ACE C18-PFP. A C18 bonded phase with unique selectivity. s Guaranteed reproducibility. lity. s Exceptional bonded phase stability

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1 ACE CPFP A C bonded phase with unique selectivity & & & & & s Guaranteed reproducibility lity s Exceptional bonded phase stability s Hydrophobic and pentafluorophenyl mixed mode interaction HPLC Columns & & & & &

2 ACE CPFP a C phase with unique selectivity Explore the Advantages of ACE CPFP 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 possible with either phase alone mproved retention of polar basic compounds for better separations Exceptional bonded phase stability for elevated temperature applications Ultra inert, ultra high purity silica, for excellent peak shape and reproducibility Ultra low bleed phase ensures UV and LC/MS compatibility Available in high throughput column dimensions Contents Page Alternate Selectivity to Standard C Columns mproving Resolution Resolution Equation mproving Resolution Selectivity or Efficiency? PFP Separation Mechanisms Applications # Substituted Methoxybenzene somers Leading C Columns # Substituted Methoxybenzene somers PFP Columns # Substituted Dinitrobenzene somers # Pharmaceutically Relevant Analytes # Structurally Diverse Analytes # Acidic Analytes # Catecholamines Comparison of Column nertness Temperature and ph Stability Reproducibility and Scalability Low Bleed for UV and LC/MS Compatibility Material Characteristics Ordering nformation Columns Method Validation Kits Australia & New Zealand contact: info@winlab.com.au or call + ()

3 ACE CPFP a C phase with unique selectivity Why do 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. t is therefore highly likely that a problem separation on one leading brand will not be significantly improved by changing to an alternate 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 alternate selectivity required for their challenging applications. How is ACE CPFP different? C bonded phases currently dominate the HPLC market, with recent surveys indicating that they are still responsible for % of all HPLC columns sold. n recent years the use of PentaFluoroPhenyl (PFP) bonded phases has grown significantly due to the alternate selectivity they provide. ACE CPFP shows alternate selectivity to C bonded columns, ACE C However, compared to C bonded phases, PFP phases have traditionally been compromised with reduced hydrophobicity, reduced stability and significant column bleed. The ACE CPFP 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 CPFP (as further detailed on page ). ACE CPFP ACE CPFP is a valuable method development tool a column combining C retention and stability with PFP selectivity R&D Team Leader, Leading Pharmaceutical Company Compounds: ),,trimethoxybenzene ),,trimethoxybenzene ),dimethoxybenzene Column: x. mm, μm Flow Rate:. ml/min Temperature: C Detection: UV, nm Mobile Phase: : v/v MeOH/HO When should use ACE CPFP? Due to their similar hydrophobic characteristics, ACE CPFP columns may be used for applications in which standard C columns would normally be considered. However, due to its integral pentafluorophenyl functionality, ACE CPFP 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 CPFP can be used to improve separations that are proving problematic on C columns. The unique ACE CPFP phase provides an alternate selectivity to C columns, but remains a valid selection for methods in which C bonded columns are specified. n many instances, the same evaluation conditions that prove unsuitable for the C column prove suitable for the CPFP column, avoiding the need for lengthy method redevelopment. Australia & New Zealand contact: info@winlab.com.au or call + ()

4 ACE CPFP a C phase with unique selectivity mprove Chromatographic Resolution The goal of chromatographic separation is to obtain adequate resolution (Rs) 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.. is desirable. The resolution equation tells us what variables affect resolution: Rs N α k Efficiency Selectivity = = = = Resolution between peaks of interest Efficiency measured by theoretical plates Selectivity the ratio of retention (k values) for two peaks Retention factor the number of column volumes required to elute a peak Retention Factor Resolution, Rs, can be increased by increasing either N, α or k. However, increasing either N or k to improve Rs suffers from quickly diminishing returns, as can be seen graphically demonstrated in Figure below. For example, Rs 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 Rs, but also with quickly diminishing returns. ncreasing k beyond a value of is usually a poor tradeoff between Rs and analysis time, as only marginal gains in Rs 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 (Rs) For a typical separation where: N =,, k =, α =. Resolution (Rs) ncreasing N, α or k increases resolution (Rs). However, as can be seen from these plots, increasing either N or k suffers from quickly diminishing returns. ncreasing 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. ncreasing α increases Rs 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. Australia & New Zealand contact: info@winlab.com.au or call + ()

5 ACE CPFP a C phase with unique selectivity mprove 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 alternate 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 (Pmax = bar), e size to Reducing particl not subμm does e separation materially improv Ef fic ncrease Selectivity (α) (change bonded phase) ed ov pr ty m ivi ct le Se m pr ov ed ie nc y ncrease Efficiency (N) (reduce particle size) Using the pow er of selectivity give s a better separation Leading <μm C Column ACE CPFP Enhanced resolution (Pmax = bar) (Pmax = bar), Sample: ) paracetamol ) hydrochlorothiazide ) methylphenylsulphoxide ) methylphenylsulphone ) aspirin ) phenacetin ),dinitrobenzene ),,trimethoxybenzene ) ethylbenzoate ) nimesulide ) ibuprofen ) indomethacin ) mefenamic acid Column Dimensions: x. mm Flow Rate:. ml/min Temperature: C Detection: UV, nm Mobile Phase: A = mm formic acid in HO and B = mm formic acid in MeOH, Gradient = to % B in minutes Reducing particle size from μm to subμm whilst retaining the C bonded phase does not materially improve the separation and additionally results in a significant pressure increase. The ACE CPFP column provides better selectivity (α) for the three critical pairs and therefore provides a superior separation compared to the subμm C column, even though the subμm 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. Australia & New Zealand contact: info@winlab.com.au or call + ()

6 ACE CPFP a C phase with unique selectivity PFP Separation Mechanisms The ACE CPFP phase exhibits multiple retention mechanisms including hydrophobic, ππ interaction, dipoledipole, 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 CPFP Hydrophobicity / ππ nteraction DipoleDipole Hydrogen Bonding Shape Selectivity ππ nteraction 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). Analyte Stationary phase 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. Dipoledipole and hydrogen bonding The carbonfluorine bonds in the PFP ring are extremely polar. Therefore, PFP phases can additionally retain analytes by dipoledipole 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 CPFP 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). Australia & New Zealand contact: info@winlab.com.au or call + ()

7 ACE CPFP a C phase with unique selectivity Application # Substituted Methoxybenzene somers Leading C Columns, Hydrophobic reference, ACE C,, Waters Sunfire. C,,, Zorbax Eclipse XDB. C,, Luna C() Enhanced resolution ACE CPFP Sample: ),,trimethoxybenzene ),,trimethoxybenzene ),dimethoxybenzene ),dimethoxybenzene ) methoxybenzene ),dimethoxybenzene ),,trimethoxybenzene ) neutral molecule (reference). Column Dimensions: x. mm Flow Rate:. ml/min Temperature: C Detection: UV, nm Mobile Phase: : v/v MeOH/HO 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 CPFP exhibits excellent resolution of all components, resulting from the integral PFP functionality contained within the unique ACE CPFP ligand. Australia & New Zealand contact: info@winlab.com.au or call + ()

8 ACE CPFP a C phase with unique selectivity Application # Substituted Methoxybenzene somers PFP Columns, Hydrophobic reference, ACE C selectivity Alternate rease icant dec but signif obicity in hydroph Loss of Hydrophobicity Typical μm PFP selectivity Alternate phobicity but hydro maintained ACE CPFP Sample: ),,trimethoxybenzene ),,trimethoxybenzene ),dimethoxybenzene ),dimethoxybenzene ) methoxybenzene ),dimethoxybenzene ),,trimethoxybenzene ) neutral molecule (reference) Column Dimensions: x. mm Flow Rate:. ml/min Temperature: C Detection: UV, nm Mobile Phase: : v/v MeOH/HO 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 alternate selectivity but at the expense of a significant decrease in hydrophobicity, which compromises the separation. The ACE CPFP maintains the hydrophobic characteristics of a C phase and provides a superior separation that can be further optimised to reduce analysis time. Australia & New Zealand contact: info@winlab.com.au or call + ()

9 ACE CPFP a C phase with unique selectivity Application # Substituted Dinitrobenzene somers Hydrophobic reference ACE C, Luna C() Enhanced resolution achieves CPFP paration baseline se omers of all is ACE CPFP Sample: ),dinitrobenzene ),dinitrobenzene ),dinitrobenzene ) neutral molecule (reference). Column Dimensions: x. mm Flow Rate:. ml/min Temperature: C Detection: UV, nm Mobile Phase: : v/v MeOH/HO This test containing aromatic dinitrobenzene isomers, performed under simple isocratic conditions, highlights that while the three phases possess similar hydrophobicities, the CPFP phase exhibits differing chromatographic selectivity (via an enhanced dipoledipole interaction) towards the dinitrobenzene isomers compared to the C phases. Both C phases fail to separate the isomers under these conditions while the CPFP achieves baseline separation of all the isomers due to its integral PFP functionality providing additional retention mechanisms. Australia & New Zealand contact: info@winlab.com.au or call + ()

10 ACE CPFP a C phase with unique selectivity Application # Pharmaceutically Relevant Analytes,,, ACE C, Typical μm PFP A typical PFP colu mn changes se lectivity but critic al pairs still result, CPFP ACE CPFP resolution achieves of all compo nents Sample: ) sulphanilamide ) nizatidine ) metronidazole ) amiloride ) hydrochlorothiazide ) caffeine ) pindolol ) metoprolol ) phenacetin ),dinitrobenzene ) hexobarbitol ) furosemide ) piroxicam ) carvedilol ) ketoprofen ) ibuprofen ) indomethacin Column Dimensions: x. mm Flow Rate:. ml/min Temperature: C Detection: UV, nm Mobile Phase: A =.% v/v formic acid in HO and B =.% v/v formic acid in MeOH Gradient = to % B in minutes The above application shows the separation of a range of pharmaceutically active analytes on an ACE C column, with sets of coeluting 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 CPFP column, enabling resolution of all components including all critical pairs previously identified. Australia & New Zealand contact: info@winlab.com.au or call + ()

11 ACE CPFP a C phase with unique selectivity Application # Structurally Diverse Analytes ACE C, ACE C PFP pulls peak s apart! ACE CPFP Sample: ) paracetamol ) hydrochlorothiazide ) methylphenylsulphoxide ) methylphenylsulphone ) aspirin ) phenacetin ),dinitrobenzene ),,trimethoxybenzene ) ethylbenzoate ) nimesulide ) ibuprofen ) indomethacin ) mefenamic acid Column Dimensions: x. mm Flow Rate:. ml/min Temperature: C Detection: UV, nm Mobile Phase: A = mm formic acid in HO and B = mm formic acid in MeOH Gradient = to % B in minutes Based upon the same ultra inert, ultra high purity silica platform as ACE C, the unique ACE CPFP phase again provides alternate selectivity, leading to superior resolution of all structurally diverse analytes, without the need for lengthy method redevelopment. Australia & New Zealand contact: info@winlab.com.au or call + ()

12 ACE CPFP a C phase with unique selectivity Application # Acidic Analytes ACE C Sample: ) benzene sulphonic acid ) hydroxybenzoic acid ) hydroxybenzoic acid ) phenol ) hydroxybenzoic acid ) sorbic acid ) benzoic acid ) dimethylphthalate ) phenylpropionic acid ) cinnamic acid ) hydroxybenzoic acid propyl ester ) neutral molecule (reference), n l retentio Additiona in s result mechanism ent er lly diff dramatica selectivity ACE CPFP Column Dimensions: x. mm, μm Flow Rate:. ml/min Temperature: C Detection: UV, nm Mobile Phase = : v/v mm KHPO (ph.) in HO/MeOH The additional retention mechanism provided by the ACE CPFP phase results in dramatically different chromatographic selectivity compared to a typical C phase, including many reversals in peak elution order. Application # Catecholamines Typical μm PFP ACE CPFP interaction drophobic Added hy paration better se leads to Sample: ) norepinephrine ) levodopa ) epinephrine ) tyrosine ) dopamine Column Dimensions: x. mm, μm Flow Rate:. ml/min Temperature: C Detection: UV, nm Mobile Phase =. mm ammonium formate (ph.) in HO.. This separation of catecholamines illustrates how strong hydrophobic binding interaction on the ACE CPFP provides a better separation of levodopa and epinephrine than is achieved by a typical PFP with weak hydrophobic binding interaction. Australia & New Zealand contact: info@winlab.com.au or call + ()

13 ACE CPFP a C phase with unique selectivity Comparison of Column nertness Leading μm, small pore C column brands x. mm i.d. LC/MS compatible dimensions Basic molecule inertness test Peak efficiency and asymmetry investigation Peak Efficiency Comparison ACE CPFP, ACE CAR, ACE C Lower Tailing,, Sunfire. C HyPurity C, Zorbax XDB. C, Zorbax SB. C,, Luna C() XBridge. C, nertsil ODS,, XTerra MS. C Ascentis Express. C, Hypersil BDS C, Spherisorb ODS Higher Tailing, Symmetry. C, Hypersil ODS,,,,, N. (pyridine) (plates/m) ACE CPFP Waters XTerra MS. C Waters Symmetry. C N.(pyr) =,pl/m N.(pyr) =,pl/m N.(pyr) =,pl/m Column Dimensions: x. mm Sample: ) uracil ) pyridine ) phenol Mobile Phase: : v/v MeOH/HO Flow Rate:. ml/min Temperature: C Wavelength: nm Conclusion Significant differences in efficiency, peak shape and selectivity are seen when analysing pyridine a small highly basic molecule. ncreased 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 ACE C columns have been previously independently tested and found to be the highest efficiency, most inert columns available. The new ACE CPFP maintains this excellent performance. Comparison p Guide ud TO C REVE HPLC COLUM SED S PHA E Com a son a a on omm n y U ed C P as s a o a y h se p i c i ns h s s o p r da od g or l i e y r h bc y o p r on f o mn f c n y o a N u a C m o nd h s s o p r da od g om t l c v y h s s r u e A c dn t Sl n l t iy o p r on f o mn f c n y o B s c o p u ds a g r a on f h es c o i g o i n l c v y a d n t e s SiOH Stationary Phases Virtually Eliminate the Negative Effects of Silanols on HPLC Separations Further inertness test data is contained within the current ACE HPLC column catalogue. Additionally, a Comparison Guide to C Columns is also available, detailing material characteristics for over HPLC column brands and comparing performance with a number of test probes. Please contact your local distributor to request your copies. Australia & New Zealand contact: info@winlab.com.au or call + ()

14 ACE CPFP 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 C at ph. ACE CPFP (high purity silica) ACE C (high purity silica) Leading PFP % nitial k (high purity silica) Zorbax SBC (moderate purity silica) Waters Spherisorb ODS (low purity silica), column volumes Acidic Exposure Conditions: Mobile Phase: Flow Rate: Temperature: Time (hours) : v/v MeOH/.% TFA in HO (ph.). ml/min C Column Dimensions: x. mm Using conditions designed to accelerate column degradation, the ACE CPFP phase shows little retention loss, with lifetime equivalent to the highly robust ACE C phase, suggesting that the ACE CPFP 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 SBC 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. Australia & New Zealand contact: info@winlab.com.au or call + ()

15 ACE CPFP a C phase with unique selectivity Guaranteed Reproducibility and Fully Scalable Of equal importance to alternate 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 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 batchtobatch and columntocolumn reproducibility are guaranteed for all ACE CPFP columns. Parent Silica Batch Silane Batch Silica Batch # Silane Batch # Silica Batch # Silane Batch # Silica Batch # Silane Batch # Particle Size Column Diameter ACE CPFP LC/MS x. mm. ml/min Analytical x. mm. ml/min ACE CPFP Preparative ACE CPFP x. mm. ml/min Column: ACE CPFP, x. mm (unless specified otherwise) Sample: ) uracil ) hydroxybenzoic acid ) acetylsalicylic acid ) benzoic acid ) hydroxybenzoic acid ) ethyl paraben Mobile Phase: : v/v MeCN/.% TFA in HO Temperature: C Wavelength: nm Flow Rate:. ml/min (unless specified otherwise) The availability of μm, μm and μm 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. Australia & New Zealand contact: info@winlab.com.au or call + ()

16 ACE CPFP a C phase with unique selectivity Low Bleed for UV and LC/MS Compatibility Many phases exhibit bleed of the bonded phase, which is most clearly seen under gradient conditions when baseline stability is affected. Whilst most ultra pure C phases can be expected to give low column bleed, careful selection of an alternate 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 bleed 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 CPFP Exhibits Low Column Bleed for UV ACE C mau < mau mau Leading μm PFP, mau ACE CPFP mau < mau Compounds: ) nicotine ) benzylamine ) procainamide ) terbutaline ) phenol ) internal standard A ) internal standard B ) internal standard C ) nortriptyline Column Dimensions: x. mm Flow Rate:. ml/min Temperature: C Detection: UV, nm Mobile Phase: A = mm KHPO in HO (ph.) B = : v/v mm KHPO in HO (ph.) / MeOH Gradient: to % B in mins, hold for mins at % B This example compares the low bleed characteristics of an ultra pure ACE C column (top) with the high bleed typical of PFP columns (middle). The ACE CPFP column (bottom) shows bleed levels comparable to the ACE C, despite containing an integral PFP functionality, which provides the alternate selectivity. Comparison of UV Bleed Evaluation of Leading C Brands and Popular Alternate Bonded Phases ACE CPFP < Leading C columns < Leading phenyl columns Low Bleed Leading polar embedded columns Leading PFP columns High Bleed Column bleed at nm (mau) Further analysis of a wider range of columns under the same conditions confirms that leading high purity C column brands show similarly low levels of column bleed. However, the evaluation of alternate bonded phases traditionally recommended to change selectivity (i.e. phenyl, polar embedded and PFP surface chemistries) reveal that all these nonc columns show significantly higher bleed. The ACE CPFP combines a low bleed level (typical of the leading C column brands) with an alternate selectivity, thus providing the analyst with a valuable method development tool. Australia & New Zealand contact: info@winlab.com.au or call + ()

17 ACE CPFP a C phase with unique selectivity Low Bleed for UV and LC/MS Compatibility (cont d) When detecting by MS, the use of nonc phases has traditionally presented additional challenges to the analyst. n extreme instances, column bleed may swamp the detector signal and mask the analyte of interest. n the following example, column bleed is monitored in the minute segment of the gradient run, at which point the % organic increases to its maximum level and column bleed is therefore also highest. The MS Spectra (left series) provides an m/z breakdown of the bleed detected during this minute window, whereas the Total on Chromatogram (right series) illustrates the bleed obtained during this same minute time period. ACE CPFP Exhibits Low Column Bleed for LC/MS MS Spectra Leading Polar Embedded column Total on Chromatogram (TC) cps Leading C column Abundance Relative Abundance cps ACE CPFP cps No column (blank) cps Column Dimensions: x. mm Flow Rate:. ml/min Temperature: C Detection: +ve ES fast scan mode (Agilent MSD), full scan range m/z time (mins) m/z Mobile Phase: A =.% v/v formic acid in HO B =.% v/v formic acid in MeCN Gradient: T(mins).. %B The TC trace and MS spectra for the polar embedded column (previously seen to show significant bleed by UV detection) again shows a high level of column bleed when analysing by LC/MS. The MS spectra from a blank run (performed with no column attached) enables the background system bleed to be quantified. Both the ACE CPFP column and leading C column exhibit bleed levels similar to the blank run, denoting that negligible column bleed is occurring. Conclusion The ACE CPFP phase may be used to provide a different selectivity to leading C columns without encountering the column bleed issues associated with many alternate (i.e. nonc) bonded phases. Australia & New Zealand contact: info@winlab.com.au or call + ()

18 ACE CPFP a C phase with unique selectivity Material Characteristics ENDCAPPED PARTCLE SZE (μm) PORE SZE (Å) SURFACE AREA (m/g) CARBON LOAD (%) MAXMUM ph RANGE Proprietary octadecyl with embedded PFP functionality Yes,,...a Octadecyl Yes,,...a PHASE FUNCTONAL GROUP CPFP C 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 LCMS Buffer Selection by John Dolan contact your distributor to request your FREE copy. ACE μm Columns (Contact your distributor for the full range of ACE μm phases available) ACE μm CPFP COLUMN LENGTH COLUMN DAMETER mm mm mm mm mm mm mm mm mm GUARD CARTRDGE.mm.mm.mm.mm.mm ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACEa ACEa ACEa ACEa ACEa ACEGD ACEGD ACEGD ACEGD ACEGD ACE μm C COLUMN LENGTH COLUMN DAMETER mm mm mm mm mm mm mm mm mm GUARD CARTRDGE.mm.mm.mm.mm.mm ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACEa ACEa ACEa ACEa ACEa ACEGD ACEGD ACEGD ACEGD ACEGD a Consider operating pressure limitations for maximum column lifetime ACE μm Columns (Contact your distributor for the full range of ACE μm phases available) ACE μm CPFP COLUMN LENGTH COLUMN DAMETER mm mm mm mm mm mm mm mm mm GUARD CARTRDGE.mm.mm.mm.mm.mm.mm.mm.mm.mm ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD ACE μm C COLUMN LENGTH COLUMN DAMETER mm mm mm mm mm mm mm mm mm GUARD CARTRDGE.mm.mm.mm.mm.mm.mm.mm.mm.mm ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD pack use with cartridge holder H and column coupler C pack use with integral microbore cartridge holder H (not mm column length) pack use with integral analytical cartridge holder H (not mm column length) pack use with semiprep cartridge holder H and column coupler C pack use with prep cartridge holder H and column coupler C When using guards, cartridge holder H and column coupler C required Australia & New Zealand contact: info@winlab.com.au or call + ()

19 ACE CPFP a C phase with unique selectivity ACE μm Columns (Contact your distributor for the full range of ACE μm phases available) ACE μm CPFP COLUMN DAMETER.mm.mm.mm.mm.mm.mm COLUMN LENGTH mm mm mm mm mm mm mm mm GUARD CARTRDGE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACEGD ACEGD ACEGD ACEGD ACEGD mm ACE ACE ACE μm C COLUMN DAMETER.mm.mm.mm.mm.mm.mm COLUMN LENGTH mm mm mm mm mm mm mm mm mm GUARD CARTRDGE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACE ACEGD ACEGD ACEGD ACEGD ACEGD pack use with integral analytical cartridge holder H (not mm column length) pack use with semiprep cartridge holder H and column coupler C pack use with prep cartridge holder H and column coupler C When using guards, cartridge holder H and column coupler C required Method Validation Kits ACE phases are widely recognized to offer outstanding reproducibility. To aid method validation, a convenient kit containing three columns of the same bonded phase and dimensions, packed with three different batches of silica is available. Method Development Kits are available for all phases and column dimensions, with the most common kits shown below..mm METHOD VALDATON KTS X.mm X.mm ACE CPFP Method Validation Kit ACE CPFP Method Validation Kit ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACE C Method Validation Kit ACE C Method Validation Kit ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK X.mm X.mm X.mm ACE CPFP Method Validation Kit ACE CPFP Method Validation Kit ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACE C Method Validation Kit ACE C Method Validation Kit ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK X.mm X.mm X.mm ACE CPFP Method Validation Kit ACE CPFP Method Validation Kit ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACE C Method Validation Kit ACE C Method Validation Kit ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK (three different batches of the same phase).mm METHOD VALDATON KTS (three different batches of the same phase).mm METHOD VALDATON KTS (three different batches of the same phase) a X.mm a a a a a a Consider operating pressure limitations for maximum column lifetime Trademarks and Registered Trademarks as appearing in this catalogue are registered with the following companies; ACE Advanced Chromatography Technologies / Ascentis Express SigmaAldrich Co. / Hypersil and HyPurity Thermo Scientific / nertsil GL Sciences / Luna Phenomenex nc. / Spherisorb, Sunfire, Symmetry, XBridge and XTerra Waters Corporation / Zorbax Eclipse XDB and Zorbax StableBond Agilent Technologies. Comparative separations may not be representative of all applications. Australia & New Zealand contact: info@winlab.com.au or call + ()

20 Free HPLC Technical Guides and Slide Chart n riso a p ComGuide ER SE D PHA SE EV S R MN C COLU LC HP TO Ph d C Use only omm ho on C ions drop Da a ca ve Hy lat son ecif ri Re lc Sp e pa to u ra ng Com na y Phas a Ne ord for io S at ared m Pha es Co Com C ases sg Phas lum Acc red d oupe r mpa Co y nc fi ie n Ef mp Ph Ac of C on paris Cate son rd ng rd ng Acc to Si ne lum of Co or za e al ion of Ph Ac om fc ses anol vi y i A tiv r cy fo Bas en Acco d ng y c Co lan o Si To receive your FREE copies of these guides or the HPLC Column slide chart contact your local distributor or info@acehplc.com ACE products are available through our international network of distributors Distributed in Australia and New Zealand WNLAB PTY LTD PO Box, Brendale, Queensland, Australia Ph: + () sales@winlab.com.au Web: Advanced Chromatography Technologies, Berry Street, Aberdeen, AB HF, Scotland Tel: + () Fax: + () info@acehplc.com P

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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