ACE C8AR A C8 bonded phse with unique selectivity Gurnteed reproducibility Exceptionl bonded phse stbility Hydrophobic nd romtic mixed mode interction HPLC Columns
ACE C8AR C8 phse with unique selectivity Explore the Advntges of ACE C8AR Provides lternte selectivity to stndrd C8 columns dditionlly recommended for compounds with romtic functionlity Ultr inert, ultr high purity silic, for excellent pek shpe nd reproducibility Comptible with highly queous mobile phses to enble the retention nd seprtion of polr compounds Exceptionl bonded phse stbility for elevted temperture pplictions Ultr low bleed phse ensures UV nd LC/MS comptibility Avilble in high throughput column dimensions Contents Pge Alternte Selectivity to Stndrd C8 Columns............................. Comprison with Leding C8 Columns Wter Soluble Vitmins........................................ Aromtic Nitrobenzenes....................................... Sulphur Compounds........................................... Anlgesics.................................................... Comprison of Column Inertness...........................................6 High Aqueous Comptibility................................................7 Temperture nd ph Stbility..............................................8 Reproducibility nd Sclbility..............................................9 Low Bleed for UV nd LC/MS Comptibility............................ Mteril Chrcteristics................................................... Ordering Informtion Columns................................................ Method Vlidtion Column Kits................................ Austrli & New Zelnd contct: info@winlb.com.u or cll +6 ()7 9 ACE Ultr Inert Bse Dectivted HPLC Columns www.cehplc.com
ACE C8AR C8 phse with unique selectivity Why do I need nother new C8 phse? The use of n ultr pure, ultr inert silic hs mny recognised benefits, including improved reproducibility, lifetime nd chromtogrphic performnce (prticulrly with bsic molecules). However, since the ultr inert silic surfce effectively no longer contributes to the seprtion, C8 columns mnufctured with high purity silics show ner identicl selectivity. It is therefore highly likely tht problem seprtion on one leding brnd will not be significntly improved by chnging to n lternte mnufcturer s equivlent product. For mny yers, experienced chromtogrphers hve been seeking phses with the proven performnce nd reproducibility benefits shown by such leding C8 column brnds, but which dditionlly provide the lternte selectivity required for their chllenging pplictions. How is ACE C8AR different? C8 bonded phses currently dominte the HPLC mrket, with recent surveys indicting tht they re still responsible for 6% of ll HPLC columns sold. However, in recent yers the use of Phenyl bonded phses hs grown significntly, due to the lternte selectivity they provide. It is estimted tht Phenyl phses now ccount for % of HPLC column sles, mking them the lrgest subctegory fter C8 phses. The ACE C8AR phse utilises specilly developed lignd combining C8 chin with integrl phenyl functionlity, thus combining the benefits of both C8 nd Phenyl chrcteristics into single phse. Bsed upon the sme ultr inert, ultr pure, ultr reproducible silic pltform s ACE C8, the unique ACE C8AR phse provides n lternte selectivity to C8 columns. ACE C8AR shows lternte selectivity to C8 bonded columns,, ACE C8 ACE C8AR 6 7 We required C8 phse tht provided different chromtogrphy nd improved resolution ACE C8AR delivered Compounds:) cetmidophenol ) minobenzoic cid ) hydroxybenzoic cid ) cffeine Column: x.6mm, μm Flow Rte:.ml/min Temperture: C Detection: UV, nm Mobile Phse: A..% v/v formic cid in HO B..% v/v formic cid in MeCN Grdient: T(mins) 9 %B Senior Anlyst, Leding Phrmceuticl Compny When should I use ACE C8AR? Due to their similr hydrophobic chrcteristics, ACE C8AR columns my be used for pplictions in which stndrd C8 columns would normlly be considered. However, due to its integrl phenyl functionlity, ACE C8AR is dditionlly recommended for seprtions tht involve compounds contining romtic functionlity. As the pplictions contined within this booklet demonstrte, ACE C8AR cn be used to improve seprtions tht re proving problemtic on C8 columns. The unique ACE C8AR phse provides n lternte selectivity to C8 columns, but remins vlid selection for methods in which C8 bonded columns re specified. In mny instnces, the sme evlution conditions tht prove unsuitble for the C8 column prove suitble for the C8AR column, voiding the need for lengthy method redevelopment. Austrli & New Zelnd contct: info@winlb.com.u or cll +6 ()7 9 www.cehplc.com ACE Ultr Inert Bse Dectivted HPLC Columns
ACE C8AR C8 phse with unique selectivity Comprison of Column Inertness Leding μm, smll pore C8 column brnds x.mm i.d. LC/MS comptible dimensions Bsic molecule inertness test Pek efficiency nd symmetry investigtion Pek Efficiency Comprison ACE C8AR 7, ACE C8 Lower Tiling 6,, Sunfire. C8, HyPurity C8 Zorbx XDB. C8,6 Zorbx SB. C8,, Lun C8() XBridge. C8, Inertsil ODS 9,7 8, XTerr MS. C8 Ascentis Express.7 C8 7, Hypersil BDS C8,7 Spherisorb ODS Higher Tiling,9 Symmetry. C8,6 Hypersil ODS 7 6,, 8,,, N. (pyridine) (pltes/m) ACE C8AR Wters XTerr MS. C8 Wters Symmetry. C8 N.(pyr) = 7,pl/m N.(pyr) = 8,pl/m N.(pyr) =,6pl/m Column Dimensions: x.mm Smple: ) urcil ) pyridine ) phenol Mobile Phse: :6 v/v MeOH/HO Flow Rte:.ml/min Temperture: C Wvelength: nm Conclusion: Significnt differences in efficiency, pek shpe nd selectivity re seen when nlysing pyridine smll highly bsic molecule. Incresed tiling nd retention re indictive of undesirble secondry interctions between pyridine nd silnol groups on the sttionry phse surfce. These interctions cn lso result in poor column reproducibility. ACE C8 columns hve been previously independently tested nd found to be the highest efficiency, most inert columns vilble. The new ACE C8AR mintins this excellent performnce. Comprison Guide C8 REVERSED PHASE HPLC S TO Comprison Dt on Commonly Used C8 Phses I Sttionry Phse Specifictions I Phses Compred According to Reltive Hydrophobicity I Comprison of Column Efficiency for Neutrl Compound I Phses Compred According to Metl Activity I Phses Grouped According to Silnol Activity I Comprison of Column Efficiency for Bsic Compounds SiOH ACE Sttionry Phses Virtully Eliminte the Negtive Effects of Silnols on HPLC Seprtions Further inertness test dt is contined within the current ACE HPLC column ctlogue. Additionlly, Comprison Guide to C8 Columns is lso vilble, detiling mteril chrcteristics for over HPLC column brnds nd compring performnce with number of test probes. Plese contct your locl distributor to request your copies. I Ctegoriztion of Phses According to Silnol Activity nd Inertness Austrli & New Zelnd contct: info@winlb.com.u or cll +6 ()7 9 6 ACE Ultr Inert Bse Dectivted HPLC Columns www.cehplc.com
ACE C8AR C8 phse with unique selectivity Comptible with Highly Aqueous Mobile Phses Whilst primrily designed to show lternte selectivity to stndrd C8 columns, ACE C8AR is lso resistnt to retention loss in % queous mobile phses, nd my be used with fst grdients where the requirement for rpid reequilibrtion nd resistnce to retention loss re essentil. Mximum Reproducibility under High Aqueous Conditions This retention loss ws originlly believed to be due to Phse Collpse, whereby the bonded phse lignds become mtted down nd thus offer reduced interction with the smple. However, more recent studies suggest tht Pore DeWetting is ctully occurring. The mobile phse is excluded from the pores under highly queous conditions nd the retention loss is due to loss of ccessible surfce re. Reproducible Chromtogrphy with % Aqueous Mobile Phse ACE C8AR 8 Reltive Retention Fctor % When seprting very polr, wter soluble compounds, highly queous (>9%) mobile phses re often required to chieve sufficient retention. However, operting conventionl C8 column under such conditions cn led to poor chromtogrphic reproducibility. Over time, peks will elute with shorter nd shorter retention times nd resolution between peks will deteriorte. 6 Leding C8 8 8 7 Time (hours) The integrl phenyl functionlity of the ACE C8AR protects ginst pore dewetting nd subsequent retention loss, resulting in highly reproducible chromtogrphy even under highly queous conditions. Ultr Inert Silic for Excellent Chromtogrphy Mny columns designed for use under high queous conditions re compromised by the low purity silic employed nd exhibit poor pek shpe with polr bsic molecules. This results in poor chromtogrphy nd ultimtely leds to poor column reproducibility. ACE C8AR columns re mnufctured from the sme ultr inert, high purity silic s ll ACE phses ensuring excellent chromtogrphy nd excellent reproducibility re obtined. Leding Competitor Comprison Hydrophilic Bses ACE C8AR ZORBAX SBAq Smple: ) nicotine ) benzylmine ) procinmide ) slbutmol ) phenol Mobile Phse:.:96.7 MeOH/mM KHPO (ph.7) Dimensions: x.6mm i.d. Flow Rte:.ml/min, Temperture: 6 C, Wvelength: nm The Zorbx SBAq column shows drmtic loss of performnce for peks nd, wheres the ultr inert ACE C8AR column mintins excellent pek shpe with hydrophilic bses. Austrli & New Zelnd contct: info@winlb.com.u or cll +6 ()7 9 www.cehplc.com ACE Ultr Inert Bse Dectivted HPLC Columns 7
ACE C8AR C8 phse with unique selectivity Temperture nd ph Stbility At low ph, column deteriortion is cused by hydrolysis of the bonded phse, with decrese in retention observed. The nture of the bonded phse, the purity of the silic surfce nd bonding density re ll criticl prmeters. The use of lower purity silic, shorter lignd nd lower bonding density re ll fctors tht will contribute to ccelerted lignd clevge nd reduced column lifetime. Accelerted Column Stbility Study 8 C t ph.9 ACE C8 (high purity silic) ACE C8AR (high purity silic) 8 Zorbx SBC8 % Initil k' 6 (moderte purity silic) Wters Spherisorb ODS (low purity silic) Leding Phenyl (high purity silic), column volumes Acidic Exposure Conditions: Mobile Phse: Flow Rte: Temperture: Time (hours) :9 MeOH/.% TFA in HO (ph.9).ml/min 8 C Column Dimensions: x.mm Using conditions designed to ccelerte column degrdtion, the ACE C8AR phse shows little retention loss, with lifetime equivlent to the highly robust ACE C8 phse. Both phses re mnufctured from the sme ultr pure silic, nd outlst the Zorbx SB C8, phse previously recognised to provide excellent stbility for high temperture nd low ph pplictions. As expected, C8 bonded column bsed upon low purity silic (Wters Spherisorb ODS) shows gretly reduced lifetime under these ccelerted conditions. Of prticulr note is the result compring the lifetime of leding phenyl column to the ACE C8AR. Despite the use of n ultr pure silic, the lifetime of the phenyl column is diminished compred with the ACE C8AR, suggesting tht the ACE C8AR my be suitble for pplictions in which phenyl columns re seen to exhibit reduced lifetime. Austrli & New Zelnd contct: info@winlb.com.u or cll +6 ()7 9 8 ACE Ultr Inert Bse Dectivted HPLC Columns www.cehplc.com
ACE C8AR C8 phse with unique selectivity Gurnteed Reproducibility nd Fully Sclble Of equl importnce to lternte selectivity is excellent reproducibility. Vritions between different btches of sttionry phse re the most common cuse of customer concern. ACE sttionry phses virtully eliminte the unpredictble negtive effects of silnols on HPLC seprtions by mintining rigid control of the complete mnufcturing process nd estblishing tight specifictions for purity, selectivity, retention, efficiency nd symmetry. Therefore, s demonstrted in the figure below bsolute btchtobtch nd columntocolumn reproducibility re gurnteed for ll ACE C8AR columns. Prent Silic Btch Silne Btch Silic Btch # Silne Btch # Silic Btch # Silne Btch # Silic Btch # Silne Btch # Prticle Size Column Dimeter LC/MS ACE C8AR x.mm.ml/min Anlyticl x.6mm.ml/min ACE C8AR Preprtive ACE C8AR x.mm.ml/min Column: ACE C8AR, x.6mm (unless specified otherwise) Smple: ) urcil ) hydroxybenzoic cid ) cetylslicylic cid ) benzoic cid ) hydroxybenzoic cid 6) ethyl prben Mobile Phse: :6 MeCN/.%TFA in HO Temperture: C Wvelength: nm Flow.ml/min (unless specified otherwise) The vilbility of μm, μm nd μm prticle sizes combined with rnge of column dimensions from cpillry through to preprtive scle ensures tht methods cn be reproducibly scled up or down. The chromtogrms bove demonstrte the excellent reproducibility chieved when silic btch nd silne btch re chnged, nd the reproducible sclbility obtined when chnging prticle size nd column dimeter. Austrli & New Zelnd contct: info@winlb.com.u or cll +6 ()7 9 www.cehplc.com ACE Ultr Inert Bse Dectivted HPLC Columns 9
ACE C8AR C8 phse with unique selectivity Mteril Chrcteristics ENDCAPPED PARTICLE SIZE (μm) PORE SIZE (Å) SURFACE AREA (m/g) CARBON LOAD (%) MAXIMUM ph RANGE Proprietry octdecyl with embedded phenyl functionlity Yes,,... Octdecyl Yes,,... PHASE FUNCTIONAL GROUP C8AR C8 For optimum column lifetime, ph rnge of 8 is recommended. To increse column lifetime t high ph, orgnic buffers, low buffer concentrtions, high % orgnic solvent nd low tempertures must be considered. Further informtion is contined within A Guide to HPLC nd LCMS Buffer Selection by John Doln contct your distributor to request your FREE copy. ACE μm Columns (Contct your distributor for the full rnge of ACE μm phses vilble) ACE μm C8AR mm mm mm mm 7 mm mm mm mm mm.mm.mm.mm.mm.6mm ACE96 ACE96 ACE966 ACE9 ACE9 ACE9 ACE96 ACE9 ACE9 ACE9 ACE9 ACE96 ACE9 ACE9 ACE9 ACE9 ACE96 ACE97 ACE97 ACE97 ACE97 ACE976 ACE9 ACE9 ACE9 ACE9 ACE96 ACE9 ACE9 ACE9 ACE9 ACE96 ACE9 ACE9 ACE9 ACE9 ACE96 ACE9 ACE9 ACE9 ACE9 ACE96 ACE9GD ACE9GD ACE9GD ACE9GD ACE9GD ACE μm C8 mm mm mm mm 7 mm mm mm mm mm.mm.mm.mm.mm.6mm ACE6 ACE6 ACE66 ACE ACE ACE ACE6 ACE ACE ACE ACE ACE6 ACE ACE ACE ACE ACE6 ACE7 ACE7 ACE7 ACE7 ACE76 ACE ACE ACE ACE ACE6 ACE ACE ACE ACE ACE6 ACE ACE ACE ACE ACE6 ACE ACE ACE ACE ACE6 ACEGD ACEGD ACEGD ACEGD ACEGD Consider operting pressure limittions for mximum column lifetime ACE μm Columns (Contct your distributor for the full rnge of ACE μm phses vilble) ACE μm C8AR mm mm mm mm 7 mm mm mm mm mm.mm.mm.mm.mm.6mm 7.7mm.mm.mm.mm ACE96 ACE96 ACE966 ACE9 ACE9 ACE9 ACE96 ACE9 ACE9 ACE9 ACE9 ACE96 ACE9 ACE9 ACE9 ACE9 ACE96 ACE98 ACE9 ACE9 ACE9 ACE97 ACE97 ACE97 ACE97 ACE976 ACE978 ACE97 ACE97 ACE97 ACE9 ACE9 ACE9 ACE9 ACE96 ACE98 ACE9 ACE9 ACE9 ACE9 ACE9 ACE9 ACE9 ACE96 ACE98 ACE9 ACE9 ACE9 ACE9 ACE9 ACE9 ACE96 ACE98 ACE9 ACE9 ACE9 ACE9 ACE9 ACE9 ACE9 ACE96 ACE98 ACE9 ACE9 ACE9 ACE9GD ACE9GD ACE9GD ACE9GD ACE9GD ACE9GD ACE9GD ACE9GD ACE9GD ACE μm C8 mm mm mm mm 7 mm mm mm mm mm.mm.mm.mm.mm.6mm 7.7mm.mm.mm.mm ACE6 ACE6 ACE66 ACE ACE ACE ACE6 ACE ACE ACE ACE ACE6 ACE ACE ACE ACE ACE6 ACE8 ACE ACE ACE ACE7 ACE7 ACE7 ACE7 ACE76 ACE78 ACE7 ACE7 ACE7 ACE ACE ACE ACE ACE6 ACE8 ACE ACE ACE ACE ACE ACE ACE ACE6 ACE8 ACE ACE ACE ACE ACE ACE ACE6 ACE8 ACE ACE ACE ACE ACE ACE ACE ACE6 ACE8 ACE ACE ACE ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD ACEGD pck use with crtridge holder H nd column coupler C pck use with integrl microbore crtridge holder H (not mm column length) pck use with integrl nlyticl crtridge holder H (not mm column length) 6 pck use with semiprep crtridge holder H nd column coupler C pck use with prep crtridge holder H6 nd column coupler C When using gurds, crtridge holder H nd column coupler C required Austrli & New Zelnd contct: info@winlb.com.u or cll +6 ()7 9 ACE Ultr Inert Bse Dectivted HPLC Columns www.cehplc.com
ACE C8AR C8 phse with unique selectivity ACE µm Columns (Contct your distributor for the full rnge of ACE µm phses vilble) ACE µm C8AR.6mm 7.7mm.mm.mm.mm.mm mm mm mm mm 7 mm mm mm mm mm ACE966 ACE96 ACE96 ACE96 ACE98 ACE9 ACE9 ACE9 ACE976 ACE978 ACE97 ACE97 ACE97 ACE96 ACE98 ACE9 ACE9 ACE9 ACE96 ACE98 ACE9 ACE9 ACE96 ACE98 ACE9 ACE9 ACE9 ACE96 ACE98 ACE9 ACE9 ACE9 ACE9GD ACE9GD ACE9GD ACE9GD ACE9GD mm mm mm mm 7 mm mm mm mm mm ACE66 ACE6 ACE6 ACE6 ACE8 ACE ACE ACE ACE76 ACE78 ACE7 ACE7 ACE7 ACE6 ACE8 ACE ACE ACE ACE6 ACE8 ACE ACE ACE6 ACE8 ACE ACE ACE ACE6 ACE8 ACE ACE ACE ACEGD ACEGD ACEGD ACEGD ACEGD ACE µm C8.6mm 7.7mm.mm.mm.mm.mm pck use with integrl nlyticl crtridge holder H (not mm column length) pck use with semiprep crtridge holder H nd column coupler C 6 pck use with prep crtridge holder H6 nd column coupler C When using gurds, crtridge holder H nd column coupler C required Method Vlidtion Kits ACE phses re widely recognized to offer outstnding reproducibility. To id method vlidtion, convenient kit contining three columns of the sme bonded phse nd dimensions, pcked with three different btches of silic is vilble. Method Development Kits re vilble for ll phses nd column dimensions, with the most common kits shown below..mm METHOD VALIDATION KITS X.mm X.mm X.mm ACE C8AR Method Vlidtion Kit ACE C8AR Method Vlidtion Kit ACE9MVK ACE9MVK ACE9MVK ACE9MVK ACE9MVK ACE9MVK ACE C8 Method Vlidtion Kit ACE C8 Method Vlidtion Kit ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK X.mm X.mm X.mm ACE C8AR Method Vlidtion Kit ACE C8AR Method Vlidtion Kit ACE9MVK ACE9MVK ACE9MVK ACE9MVK ACE9MVK ACE9MVK ACE C8 Method Vlidtion Kit ACE C8 Method Vlidtion Kit ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK ACEMVK X.6mm X.6mm X.6mm ACE C8AR Method Vlidtion Kit ACE C8AR Method Vlidtion Kit ACE96MVK ACE96MVK ACE96MVK ACE96MVK ACE96MVK ACE96MVK ACE C8 Method Vlidtion Kit ACE C8 Method Vlidtion Kit ACE6MVK ACE6MVK ACE6MVK ACE6MVK ACE6MVK ACE6MVK (three different btches of the sme phse).mm METHOD VALIDATION KITS (three different btches of the sme phse).6mm METHOD VALIDATION KITS (three different btches of the sme phse) Consider operting pressure limittions for mximum column lifetime Trdemrks nd Registered Trdemrks s ppering in this ctlogue re registered with the following compnies; ACE Advnced Chromtogrphy Technologies / Ascentis SigmAldrich Co. / Hypersil nd HyPurity Thermo Scientific / Inertsil GL Sciences / Lun Phenomenex Inc. / Spherisorb, Sunfire, Symmetry, XBridge nd XTerr Wters Corportion / Zorbx Agilent Technologies. Comprtive seprtions my not be representtive of ll pplictions. Distributed in Austrli nd NZ by WINLAB PTY LTD PO Box 7, Brendle, Queenslnd, Austrli Ph: +6 ()7 9 Emil: sles@winlb.com.u Web: www.winlb.com.u www.cehplc.com ACE Ultr Inert Bse Dectivted HPLC Columns