HASE Further inertness test data is contained within the ACE HPLC column catalogue and other ACE literature. Additionally, a Comparison Guide to C Columns is also available, detailing material characteristics for over 0 HPLC column brands and comparing performance with a number of test probes. Please contact your local distributor to request your copies. ACE Ultra Inert Base Deactivated HPLC Columns

14 ACE C-PFP a C phase with unique selectivity Excellent Temperature and ph Stability At low ph, column deterioration is caused by hydrolysis of the bonded phase, with a decrease in retention observed. The nature of the bonded phase, the purity of the silica surface and bonding density are all critical parameters. The use of a lower purity silica, a shorter ligand and a lower bonding density are all factors that will contribute to accelerated ligand cleavage and reduced column lifetime. Accelerated Column Stability Study - 0 C at ph. 00 ACE C-PFP (high purity silica) ACE C (high purity silica) 0 Conventional PFP (high purity silica, propyl spacer) % Initial k 0 Zorbax SB-C (moderate purity silica) 0,000 column volumes Waters Spherisorb ODS (low purity silica) Time (hours) Acidic Exposure Conditions: Mobile Phase: : v/v MeOH/0.% TFA in H O (ph.) Flow Rate: 0.0 ml/min Temperature: 0 C Column Dimensions: 0 x. mm Using conditions designed to accelerate column degradation, the ACE C-PFP phase shows little retention loss, with lifetime equivalent to the highly robust ACE C phase, suggesting that the ACE C-PFP may be suitable for applications in which PFP columns exhibit reduced lifetime. As expected, a C bonded column based upon a moderate purity silica (Zorbax SB-C a phase previously recognised to provide excellent stability for high temperature and low ph applications) and a low purity silica (Waters Spherisorb ODS) show significantly reduced lifetimes. Please see page for acknowledgement of trademarks. Comparative data may not be representative of all applications. Phenomenex columns were not used in the above comparison. ACE Ultra Inert Base Deactivated HPLC Columns

15 ACE C-PFP a C phase with unique selectivity Guaranteed Reproducibility and Fully Scalable Of equal importance to alternative selectivity is excellent reproducibility. Variations between different batches of stationary phase are the most common cause of customer concern. ACE stationary phases virtually eliminate the unpredictable negative effects of silanols on HPLC and UHPLC separations by maintaining a rigid control of the complete manufacturing process and establishing tight specifications for purity, selectivity, retention, efficiency and asymmetry. Therefore, as demonstrated in the figure below, absolute batch-to-batch and column-to-column reproducibility are guaranteed for all ACE C-PFP columns. Silica Batch # Parent Silica Batch Silane Batch Silane Batch # Silica Batch # Silane Batch # Silica Batch # Silane Batch # ACE C-PFP Particle Size Column Diameter LC/MS 0 x. mm 0. ml/min ACE C-PFP Analytical 0 x. mm.00 ml/min ACE 0 C-PFP Preparative 0 x. mm. ml/min Column: ACE C-PFP, 0 x. mm (unless specified otherwise) Sample: ) uracil ) -hydroxybenzoic acid ) acetylsalicylic acid ) benzoic acid ) -hydroxybenzoic acid ) ethyl paraben Mobile Phase: : v/v MeCN/0.% TFA in H O Temperature: C Wavelength: nm Flow Rate:.00 ml/min (unless specified otherwise) The availability of µm, µm, µm and 0µm particle sizes combined with a range of column dimensions from capillary through to preparative scale ensures that methods can be reproducibly scaled up or down. The chromatograms above demonstrate the excellent reproducibility achieved when silica batch and silane batch are changed, and the reproducible scalability obtained when changing particle size and column diameter. ACE Ultra Inert Base Deactivated HPLC Columns

16 ACE C-PFP a C phase with unique selectivity Low Bleed for UV and LC/MS Compatibility Many phases exhibit bleed of the bonded phase, which can be most clearly seen under gradient conditions when baseline stability is affected. Whilst most ultra pure C phases would be expected to give low column bleed, careful selection of an alternative selectivity bonded phase is required, to ensure that column bleed does not cause unforeseen problems when analysing at low UV wavelengths or by LC/MS. Note, absolute column bleeds depends on a number of factors and may vary from day to day and system to system. Therefore it is important, when comparing column bleed, to do so under controlled conditions. ACE C-PFP Exhibits Low Column Bleed for UV 0 ACE C 00 mau <0 mau mau Conventional µm PFP, 0 mau mau ACE C-PFP Compounds: ) nicotine ) benzylamine ) procainamide ) terbutaline ) phenol ) internal standard A ) internal standard B ) internal standard C ) nortriptyline Column Dimensions: 0 x. mm Flow Rate:.00 ml/min Temperature: 0 C Detection: UV, nm Mobile Phase: A = 0 mm KH PO in H O (ph.) B = : v/v 0 mm KH PO in H O (ph.) / MeOH Gradient: to % B in 0 mins, hold for mins at % B Comparative data may not be representative of all applications. Phenomenex columns were not used in the above comparison. <0 mau This example compares the low bleed characteristics of an ultra pure ACE C column (top) with the high bleed typical of a conventional PFP column (middle). The ACE C-PFP column (bottom) shows improved bleed levels compared to the PFP column and offers bleed levels comparable to the ACE C column, despite containing an integral PFP functionality which provides the alternative selectivity. For MS detection, the use of non-c phases has traditionally presented additional challenges and in extreme instances, column bleed can swamp the detector signal and mask the analyte of interest. TIC trace and MS spectra comparisons also show that ACE C-PFP columns provide extremely low levels of bleed, comparable to levels from a blank column run (with no column attached) and to bleed levels from leading C columns. Other typical bonded phases that might be considered to provide alternative selectivity to C columns (i.e. conventional phenyl, polar embedded, PFP and AQ type surface chemistries) would all be expected to show higher levels of bleed. Conclusion The ACE C-PFP phase may be used to provide alternative selectivity to leading C columns without encountering the column bleed issues associated with many alternative (i.e. non-c) bonded phases. ACE Ultra Inert Base Deactivated HPLC Columns

17 ACE C-PFP a C phase with unique selectivity ACE Excel UHPLC columns ACE Excel UHPLC C-PFP Column Advantages High efficiency µm, µm and µm particles Compatible with all UHPLC and HPLC systems Exceptional reproducibility and column lifetime Fully scalable with other ACE particle sizes Rugged and reliable day-to-day performance Robust up to,000 bar (,000psi) ACE C-PFP is available as Excel UHPLC high efficiency µm, µm and µm particle size columns, for demanding UHPLC applications. Selectivity is unchanged from the µm, µm and 0µm ACE HPLC C-PFP columns, making scale-up from UHPLC to HPLC, or vice versa, simple and reproducible. ACE Excel UHPLC C-PFP columns are fully compatible with all commercial UHPLC systems and are stable up to,000 bar (,000psi). The low dead volume dual compatible UHPLC/HPLC Excel column hardware has been optimised to take full advantage of the low dispersion of modern UHPLC instruments. Additionally, all ACE Excel UHPLC columns are manufactured using a proprietary HSC TM (High Stability Column) manufacturing process that results in ultra robust UHPLC columns. To further extend ACE C-PFP column lifetimes at UHPLC column pressures, ACE UHPLC pre-column filters are recommended (see page ). ACE columns have led the way in offering C bonded phases with the advantage of extra selectivity. The ACE C-PFP and other ACE extra selectivity phases, such as the C-AR, C-Amide, CN-ES and SuperC have proved to be extremely valuable tools for achieving separations that may not be possible with standard C bonded phases. These extra selectivity phases are not meant to replace standard C bonded phases, but rather complement them and so provide chromatographers with powerful, additional mechanisms of separation that can be used to achieve better overall chromatographic results. The availability of ACE Excel UHPLC columns means that chromatographers can combine the power of bonded phase selectivity with the efficiency and speed of UHPLC, providing chromatographers with more choices to achieve better results. ACE Excel delivers excellent resolution and peak shape Application #0 Pharmaceuticals and Related Compounds by UHPLC Mobile Phase: A = mm formic acid in H O and B = mm formic acid in MeOH Gradient: to 00% B in minutes Flow Rate: 0. ml/min Temperature: 0 C Detection: UV, nm Column Dimensions: 0 x.mm ACE Excel C Zorbax Eclipse. XDB C Waters ACQUITY. BEH C Phenomenex Kinetex. C ACE Excel C-PFP. paracetamol. hydrochlorothiazide. methylphenylsulphoxide. methylphenylsulphone. aspirin. phenacetin.,-dinitrobenzene Comparative data may not be representative of all applications. Please see p. for acknowledgement of trademarks..,,-trimethoxybenzene. ethylbenzoate 0. nimesulide. ibuprofen. indomethacin. mefenamic acid P max = 0 bar P max = bar P max = 0 bar The above example highlights that these leading C brands provide similar selectivity and fail to fully resolve the components under these conditions, with or more critical pairs non baseline resolved in each case. The unique selectivity of the ACE Excel C-PFP phase enables improved separation. P max = bar P max = bar ACE Ultra Inert Base Deactivated HPLC Columns

18 ACE C-PFP a C phase with unique selectivity Material Characteristics PHASE FUNCTIONAL GROUP ENDCAPPED PARTICLE PORE SIZE (µm) SIZE (Å) SURFACE AREA (m /g) CARBON LOAD (%) RECOMMENDED ph RANGE USP LISTING ACE C-PFP Proprietary octadecyl with Yes,,, a L embedded PFP functionality a For optimum column lifetime, a ph range of - is recommended. To increase column lifetime at high 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. UHPLC and HPLC Column Options ACE C-PFP columns are offered in different hardware formats. ACE Excel UHPLC columns are recommended for more demanding UHPLC applications where high pressures and/or flow rates are typically used. ACE HPLC columns are available in a wider range of dimensions from capillary to preparative scale. Identical column selectivity is achieved with both hardwares. ACE Excel C-PFP UHPLC Columns ACE Excel C-PFP UHPLC columns are available with µm, µm and µm particles, using unique Excel hardware specially optimised to take full advantage of low dispersion modern UHPLC instruments. All ACE Excel UHPLC columns are manufactured using a proprietary HSC (High Stability Column) manufacturing process that results in ultra robust UHPLC columns rated for use at pressures up to,000bar/,000psi. ACE Excel µm C-PFP UHPLC/HPLC Columns (supplied in dual compatible UHPLC/HPLC hardware format with 000bar/000psi pressure limit) COLUMN DIAMETER COLUMN LENGTH 0 mm 0 mm mm 0 mm mm 00 mm mm 0 mm PRE-COLUMN FILTER.mm EXL-00-00U EXL-00-00U EXL-00-0U EXL-00-00U EXL-00-0U EXL-00-00U EXL-00-0U EXL-00-0U EXL-PCF0.0mm EXL-00-00U EXL-00-00U EXL-00-0U EXL-00-00U EXL-00-0U EXL-00-00U EXL-00-0U EXL-00-0U EXL-PCF0.mm EXL-00-0U EXL-00-0U EXL-00-U EXL-00-0U EXL-00-U EXL-00-0U EXL-00-U EXL-00-U EXL-PCF0 ACE Excel µm C-PFP UHPLC/HPLC Columns (supplied in dual compatible UHPLC/HPLC hardware format with 000bar/000psi pressure limit) COLUMN DIAMETER COLUMN LENGTH 0 mm 0 mm mm 0 mm mm 00 mm mm 0 mm 0 mm PRE-COLUMN FILTER.mm EXL-0-00U EXL-0-00U EXL-0-0U EXL-0-00U EXL-0-0U EXL-0-00U EXL-0-0U EXL-0-0U EXL-0-0U EXL-PCF0.0mm EXL-0-00U EXL-0-00U EXL-0-0U EXL-0-00U EXL-0-0U EXL-0-00U EXL-0-0U EXL-0-0U EXL-0-0U EXL-PCF0.mm EXL-0-0U EXL-0-0U EXL-0-U EXL-0-0U EXL-0-U EXL-0-0U EXL-0-U EXL-0-U EXL-0-U EXL-PCF0 ACE Excel µm C-PFP UHPLC/HPLC Columns (supplied in dual compatible UHPLC/HPLC hardware format with 000bar/000psi pressure limit) COLUMN DIAMETER COLUMN LENGTH 0 mm 0 mm mm 0 mm mm 00 mm mm 0 mm 0 mm PRE-COLUMN FILTER.mm EXL-0-00U EXL-0-00U EXL-0-0U EXL-0-00U EXL-0-0U EXL-0-00U EXL-0-0U EXL-0-0U EXL-0-0U EXL-PCF0.0mm EXL-0-00U EXL-0-00U EXL-0-0U EXL-0-00U EXL-0-0U EXL-0-00U EXL-0-0U EXL-0-0U EXL-0-0U EXL-PCF0.mm EXL-0-0U EXL-0-0U EXL-0-U EXL-0-0U EXL-0-U EXL-0-0U EXL-0-U EXL-0-U EXL-0-U EXL-PCF0 0/pk. Compatible with all UHPLC column brands and all UHPLC instruments. For inlet connections onto a Waters Acquity system a pre-column filter incorporating the unique Waters Acquity column port profile (p/n EXL-PCF0/ACQ) is alternatively recommended. ACE Pre-column Filters for UHPLC To further extend column lifetimes under UHPLC conditions the use of ACE UHPLC Pre-column filters is recommended. ACE UHPLC Pre-column filters are pressure rated to,000bar (,000psi), can be installed simply in seconds and are compatible with all manufacturers UHPLC instruments and all brands of UHPLC columns. Please contact us for further information. ACE UHPLC Column Connectors ACE UHPLC column connectors are ultra low dispersion and compatible with all brands of UHPLC columns and all manufacturers instruments. They are pressure rated to,00 bar (,000 psi). These connectors are simple to install and are reusable. Please contact us for further information. ACE UHPLC Pre-column Filters (p/n EXL-PCF0, 0 pack and p/n EXL-PCF0/ACQ, 0 pack) ACE UHPLC Column Connector (p/n EXL-CC0, 0 pack) ACE Ultra Inert Base Deactivated HPLC Columns

19 ACE C-PFP a C phase with unique selectivity ACE C-PFP HPLC Columns ACE C-PFP HPLC columns are available with µm, µm and 0µm particles, in a wider range of dimensions than available in the ACE Excel UHPLC column format. For optimum column lifetime, a maximum operating pressure of bar/,000psi is recommended. ACE µm C-PFP HPLC Columns (supplied in HPLC hardware format with bar/000psi pressure limit) COLUMN DIAMETER COLUMN LENGTH 0 mm 0 mm mm 0 mm mm 00 mm mm 0 mm 0 mm GUARD CARTRIDGE.0mm - ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 b ACE-0-00GD.mm ACE-0-00 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 b ACE-0-00GD.0mm ACE-0-00 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 b ACE-0-00GD.0mm - - ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 b ACE-0-00GD.mm ACE-0-0 ACE-0-0 ACE-0- ACE-0-0 ACE-0- ACE-0-0 ACE-0- ACE-0- ACE-0- b ACE-0-00GD ACE µm C-PFP HPLC Columns (supplied in HPLC hardware format with bar/000psi pressure limit) COLUMN DIAMETER COLUMN LENGTH 0 mm 0 mm mm 0 mm mm 00 mm mm b Consider operating pressure limitations for maximum column lifetime 0 mm 0 mm GUARD CARTRIDGE.0mm - ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD.mm ACE-0-00 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD.0mm ACE-0-00 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD.0mm - - ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD.mm ACE-0-0 ACE-0-0 ACE-0- ACE-0-0 ACE-0- ACE-0-0 ACE-0- ACE-0- ACE-0- ACE-0-00GD.mm ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD 0mm ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD.mm ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD 0mm ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD ACE 0µm C-PFP HPLC Columns (supplied in HPLC hardware format with bar/000psi pressure limit) COLUMN DIAMETER COLUMN LENGTH 0 mm 0 mm mm 0 mm mm 00 mm mm 0 mm 0 mm GUARD CARTRIDGE.mm ACE-0-0 ACE-0-0 ACE-0- ACE-0-0 ACE-0- ACE-0-0 ACE-0- ACE-0- ACE-0- ACE-0-00GD.mm ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD 0mm ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD.mm ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD 0mm ACE-0-00 ACE-0-0 ACE-0-00 ACE-0-0 ACE-0-0 ACE-0-0 ACE-0-00GD pack, use with cartridge holder H000 and column coupler C000 When using guards, cartridge holder H000 and column coupler C000 required pack, use with integral microbore cartridge holder H000 (not 0mm column length) pack use with semi-prep cartridge holder H000 and column coupler C000 pack, use with integral analytical cartridge holder H000 (not 0mm column length) pack use with prep cartridge holder H000 and column coupler C000 ACE C-PFP Capillary and Nano HPLC Columns ACE C-PFP capillary and nano columns are available in 0.mm (00µm), 00µm, 00µm and µm internal diameters with µm and µm particle sizes. Please enquire for further information. ACE Pre-column Filters for HPLC To further extend column lifetimes under HPLC conditions the use of ACE HPLC Pre-column filters is recommended. ACE HPLC Pre-column filters are simple to install and replace, are compatible with all HPLC column brands and systems and offer effective low cost column protection. Contact us for further details. ACE HPLC Column Connectors ACE HPLC column connectors are compatible with all brands of HPLC columns and all manufacturers instruments. Please contact us for further information. ACE 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 range of unique, highly selective phases in µm, µm, µm and 0µm particle sizes. Please enquire for further information. ACE Analytical and Microbore Pre-column Filters (p/n ACE-CS0, 0 pack and p/n ACE-HP0, 0 pack) ACE Fingertight HPLC Column Connector (p/n ACE-CC0, 0 pack) ACE and ACE Excel are trademarks of Advanced Chromatography Technologies Limited. Advanced Chromatography Technologies Limited acknowledges the registered and unregistered trademarks of Sigma-Aldrich Co., Thermo Scientific, Phenomenex Inc., GL Sciences, Waters Corporation, Agilent Technologies and has no affiliation with any of these companies. ACE Ultra Inert Base Deactivated HPLC Columns

20 ACE Extra Selectivity robust UHPLC/HPLC phases ACE C-PFP is one of a range of phases that have been specially developed to provide robust, alternative selectivity for UHPLC and HPLC method development. To find out more contact your local ACE distributor or info@ace-hplc.com ACE products are available through our international network of distributors For further information please contact Authorised Distributor: Hichrom Limited The Markham Centre, Station Road Theale, Reading, Berks, RG PE Tel: Fax: 0 sales@hichrom.co.uk Advanced Chromatography Technologies Limited, Berry Street, Aberdeen, AB HF, Scotland Tel: + (0) 0 Fax: + (0) 0 info@ace-hplc.com P V