ACE HILIC + + UHPLC and HPLC Columns. For Reproducible Polar Analyte Separations. Three HILIC Column Chemistries Available

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1 ACE HILIC UHPLC and HPLC s For Reproducible Polar Analyte Separations Three HILIC Chemistries Available

2 ACE HILIC s What is HILIC? Hydrophilic Interaction Liquid Chromatography (HILIC) was first described by Alpert*. HILIC is ideal for the separation and retention of polar species including polar neutral and polar ionised analytes. HILIC separations typically include a polar stationary phase with high organic solvent containing mobile phases. Mechanistically HILIC is complex (Fig ) and provides multiple modes of interaction between the analyte, stationary phase, eluent and water enriched layer at the stationary phase particle-eluent interface**. * A. J. Alpert, J. Chromatogr., 99 (99) 77. ** See the FREE ACE guide to reproducible HILIC method development for more information order your copy now. Charged Analyte Electrostatic Attraction Polar Neutral Analyte Neutral Analyte Partitioning H-bonding Stationary Phase Particle Electrostatic Repulsion Charged Analyte Water enriched layer Figure. Schematic of interactions between different types of polar analytes and a stationary phase in HILIC mode When Should You Consider HILIC? HILIC provides the retention and separation of hydrophilic or polar to very polar analytes not well retained in RPLC. Hydrophilic or polar to very polar analytes have log P values (measure of lipophilicity) of around zero or less (Fig A). Generally, polar analytes are suitable for HILIC if they elute before caffeine in gradient RPLC (Fig B). Figure A. Analyte suitability for HILIC from Log P Figure reproduced with permission. First published in Chromatography Today, Volume 8, Issue, November/ December HILIC Log P Scale << - - >> Glyphosate log P = -.9 Caffeine log P = -. RPLC Amitriptyline log P =.9 mv 6 Glyphosate Amitriptyline Caffeine 6 8 Figure B. Analyte suitability for HILIC from gradient RPLC : ACE Excel C8, x. mm, µm Part Number: EXL --U Mobile Phase: A = mm ammonium formate, ph. (aq) B = mm ammonium formate, ph. in 9: v/v MeCN:H O Gradient: - %B in utes Detection: ELSD Flow Rate:. ml/ Temperature: ºC Injection: µl Analysed using VWR-Hitachi Chromaster with VWR ELSD9.

3 HILIC Provides Alternative Selectivity and Retention to RPLC HILIC provides improved retention and alternative selectivity to RPLC for polar and very polar analytes. Analysis of a six component polar analyte mixture by RPLC and HILIC shows inverted elution profiles (see below).. Cytosine log P = -.. Hypoxanthine log P = Thye log P = -.9. Theobroe log P = -.8. Theophylline log P = Caffeine log P = -. RPLC: 6 Elution Order Polar analyte peak (cytosine) elutes essentially on the solvent front even at % MeCN using ACE C8 in RPLC. 6 : ACE Excel C8, x.6 mm, µm Part number: EXL --6U Mobile Phase:. % acetic acid in 9: v/v H O:MeCN Flow Rate:. ml/ Temperature: ºC Detection: nm Injection: µl HILIC: 6 Elution Order Cytosine and all polar analytes well retained with HILIC analysis using ACE HILIC-N (neutral character phase). : ACE HILIC-N (neutral character phase), x.6 mm, µm Part number: HILN--6U Mobile Phase:. % acetic acid in :9 v/v H O:MeCN Flow Rate:. ml/ Temperature: ºC Detection: nm Injection: µl Conclusions ACE HILIC columns provide alternative selectivity to ACE RPLC columns with excellent retention of polar analytes. ACE HILIC UHPLC / HPLC s

4 ACE HILIC s - Options Three ACE HILIC stationary phases have been developed to provide the solution to your HILIC separation needs. Phase Functional Group Endcapped Particle Size (µm) Pore Size (Å) Surface Area (m /g) Carbon Load (%) Recommended ph Range ACE HILIC-A Proprietary SIL No.7,, ACE HILIC-B Proprietary Aopropyl No.7,, ACE HILIC-N Proprietary Polyhydroxy No.7,, To extend column lifetime under HPLC conditions (up to psi / bar), ACE guard cartridges (/pk) or ACE pre-column filters (/pk) are recommended. For the guard system a guard cartridge holder (H) and coupler (C) are also required. To extend column lifetime under UHPLC conditions (up to psi / bar) an ACE UHPLC pre-column filter is recommended. ACE HILIC-A An acidic character phase with an ionisable negative surface charge depending upon mobile phase ph. b-blockers (ACE AN#) 6. Propranolol. Sotalol. 7.. Acebutalol. Salbutamol. Atenolol : ACE HILIC-A, x.6 mm, µm Part Number: HILA--6U Mobile Phase: mm ammonium formate ph.7 in MeCN/H O (88: v/v) Flow Rate:. ml/ Injection: µl Temperature: ºC Detection: nm ACE HILIC-B A basic character phase with an ionisable positive surface charge depending upon mobile phase ph. Succinic acid and methylmalonic acid (ACE AN#) mv 6 Solvent front. Succinic acid. Methylmalonic acid : ACE HILIC-B, x.6 mm, µm Part Number: HILB--6U Mobile Phase: mm ammonium formate ph. in MeCN/H O (9: v/v) Flow Rate:. ml/ Temperature: ºC Injection: µl Detection: ELSD (Evaporator temp: ºC, Nebuliser temp: ºC, Gas speed: SLM) ACE HILIC-N A polar neutral character phase with the capability of H-bonding amongst other mechanisms of interaction. Nucleobases and nucleosides (ACE AN#).E 8.E 6.E.E.E.E : ACE HILIC-N, x.6 mm, µm Part Number: HILN--6U Mobile Phase: mm ammonium formate ph.7 in MeCN/H O (9: v/v) Flow Rate:. ml/ Temperature: ºC Injection: µl Detection: nm Deoxyadenosine. Adenine. Adenosine

5 ACE HILIC Method Development ACE HILIC columns provide alternative selectivity to each other. The power of systematic screening of different phase chemistries for HILIC method development is seen below. Maximise your HILIC method development success by following the ACE HILIC method development protocol using three optimised ACE HILIC column chemistries protocol available in a FREE HILIC guide. Advantages of using ACE HILIC Method Development Kits ACE HILIC-A 8 7, 6, Explore Selectivity: Method Development Kits available: column kit for the same price as a single column see page 6 ACE HILIC-B Different Elution Orders and Retention Times Observed ACE HILIC-N 6, 9 7 6, p-aobenzoic acid. -Hydroxybenzoic acid. Nicotinamide. Acebutolol. Adenine 6. Mandelic acid 7. Tyrae 8. Atenolol 9. -Deoxyguanosine s: All x.6 mm, µm (Part numbers: ACE HILIC-A: HILA--6U, ACE HILIC-B: HILB--6U, ACE HILIC-N: HILN--6U) Mobile Phase: mm ammonium formate ph.7 in MeCN/H O (9: v/v) Flow Rate:. ml/ Temperature: C Detection: nm Sample: ) p-aobenzoic acid ) -Hydroxybenzoic acid ) Nicotinamide ) Acebutolol ) Adenine 6) Mandelic acid 7) Tyrae 8) Atenolol 9) -Deoxyguanosine Conclusions ACE HILIC columns provide alternative selectivity to each other ideal for HILIC method development. ACE HILIC UHPLC / HPLC s

6 ACE HILIC UHPLC / HPLC and Microbore s Part Numbers Matched guard cartridges (for HPLC columns) or prefilters (for UHPLC columns) are available please enquire. ACE HILIC Method Development Kits (Contains HILIC columns: ACE HILIC-A, ACE HILIC-B and ACE HILIC-N of specified dimensions) (UHPLC/HPLC hardware format with bar / psi pressure limit) Dimensions.7 µm µm µm. x mm MDKH-7-U MDKH--U MDKH--U. x mm MDKH-7-U MDKH--U MDKH--U. x mm MDKH-7-U MDKH--U MDKH--U. x mm MDKH-7-U MDKH--U MDKH--U. x 7 mm MDKH-7-7U MDKH--7U MDKH--7U. x mm MDKH-7-U MDKH--U MDKH--U. x mm - MDKH--U MDKH--U. x mm - MDKH--U MDKH--U. x mm - MDKH--U MDKH--U. x mm MDKH-7-U MDKH--U MDKH--U. x mm MDKH-7-U MDKH--U MDKH--U. x mm MDKH-7-U MDKH--U MDKH--U. x mm MDKH-7-U MDKH--U MDKH--U. x 7 mm MDKH-7-7U MDKH--7U MDKH--7U. x mm MDKH-7-U MDKH--U MDKH--U. x mm - MDKH--U MDKH--U. x mm - MDKH--U MDKH--U. x mm - MDKH--U MDKH--U.6 x mm - MDKH--6U MDKH--6U.6 x mm - MDKH--6U MDKH--6U.6 x mm - MDKH--6U MDKH--6U.6 x mm - MDKH--6U MDKH--6U.6 x 7 mm - MDKH--76U MDKH--76U.6 x mm - MDKH--6U MDKH--6U.6 x mm - MDKH--6U MDKH--6U.6 x mm - MDKH--6U MDKH--6U.6 x mm - MDKH--6U MDKH--6U ACE HILIC-A s (Individual HILIC s of specified dimensions) (UHPLC/HPLC hardware format with bar / psi pressure limit) Dimensions.7 µm µm µm. x mm HILA-7-U HILA--U HILA--U. x mm HILA-7-U HILA--U HILA--U. x mm HILA-7-U HILA--U HILA--U. x mm HILA-7-U HILA--U HILA--U. x 7 mm HILA-7-7U HILA--7U HILA--7U. x mm HILA-7-U HILA--U HILA--U. x mm - HILA--U HILA--U. x mm - HILA--U HILA--U. x mm - HILA--U HILA--U. x mm HILA-7-U HILA--U HILA--U. x mm HILA-7-U HILA--U HILA--U. x mm HILA-7-U HILA--U HILA--U. x mm HILA-7-U HILA--U HILA--U. x 7 mm HILA-7-7U HILA--7U HILA--7U. x mm HILA-7-U HILA--U HILA--U. x mm - HILA--U HILA--U. x mm - HILA--U HILA--U. x mm - HILA--U HILA--U.6 x mm - HILA--6U HILA--6U.6 x mm - HILA--6U HILA--6U.6 x mm - HILA--6U HILA--6U.6 x mm - HILA--6U HILA--6U.6 x 7 mm - HILA--76U HILA--76U.6 x mm - HILA--6U HILA--6U.6 x mm - HILA--6U HILA--6U.6 x mm - HILA--6U HILA--6U.6 x mm - HILA--6U HILA--6U ACE HILIC Microbore Method Development Kits (Contains HILIC Microbore columns: ACE HILIC-A, ACE HILIC-B and ACE HILIC-N of specified dimensions) (HPLC hardware format with bar/6 psi recommended pressure limit) µm µm Dimensions /6 port / port /6 port / port. x mm MDKH-- MDKH--S MDKH-- MDKH--S. x mm MDKH-- MDKH--S MDKH-- MDKH--S. x 7 mm MDKH--7 MDKH--7S MDKH--7 MDKH--7S. x mm MDKH-- MDKH--S MDKH-- MDKH--S. x mm MDKH-- MDKH--S MDKH-- MDKH--S. x mm MDKH-- MDKH--S MDKH-- MDKH--S. x mm - - MDKH-- MDKH--S. x mm MDKH-- MDKH--S MDKH-- MDKH--S. x mm MDKH-- MDKH--S MDKH-- MDKH--S. x 7 mm MDKH--7 MDKH--7S MDKH--7 MDKH--7S. x mm MDKH-- MDKH--S MDKH-- MDKH--S. x mm MDKH-- MDKH--S MDKH-- MDKH--S. x mm MDKH-- MDKH--S MDKH-- MDKH--S. x mm - - MDKH-- MDKH--S 6 FREE ACE HILIC Method Development Guide Available ACE HILIC-A Microbore s (Individual HILIC Microbore s of specified dimensions) (HPLC hardware format with bar/6 psi recommended pressure limit) µm µm Dimensions /6 port / port /6 port / port. x mm HILA-- HILA--S HILA-- HILA--S. x mm HILA-- HILA--S HILA-- HILA--S. x 7 mm HILA--7 HILA--7S HILA--7 HILA--7S. x mm HILA-- HILA--S HILA-- HILA--S. x mm HILA-- HILA--S HILA-- HILA--S. x mm HILA-- HILA--S HILA-- HILA--S. x mm - - HILA-- HILA--S. x mm HILA-- HILA--S HILA-- HILA--S. x mm HILA-- HILA--S HILA-- HILA--S. x 7 mm HILA--7 HILA--7S HILA--7 HILA--7S. x mm HILA-- HILA--S HILA-- HILA--S. x mm HILA-- HILA--S HILA-- HILA--S. x mm HILA-- HILA--S HILA-- HILA--S. x mm - - HILA-- HILA--S ACE HILIC Method Development Guide A Step by Step Approach to Developing Reproducible HILIC Methods Contact us for further details.

7 ACE HILIC UHPLC / HPLC and Microbore s Part Numbers continued... ACE HILIC-B s (Individual HILIC s of specified dimensions) (UHPLC/HPLC hardware format with bar / psi pressure limit) Dimensions.7 µm µm µm. x mm HILB-7-U HILB--U HILB--U. x mm HILB-7-U HILB--U HILB--U. x mm HILB-7-U HILB--U HILB--U. x mm HILB-7-U HILB--U HILB--U. x 7 mm HILB-7-7U HILB--7U HILB--7U. x mm HILB-7-U HILB--U HILB--U. x mm - HILB--U HILB--U. x mm - HILB--U HILB--U. x mm - HILB--U HILB--U. x mm HILB-7-U HILB--U HILB--U. x mm HILB-7-U HILB--U HILB--U. x mm HILB-7-U HILB--U HILB--U. x mm HILB-7-U HILB--U HILB--U. x 7 mm HILB-7-7U HILB--7U HILB--7U. x mm HILB-7-U HILB--U HILB--U. x mm - HILB--U HILB--U. x mm - HILB--U HILB--U. x mm - HILB--U HILB--U.6 x mm - HILB--6U HILB--6U.6 x mm - HILB--6U HILB--6U.6 x mm - HILB--6U HILB--6U.6 x mm - HILB--6U HILB--6U.6 x 7 mm - HILB--76U HILB--76U.6 x mm - HILB--6U HILB--6U.6 x mm - HILB--6U HILB--6U.6 x mm - HILB--6U HILB--6U.6 x mm - HILB--6U HILB--6U ACE HILIC-N s (Individual HILIC s of specified dimensions) (UHPLC/HPLC hardware format with bar / psi pressure limit) Dimensions.7 µm µm µm. x mm HILN-7-U HILN--U HILN--U. x mm HILN-7-U HILN--U HILN--U. x mm HILN-7-U HILN--U HILN--U. x mm HILN-7-U HILN--U HILN--U. x 7 mm HILN-7-7U HILN--7U HILN--7U. x mm HILN-7-U HILN--U HILN--U. x mm - HILN--U HILN--U. x mm - HILN--U HILN--U. x mm - HILN--U HILN--U. x mm HILN-7-U HILN--U HILN--U. x mm HILN-7-U HILN--U HILN--U. x mm HILN-7-U HILN--U HILN--U. x mm HILN-7-U HILN--U HILN--U. x 7 mm HILN-7-7U HILN--7U HILN--7U. x mm HILN-7-U HILN--U HILN--U. x mm - HILN--U HILN--U. x mm - HILN--U HILN--U. x mm - HILN--U HILN--U.6 x mm - HILN--6U HILN--6U.6 x mm - HILN--6U HILN--6U.6 x mm - HILN--6U HILN--6U.6 x mm - HILN--6U HILN--6U.6 x 7 mm - HILN--76U HILN--76U.6 x mm - HILN--6U HILN--6U.6 x mm - HILN--6U HILN--6U.6 x mm - HILN--6U HILN--6U.6 x mm - HILN--6U HILN--6U ACE HILIC-B Microbore s (Individual HILIC Microbore s of specified dimensions) (HPLC hardware format with bar/6 psi recommended pressure limit) Dimensions µm µm /6 port / port /6 port / port. x mm HILB-- HILB--S HILB-- HILB--S. x mm HILB-- HILB--S HILB-- HILB--S. x 7 mm HILB--7 HILB--7S HILB--7 HILB--7S. x mm HILB-- HILB--S HILB-- HILB--S. x mm HILB-- HILB--S HILB-- HILB--S. x mm HILB-- HILB--S HILB-- HILB--S. x mm - - HILB-- HILB--S. x mm HILB-- HILB--S HILB-- HILB--S. x mm HILB-- HILB--S HILB-- HILB--S. x 7 mm HILB--7 HILB--7S HILB--7 HILB--7S. x mm HILB-- HILB--S HILB-- HILB--S. x mm HILB-- HILB--S HILB-- HILB--S. x mm HILB-- HILB--S HILB-- HILB--S. x mm - - HILB-- HILB--S ACE HILIC-N Microbore s (Individual HILIC Microbore s of specified dimensions) (HPLC hardware format with bar/6 psi recommended pressure limit) Dimensions µm µm /6 port / port /6 port / port. x mm HILN-- HILN--S HILN-- HILN--S. x mm HILN-- HILN--S HILN-- HILN--S. x 7 mm HILN--7 HILN--7S HILN--7 HILN--7S. x mm HILN-- HILN--S HILN-- HILN--S. x mm HILN-- HILN--S HILN-- HILN--S. x mm HILN-- HILN--S HILN-- HILN--S. x mm - - HILN-- HILN--S. x mm HILN-- HILN--S HILN-- HILN--S. x mm HILN-- HILN--S HILN-- HILN--S. x 7 mm HILN--7 HILN--7S HILN--7 HILN--7S. x mm HILN-- HILN--S HILN-- HILN--S. x mm HILN-- HILN--S HILN-- HILN--S. x mm HILN-- HILN--S HILN-- HILN--S. x mm - - HILN-- HILN--S Important Note: ACE microbore columns (. mm id and. mm id) are available with either standard /6 (- thread) connections or / (6- thread) connections. For use with Eksigent micro and nano LC systems, order columns with / connections and use either ACE 6- fittings (part number ACE- MC, pack) or Eksigent 6- fittings (part number 96). For /6 HPLC column connections up to 6 psi, PEEK /6 fingertight fittings (part number ACE-CC, pack) are recommended. For / Microbore HPLC column connections up to 6 psi, PEEK / (6- thread) fingertight fittings (part number ACE-MC, pack) are recommended. For /6 UHPLC column connections up to psi, reuseable /6 fittings (part number EXL-CC, pack) are recommended. To further extend UHPLC and HPLC column lifetimes, ACE pre-column filters are recommended. For further details please contact your distributor or visit ACE HILIC UHPLC / HPLC s 7

8 ILIC H E AC pment evelo thod D Me by A Step FREE proach Step Ap E d AC o Meth ucible prod ping Re lo to Deve Guide ethods HILIC M A practical introduction to developing and optimising HILIC separations with example HILIC applications HILIC pment lo Deve de Gui ble Availa at chure o r b e h load t.com Down.ace-hplc www Free RPLC columns! Explore selectivity with ACE UHPLC and HPLC Method Development Kits ACE HILIC columns are available through our international distributor network ACE rs Reve ed-p Meth hase o lopm d ent K its: Up to co the pr lumns for ice of! Deve P Advanced Chromatography Technologies Ltd, Berry Street, Aberdeen, AB HF, Scotland 8 Tel : () 7 Fax: () info@ace-hplc.com

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