Discover the Advantages of HALO and HALO BioClass Fused-Core Columns

Transcription

1 Discover the Advantages of HALO and HALO BioClass Fused-Core Columns

2 BIOCLASS COLUMNS Table of Contents Milestones in the Development of Fused-Core Particles Superior Performance of HALO Fused-Core Columns Key Advantages of HALO Fused-Core Columns HALO: Dependable Quality and Reproducibility Selecting the Appropriate Pore Size HALO Columns for Small Molecule Separations Reversed-Phase Separations with HALO HILIC Separations with HALO HALO Columns HALO. Columns HALO Columns HALO BioClass Columns HALO Protein Columns HALO Peptide Columns HALO Glycan Columns HALO UHPLC and HPLC Guard Columns Column Part Number Listing HALO and Fused-Core are registered trademarks of Advanced Materials Technology, Inc. MILESTONES IN THE DEVELOPMENT OF FUSED-CORE PARTICLES mid s approximately yrs Superficially porous particles (SPPs) were first proposed by Golay. Horvath, Preiss, and Lipsky described a nucleotides separation using ~ μm cores with a thin layer of anion exchange resin; called the particles pellicular referring to the thin, skin-like coating. Kirkland, Zipax - Silica sol on glass beads that were ~ μm particle size. Kirkland, Permaphase permanently bonded stationary phase on SPP. Interest in SPPs dwindled due to the low surface areas and lower efficiencies, slower speeds of these column supports compared to non-core particles that could be made in smaller sizes. Kirkland describes μm SPPs with Å pores. Agilent introduces μm Poroshell based on Kirkland s work. Kirkland, Langlois and DeStefano, working at Advanced Materials Technology, Inc. described HALO particles with a. μm core and a. μm thick shell for a total particle size of. μm. Summary Dr. Joseph (Jack) Kirkland has been intimately involved in the development of HPLC packings, including porous and Fused-Core (SPP), throughout his distinguished career SPPs were among the first packings developed for HPLC and have become important again after a -year hiatus AMT celebrates its tenth year in business and continues to introduce innovative solutions for challenging applications. In its decade in business, AMT has strengthened its capabilities by investing in superior talent and expertise in the area of liquid chromatography. Advanced Materials Technology (AMT) was the first company to commercialize Fused-Core particles smaller than μm in Columns packed with these. μm particles have created a revolution in HPLC technology - Performance is comparable to the performance of sub- μm non-core particles, but with half the back pressure Zipax Poroshell HALO. HALO Horvath pellicular - Analysts can obtain very high efficiencies and faster separations using their existing HPLC instruments, which may be limited to bar AMT continues to be a leader in the development and commercialization of novel packing materials for small and large molecules (HALO, ; BioClass, ) Poroshell is a trademark of Agilent Technologies

3 SUPERIOR PERFORMANCE OF HALO FUSED-CORE COLUMNS: HALO FUSED-CORE COLUMNS HALO μm columns will deliver reliable high speed and high resolution separations at pressures lower than most non-core sub- μm columns. HALO. μm columns can meet or exceed the performance of most non-core sub- μm columns at pressures one-third to one-half the back pressure under the same conditions. Figure A. Scanning Electron Microscope (SEM) image of HALO particles. The particle size distribution is very narrow due to the separate sizing steps for solid cores and finished Fused-Core silica particles. Figure C. van Deemter Plot of Plate Height vs. Linear Velocity (flow rate) Effect of Particle Size and Type Columns: x. mm, Non-core C, μm; Non-core C,. μm; Non-core C,. μm; HALO C,. μm Solute: naphthalene; mobile phase: % ACN/% water, C HALO μm columns will match the performance of totally porous μm columns at roughly half the back pressure under the same conditions. Some of the early and current explanations for the excellent performance of Fused-Core columns are described below. Early Explanations for Superior Performance Faster Mass Transfer due to a thin porous bonded-phase layer exterior to particle s solid silica core More Uniform and Stable Column beds due to very narrow particle size distribution (~ % RSD vs. ~% RSD for non-core particles) Current Understanding of SPP Performance (Figure C) The superior performance of Fused-Core SPP columns is now believed to be due to: Reduction in eddy diffusion - % smaller van Deemter A term due to more uniform analyte flow paths through the column bed Figure B. SEM image of a focused-ion-beamcleaved HALO Protein. μm silica particle. This cut-away view shows the solid core with its very thin. μm outer porous layer. Plate Height, micrometers. μ μm non-core. μm non-core Much lower longitudinal broadening, flat van Deemter plot and higher optimum linear velocity (flow rate) - Due to the presence of the particle s solid core ( % smaller van Deemter B term ). μm non-core Much smaller reduced plate heights and high efficiencies for SPP columns due to smaller van Deemter A and B terms for SPP particles H = A B μ + + Cμ van Deemter Equation Mobile Phase Velocity, mm/sec H = height equivalent to theoretical plate A = eddy diffusion term B = longitudinal diffusion term C = resistance to mass transfer term μ = mobile phase linear velocity (L/t ) *G.J. Kennedy, J.H. Knox, J. Chromatogr. Sci ().

4 KEY ADVANTAGES OF HALO FUSED-CORE COLUMNS HALO FUSED-CORE PERFORMANCE High Speed Separations (Figures D and E) Smaller reduced plate heights lead to high efficiencies; narrower and taller peaks, for improved resolution and lower detection limits (LODs and LOQs) Flat van Deemter plot and higher linear velocity optimum (Figure C, page ) allow higher flow rates with minimal column efficiency loss High Resolution Separations (Figure F) High efficiency with longer geometries (,, mm) provides greater resolving power for challenging applications Lower back pressure permits columns to be used in series for the most demanding UHPLC and HPLC separations Excellent Ruggedness and Reproducibility Less plugging, longer usable column lifetime and greater uptime due to larger porosity frits (vs. sub- μm totally porous (non-core) columns) - μm frits for HALO. and HALO - μm frits for HALO vs... μm frits for sub- μm non-core columns Excellent column-to-column and lot-to-lot reproducibility (Page ) PERFORMANCE VS. NON-CORE COLUMNS HALO Superior efficiency compared to many popular sub- μm non-core columns ~ % lower back pressure than most commercially available sub- μm non-core columns HALO. Comparable resolution and peak capacity to sub- μm non-core columns at half of the back pressure under the same conditions, or Two-fold higher throughput at twice the flow rate vs. sub- μm non-core columns at similar pressures HALO Provides comparable efficiency and resolution to μm non-core columns for HPLC separations at % lower back pressure HALO BioClass Comparable to or better than performance of sub- μm non-core columns for bioseparations at / to / of the back pressure Outperforms legacy non-core columns in terms of peak shape, peak capacity and recovery ULTRAFAST PEPTIDE SEPARATION Figure D. Separation of a -peptide mixture is accomplished in less than one minute using a. x mm, HALO. Peptide ES-C column using a delay-volumeminimized and-optimized Agilent system. ULTRAFAST BALLISTIC GRADIENT USING HALO Figure E. Many researchers have found HALO columns in. mm ID to be very useful for highthroughput, ballistic separations by LC and LC-MS. CARBONYL-DNPH HIGH RESOLUTION SEPARATION Column:. x mm, HALO Peptide ES-C Part Number: - Mobile Phase A: water/.% TFA; Mobile Phase B: / ACN/water/.% TFA Gradient: Hold at.% B until. min.,.% to % B from.. min. Flow Rate:. ml/min. Temperature: Pressure: bar LC System: Agilent SL. Gly-Tyr. Val-Tyr-Val. Angiotensin / (-) amide. Met-enk. Angiotensin / (-) amide. Angiotensin II. Leu-Enk. Ribonuclease A. Angiotensin (-) (mouse). Porcine Insulin Detection: nm Injection: μl Column:. x mm, HALO C Part Number: - Mobile Phase A: Water/.% TFA Mobile Phase B: Acetonitrile/.% TFA Gradient: -% B in sec. Flow Rate: ml/min. Pressure: bar Detection: UV nm, PDA Injection Volume:. μl Flow Cell: μl micro LC System: Shimadzu Nexera Time (sec.). Atenolol. Pindolol. Propranolol. Indoprofen. Naproxen. Coumatetralyl Figure F. Environmental samples can be quite complex as demonstrated by this gradient separation dinitrophenylhydrazone (DNPH) carbonyl compound derivatives using a. x mm, HALO C column. Column:. x mm, HALO C, μm, Å Part Number: - Mobile Phase A: HO Mobile Phase B: / ACN/THF. Formaldehyde-,-DNPH. Acetaldehyde-,-DNPH. Acetone-,-DNPH. Acrolein-,-DNPH Gradient: Time %B. Propionaldehyde-,-DNPH - min.. Crotonaldehyde-,-DNPH - min. - Flow Rate:. ml/min. Temperature: Detection: nm Peak Capacity: Max. Pressure: bar Injection Volume:. μl. Butyraldehyde-,-DNPH. Benzaldehyde-,-DNPH. Cyclohexanone-,-DNPH. Isovaleraldehyde-,-DNPH. Valeraldehyde-,-DNPH. o-tolualdehyde-,-dnph. m-tolualdehyde-,-dnph. p-tolualdehyde-,-dnph. Hexaldehyde-,-DNPH.,-Dimethylbenzaldehyde-,-DNPH. Heptaldehyde-,-DNPH. Octyl aldehyde-,-dnph. Nonanal-,-DNPH. Decyl aldehyde-,-dnph, -

5 HALO: DEPENDABLE QUALITY AND REPRODUCIBILITY HALO COLUMNS FOR SMALL AND LARGE MOLECULES: SELECTING THE APPROPRIATE PORE SIZE Figure G. Consistent reproducible performance from column to column and lot to lot is ensured because of well-designed processes and practices in the manufacture of HALO Fused-Core particles, HALO phases and HALO columns. Representative chromatograms of QA and QC tests are shown below, along with a historical plot of selectivity between a neutral and basic probe. Advanced Materials Technology incorporates the know-how from its principal scientists, each with over years of experience in liquid chromatography, particle synthesis and column manufacturing to bring high quality, innovative products to our customers. butyl paraben uracil propranolol naphthalene QA TEST. x mm, HALO. μm : CH OH/ mm phosphate (ph ). ml/min., C acenaphthene amitriptyline uracil phenol QC TEST -Cl--NO ² -benzene. x mm, HALO. μm : CH CN/water. ml/min., C naphthalene How to Choose the Right Pore Size? Match the column pore size according to the range of molecular weights (MWs) of the analytes in your sample (Table A) Small molecules (< Da) are usually analyzed using HALO Ångstrom columns - Packing materials with smaller pores have greater surface area, which allows improved retention and loading capacity for lower MW analytes (Page ) - When an analyte is too large for the pores, restricted diffusion can occur, which can lead to peak broadening and reduced retention (Figure I) For macrocyclic antibiotics and biomolecules such as peptides and proteins, use larger pore sizes such as Å HALO Peptide and Å HALO Protein columns (Page ) MW Range Table A. Guidance for Pore Size Selection Pore Size (Å) < Da HALO Column Family HALO Å (small molecules) Products HALO HALO. HALO < kda* HALO BioClass HALO Glycan Da < MW < kda HALO BioClass HALO Peptide HALO. Peptide HALO Peptide kda < MW < kda HALO BioClass HALO Protein * for glycans, glycopeptides and glycoproteins RESTRICTED DIFFUSION FOR HIGHER MW POLYPEPTIDES ON HALO Å -... Figure H. The selectivity value, alpha (k ami /k acenaph ) is plotted versus lot number for the amitriptyline/acenaphthene pair of individual HALO C lots from to. The vertical error bars show the bounds for two standard deviations about the mean value of.. The reproducible selectivity is indicative of processes that are well-controlled, which can produce reliable, high-quality columns. REPRODUCIBLE PERFORMANCE OVER TIME Alpha, K ami /K acenaph Selectivity for amitriptyline/acenaphthene ( ) Lot Number Figure I. In this example, broad peaks are observed in the upper chromatogram for bovine and human insulin, cytochrome C and lysozyme (MWs > ~ Da) chromatographed using a HALO Ångstrom column.this broadening is due to restricted diffusion using the smaller pore column. However, those same four peaks are much narrower and taller in the lower chromatogram using the Ångstrom HALO Peptide ES-C column. Peptide or Polypeptide PW. HALO Å PW. HALO Å MW (kda) Leucine enkephalin... Bovine insulin... Human insulin... Cytochrome C... Lysozyme... Halo. C Å Halo. Peptide ES-C Å Cyt C Cyt C Peptides (in RT order): Leu-Enk, Bovine Insulin, Human Insulin, Cytochrome C, Lysozyme Lyso Lyso Column:. x mm Part Number: HALO. C: - HALO. Peptide ES-C: - Mobile Phase A: / water/acetonitrile/.% TFA Mobile Phase B: / water/acetonitrile/.% TFA Gradient: % B to % B in min. Flow Rate:. ml/min. Temperature: Detection: UV nm, VWD Injection Volume:. μl LC System: Agilent

6 HALO COLUMNS FOR SMALL MOLECULE ANALYSES where Of the three variables in the general resolution equation, including efficiency (N) and retention(k), selectivity (α) is the most powerful parameter for adjusting and improving resolution between peaks in a chromatographic separation. EFFICIENCY SELECTIVITY RETENTION k = (k +k ), α = k N and = L = L H h x dp k [ ] Rs ( = N ) [ ] x (α ) x k α (+k) Moreover, column phase selectivity is one of the four most powerful and useful parameters for adjusting HPLC separation selectivity (see Table B). For ionizable analytes, mobile phase ph is, by far, the most effective parameter. However, column stationary phase is comparable to organic modifier choice (acetonitrile vs. methanol) and percent organic modifier/gradient steepness in its ability to change relative retention for UHPLC and HPLC separations. HALO columns are available in different stationary phases to use for method development and to apply for various types of analyses. The HALO phases that are available for reversedphase separations of small molecules are shown in Table C, and the phases are listed according to their differences in selectivity compared to HALO C at both ph. and ph. For example, if you were looking for a column with a different selectivity to a HALO C column at low ph, you might consider Table C and select a HALO PFP column as one most likely to be orthogonal to C. However, the other available HALO phases (Phenyl-Hexyl, RP-Amide, ES-CN) also retain and separate analytes via retention mechanisms different from HALO C and HALO C, so it might be prudent to consider one or more of the former phases as part of a comprehensive column screening or method development strategy (Figure J). Table B. Parameters That Affect HPLC Selectivity in Order of Increasing Effectiveness (Refs. and ) HPLC Parameter Mobile phase ph (ionizable analytes only) Organic modifier choice Percent organic modifier or gradient steepness Column stationary phase Column temperature Buffer choice Buffer concentration Effectiveness for Changing Selectivity Most Effective Least Effective HALO, HALO. and HALO columns for small molecule analyses are available in the following phases: Reversed Phase C C RP-Amide Phenyl-Hexyl ES-CN PFP Hydrophilic Interaction (HILIC) and Normal Phase HILIC (silica) Penta-HILIC Complete descriptions of the various phases are provided in Table D, along with physical and chemical properties, target analytes and best applications for each phase (Table E). See pages - for additional information about and applications for HALO HILIC and Penta-HILIC. Table C. Relative Reversed-Phase Orthogonality of HALO Phases vs. HALO C at Low and Mid ph (Refs. -) ph. ph Most Similar HALO C HALO C HALO C HALO Phenyl-Hexyl HALO ES-CN HALO RP-Amide HALO C HALO PFP HALO Phenyl-Hexyl HALO ES-CN Most Orthogonal HALO PFP HALO RP-Amide Figure J. Example Strategy for Comprehensive Method Development Using Multiple HALO Stationary Phases and Column/Condition Screening, Followed by Optimization of Gradient Time, Temperature and ph HALO C HALO ES-CN HALO RP-Amide Screening gradients Select phase, ph, solvent Optimize t G, T, ph -% CH ³ CN, low ph -% CH ³ OH, low ph -% CH ³ CN, mid ph -% CH ³ OH, mid ph

7 HALO COLUMNS FOR SMALL MOLECULE SEPARATIONS Table D. HALO Small Molecule Column Specifications HALO COLUMNS FOR SMALL MOLECULE SEPARATIONS Table E. HALO Phases: Features and Benefits, Target Analytes and Best Applications C C Phenyl-Hexyl PFP ES-CN RP-Amide HILIC Penta-HILIC USP Designation L L L L L L L Pending Particle Size(s) (μm) Carbon Load (%) N.A.... Surface Area (m /g) Low ph Limit High ph Limit Max. Temp. Lower ph Limit Max. Temp. Upper ph Limit Endcapped Yes Yes Yes Yes Yes Yes N.A. No Phase Structure Phase Structure C C ES-CN RP-Amide C C Phenyl-Hexyl PFP ES-CN RP-Amide HILIC Bonded Phase Features and Benefits Target Analytes Best Applications C (octadecyldimethylsilane) C (octyldimethylsilane) Phenyl-Hexyl (phenylhexyldimethylsilane) PFP (pentafluorophenylpropylsilane) ES-CN (diisopropylcyanopropylsilane) C Amide None (Bare Silica) Excellent performance for broad range of analyte polarities Excellent performance for broad range of analyte polarities Complementary selectivity to alkyl phases Enhanced selectivity for aromatic compounds Complementary selectivity to alkyl phases Enhanced selectivity for stereoisomers Can be used in RPLC and HILIC modes Complementary selectivity to alkyl phases More retention for polar analytes and much less retention for non-polar analytes Complementary selectivity to alkyl phases Enhanced stability for minimum bleed and long life Can be used in HILIC and normal-phase modes Diverse analytes ranging from polar to non-polar Uncharged acids and bases, uncharged ion pairs Diverse analytes ranging from polar to non-polar Uncharged acids and bases, uncharged ion pairs Electron-poor molecules, aromatic or unsaturated compounds (ketones, nitriles, alkenes) Electron-rich compounds, aromatics, unsaturated compounds with double and/or triple bonds Polar and very polar bases, acids and neutrals Very hydrophobic analytes retained too strongly on C phase Alcohols, Acids, Phenols, Catechins Polar and very polar bases, acids and neutrals, especially with log P<. Analytes differing by an aliphatic or aromatic group Analytes differing by an aliphatic or aromatic group Aromatic molecules with electron-withdrawing groups (NO, COOH, COOR, halogens), heterocycles, benzodiazepines, highly aqueous conditions Basic analytes at low ph, stereoisomers, steroids, taxanes, substituted aromatics, highly aqueous conditions Aromatic molecules with electron-withdrawing groups (NO, COOH, COOR, halogens), heterocycles, benzodiazepines, highly aqueous conditions Acidic and basic analytes, heterocycles, proton donors and acceptors, highly aqueous conditions Enhanced sensitivity and peak shape for LC-MS, analyses of basic analytes in HILIC mode, normal-phase analysis with non-aqueous mobile phases Phenyl-Hexyl HILIC Penta-HILIC proprietary penta-hydroxy ligand Ideal for separation of highly polar compounds that are poorly retained in RPLC Polar analytes with log P values near or less than Polar acids, bases and zwitterions that are not retained or are poorly retained with RPLC PFP Penta-HILIC

8 REVERSED-PHASE SEPARATIONS WITH HALO To illustrate the selectivity differences among the various HALO RPLC phases, the following examples are provided. BENZODIAZEPINES ON HALO FUSED-CORE BONDED PHASES HALO C VS. RP-AMIDE FOR PHENOLICS Column:. x mm Part Numbers: -, -, -, - Mobile Phase A: mm Ammonium Acetate Mobile Phase B: Acetonitrile Gradient: - % B in. min. Flow Rate:. ml/min; Detection: UV nm Injection volume: μl Pressure: bar. Oxazepam. Lorazepam. Nitrazepam. Alprazolam. Clonazepam. Temazepam. Flunitrazepam. Diazepam Figure M. HALO RP-Amide provides greater retention and resolution compared to HALO C for this phenol mixture. HALO RP-AMIDE Figure K. HALO Phenyl-Hexyl is the most retentive phase for these anti-anxiety drugs due to its propensity for interactions.,, HALO Phenyl-Hexyl HALO C HALO RP-Amide HALO C Column:. x mm, HALO. μm RP-Amide and HALO C Part Number: - and - Mobile Phase: /: A/B A=. M Potassium phosphate, ph =. B= Acetonitrile Flow Rate:. ml/min.. Uracil. Catechol.,,-Trinitrophenol.,,-Trichlorophenol., -Biphenol., -Dihydroxyacetophenone. -Chlorophenol, HALO PFP AROMATIC AND NITROAROMATIC COMPOUNDS Figure L. HALO C and HALO ES-CN columns may be used as orthogonal confirmatory columns for explosives analysis. Column:. x mm, HALO C, ES-CN Part Numbers: C=-, ES-CN=- Mobile Phase: /-A/B for C, /-A/B for ES-CN A= water B= methanol Flow Rate:. ml/min. Pressure: About bar i i Temperature: Detection: UV mn, VWD Injection Volume: Sample Solvent: water/methanol : (v/v) Response Time:. sec. Flow Cell: LC System: Shimadzu Prominence UFLC XR Extra Column Volume: HALO C HALO ES-CN. Resorcinol. Benzyl alcohol. Phenylacetonitrile. -Indanol.,-DNT.,-DNT.,-DNT. Anisole.-Chloro--nitrobenzene. Toluene DNT = dinitrotoluene i = impurity SEPARATION OF STRUCTURALLY SIMILAR STEROIDS ON HALO C AND PFP Figure N. HALO PFP delivers improved resolution and different elution order compared to HALO C for this mixture of steroids. HALO C HALO PFP, Column:. x mm, HALO C. Prednisone. x mm, HALO PFP. Cortisone Part Number: C: -. Prednisolone PFP: -. Hydrocortisone Mobile Phase: /: water/methanol Flow Rate:. ml/min. Pressure: about bar Detection: UV nm, VWD Injection Volume: Sample Solvent: % methanol in water Response Time:. sec. Flow Cell:. μl semi-micro LC System: Shimadzu Prominence UFLC XR ECV: i

9 HILIC SEPARATIONS WITH HALO NUCLEOSIDES AND NUCLEOBASES ON HALO PENTA-HILIC Figure P. These nucleosides and nucleobases are separated in under minutes using a HALO. Penta-HILIC column. Hydrophilic interaction liquid chromatography (HILIC) is a useful UHPLC and HPLC mode for the following situations: Polar analytes that are poorly or not retained in RPLC Basic analytes that have poor peak shape (overloading) and/or poor retention at low ph in RPLC Analytes that have log P values near or less than zero When conditions orthogonal to RPLC mode are needed (elution order change) HALO columns are currently available in two different phases for HILIC separations (see Tables D and E, pages and, for properties, features and benefits): HALO HILIC HALO Penta-HILIC HALO HILIC is a Fused-Core silica phase that can be used either in HILIC mode or in normal-phase mode with waterimmiscible solvents (NPLC). RETENTION ORDER REVERSAL AND IMPROVED RETENTION WITH HILIC HALO Penta-HILIC is a bonded silica phase, which has a highly polar ligand with hydroxyl groups tethered via novel proprietary linkage chemistry to Fused-Core silica particles. Some Typical Analytes for HILIC Separations Basic pharmaceuticals Peptides Polar organic acids Catecholamines and other neurotransmitters Nucleosides and nucleobases Drug glycoside and glycuronide metabolites Mono-, di-, tri- and other oligosaccharides Opiates Glycosylceramides Polar triazines and pyrimidines Analytes from metabolomic profiling For more information on HILIC separations, please see references - on page. Absorbance ( mm) - CEPHALOSPORINS ON HALO PENTA-HILIC AND HALO HILIC Column:. x mm, HALO Penta-HILIC Part Number: - Mobile Phase A: mm Ammonium formate in % Acetonitrile/% water Mobile Phase B: mm Ammonium Formate in % Acetonitrile/% water Gradient: Time %B - min. - min. - Flow Rate:. ml/min. Temperature: Detection: UV nm, VWD Injection volume:. μl Pressure: about bar. Thymine. Uracil. Thymidine. -Deoxyadenosine. Adenine. Uridine. Adenosine. Hypoxanthine. Xanthine. Cytosine. -Deoxycytidine. Guanine. -Deoxyguanosine. Cytidine. Guanosine Figure Q. HALO Penta-HILIC shows increased retention and different selectivity vs. HALO HILIC for these cephalosporins. nm, HALO Penta-HILIC HALO HILIC Column:. x mm Part Number: HALO Penta-HILIC: - HALO HILIC: - Mobile Phase: A= / ACN/H with mm NH formate, ph=. (adj.) Mobile Phase: B= / ACN/H with mm NH formate, ph=. (adj.) Gradient: HALO Penta-HILIC: -% B in min. HALO HILIC: -% B in min. Flow Rate:. ml/min. Pressure: bar with HALO Penta-HILIC bar with HALO HILIC Detection: UV nm, VWD Injection Volume:. μl Sample Solvent: / ACN/water Flow Cell: μl semi-micro LC System: Agilent. Cephalothin. Cefoxitin. Cefotaxime. Cefazolin. Cefaclor. Cephalexin. Cephradine. Cefadroxil. Ceftazidime. Cephalosporin C Figure O. You can often obtain a complete reversal in elution order and different selectivity using HILIC mode compared to reversed-phase mode under the same or appropriate conditions. adenosine + cytosine acenaphthene acenaphthene adenosine cytosine.. Column:. x mm Part Number:. μm HALO C: - Mobile Phase A: / ACN/. M Ammonium Formate Flow Rate:. ml/min. ph:. Column:. x mm Part Number:. μm HALO HILIC: - Mobile Phase A: / ACN/. M Ammonium Formate Flow Rate:. ml/min ph:. REVERSED-PHASE SEPARATION OF CEPHALOSPORINS USING HALO ES-CN nm Figure R. HALO HILIC and Penta-HILIC columns often offer an orthogonal separation relative to reversed-phase separations, as shown here for HALO ES-CN for a subset of the same cephalosporins shown in Figure Q Column:. x mm. μm HALO ES-CN Part Number: - Mobile Phase A:. M Phosphate buffer, ph. (adj.) Mobile Phase B: Methanol Gradient: % B to % B in. min. Flow Rate:. ml/min. Temperature: Detection: UV nm, VWD Injection Volume:. μl Sample Solvent: /: Water/Methanol Response Time:. sec. Flow Cell:. μl semi-micro LC System: Shimadzu Prominence UFLC XR ECV: about μl Pressure: bar at start of gradient. Cefadroxil. Ceftazidime. Cefaclor. Cephalexin. Cephradine. Cefotaxime. Cefoxitin. Cefazolin

10 HALO (UHPLC) Highest UHPLC performance possible without the disadvantages of sub- μm columns HALO FAST LOCAL ANESTHETIC SEPARATION USING HALO PENTA-HILIC Figure T. This mixture of five local anesthetics is resolved isocratically in. minutes using a HALO Penta-HILIC column. Use when the highest efficiency is needed Excellent for fast method development and column/ condition screening Best performance obtained with instrumentation having extracolumn volume (IBW< μl) Ruggedness for R&D μm inlet frit Pressure limit, bar/, psi Note: IBW is instrumental band width and is a measure of extracolumn dispersion. ULTRA-FAST SEPARATION OF ANTICOAGULANTS USING HALO C Solid Core Shell with Å pores. μm. μm Figure S. This separation of anticoagulants is completed in one minute using a short. x mm HALO C column using a Shimadzu Nexera UHPLC system. μm mm (mau) STEROID SEPARATION USING HALO PFP Column:. x mm, HALO Penta-HILIC Part Number: - Isocratic: /: ACN/water with mm Ammonium Formate buffer, ph Flow Rate:. ml/min. Detection: UV nm, photodiode array detector Injection Volume:. μl Sample Solvent: / ACN/. M ammonium formate buffer ph Data Rate: Hz Response Time:. sec. Flow Cell:. μl semi-micro Pressure: bar LC System: Agilent SL. Benzocaine. Lidocaine. Tetracaine. Procaine. Procainamide Absorbance ( mm) Column:. x mm, HALO C Part Number: - Mobile Phase A: mm Formic acid Mobile Phase B: / Acetonitrile/Methanol Gradient: Time %B Flow Rate:. ml/min. Detection: nm Injection Volume:. μl Maximum Pressure: bar. Uracil (t ).,-Dihdroxycoumarin. -Hydroxycoumarin. Coumarin. -Chloro--hydroxycoumarin. Warfarin. Coumatetralyl. Coumachlor Figure U. HALO PFP columns often show excellent selectivity for steroids. HALO PFP is able to readily separate a mixture of steroids in less than minutes in gradient mode. mm (mau) Column:. x mm, HALO PFP Part Number: - Mobile Phase A: water Mobile Phase B: methanol Gradient: Time %B min. min. min. Flow Rate:. ml/min. Pressure: bar initial Detection: UV nm, VWD Injection volume: Sample Solvent: methanol Response Time:. sec. Flow Cell: ECV: LC System: Shimadzu Prominence UFLC XR. Uracil. Hydrocortisone. Prednisolone. Cortisone. Prednisone. Dexamethasone. ß-Estradiol. Estrone. Halcinonide See page for full list of HALO part numbers.

11 HALO. (UHPLC AND HPLC) HALO. Reliable, efficient performance with lower back pressure compared to all sub- μm columns Use for high speed or high resolution with UHPLC or HPLC applications Excellent for R&D and routine analyses μm inlet frit Pressure limit, bar/ psi ULTRAFAST SEPARATION OF STATIN DRUGS HIGH RESOLUTION SEPARATION OF EXPLOSIVES Figure W. In this example, a. x mm HALO. C column is used to resolve explosives in minutes. This separation is quite sensitive to temperature, and was optimized using gradient time x temperature (t G x T) computer modelingand simulation using DryLab software. nm - Shell with Å pores Column:. x mm, HALO C,. μm Part Number: - Mobile Phase A: Water Mobile Phase B: Methanol Gradient: Time %B... Flow Rate:. ml/min. Temperature: Detection: UV nm, VWD Injection Volume: μl Sample Solvent: /: Water/methanol Response Time:. sec. Data rate: Hz Pressure: bar to start, max. bar Flow Cell:. μl semi-micro LC System: Shimadzu Prominence UFLC XR ECV: about μl. μm Figure V. These common statin drugs are separated in minute using a -mm HALO Phenyl-Hexyl column Column:. x mm, HALO. Phenyl-Hexyl Part Number: - Mobile Phase: /: A/B A:. M formic acid in water B: Acetonitrile Flow Rate:. ml/min. Pressure: Bar Detection: UV nm, VWD Injection Volume:. μl Sample Solvent: / methanol/water ( mm formic acid) Response Time:. sec. Flow Cell:. μl semi-micro LC System: Shimadzu Prominence UFLC XR ECV:. μm. Pravastatin. Atorvastatin. Mevastatin. Simvastatin. μm. HMX. RDX.,,-Trinitrotoluene.,-Dinitrotoluene.,-Dinitroaniline. Nitrobenzene. Nitroglycerin. Tetryl. -Amino-,-Dinitrotoluene. -Amino-,-Dinitrotoluene.,-Dinitrotoluene.,-Dinitrotoluene. -Nitrotoluene. -Nitrotoluene. -Nitrotoluene. PETN (pentaerythritol tetranitrate) ULTRAFAST SEPARATION OF TRICYCLIC ANTIDEPRESSANTS Figure X. These basic tricyclic antidepressants are separated in less than two minutes, with excellent peak shape, using a HALO Penta-HILIC column. nm HIGH RESOLUTION OF NEONICOTINOIDS ON HALO. ES-CN Column:. x mm, HALO Penta HILIC Part Number: - Mobile Phase: /: A/B A:. M Ammonium formate, ph=. (adj.) B: Acetonitrile Flow Rate:. ml/min. Detection: UV nm, VWD Injection Volume:. μl Sample Solvent: /: Water/acetonitrile Response Time:. sec. Maximum Pressure: Bar Flow Cell:. μl semi-micro LC System: Shimadzu Prominence UFLC XR ECV: about μl. Trimipramine. Amitriptyline. Doxepin. Nortriptyline. Amoxapine Figure Y. Six neonicotinoids are separated using a HALO. ES-CN column. The sub- μm Fused-Core silica-based packing allows rapid separations at modest pressures. nm Column:. x mm, HALO ES-CN Part Number: - Mobile Phase: /: A/B A:.% Formic acid in water B: Acetonitrile Flow Rate:. ml/min. Pressure: Bar Detection: UV nm, VWD Injection Volume:. μl Sample Solvent: Acetonitrile Response Time:. sec. Flow Cell:. μl semi-micro LC System: Shimadzu Prominence UFLC XR ECV:. Nitenpyram. Thiamethoxam. Clothianidin. Imidacloprid. Acetamiprid. Thiacloprid See page for full list of HALO. part numbers.

12 BENZODIAZEPINE SEPARATION USING HALO PFP HALO (HPLC) Performance of μm non-core column at μm column pressures Ideal for: QC laboratories Dirty samples High throughput, ballistic gradient and isocratic applications High resolution at HPLC back pressures (using columns in series) μm inlet frit Pressure limit, bar/ psi FAST, HIGH RESOLUTION GRADIENT FLAVONOID SEPARATION Figure Z. This mixture of flavonoids is baseline resolved in less than minutes using a. x mm HALO C column with a fast.-ml/min. flow rate with an LC-MS-compatible mobile phase. Volts.... HALO Shell with Å pores μm SAMPLE:. μm. μm Column:. x mm, HALO C Part Number: - Flow Rate:. ml/min. Gradient: % CH CN for. min. -% CH CN/ mm NHCOO (.% HCOOH) in min. Max. Pressure: bar ANALYTES:. Hesperidin. Myricetin. Quercetin. Naringenin. Apigenin. Hesperetin. Kaempferol. Biochanin Figure AA. These six benzodiazepine drugs are separated in minutes with better performance than a μm non-core column at ½ the pressure. LC-MS ANALYSIS OF STEVIA GLYCOSIDES USING HALO PENTA-HILIC Figure BB. LC-MS analysis of stevia glycosides from a Stevia natural sweetener extract is easily accomplished using the HALO Penta-HILIC column due to its unique bonded phase containing five OH groups. POLAR AROMATIC COMPOUNDS ON HALO RP-AMIDE Figure CC. HALO RP-Amide shows excellent resolution and peak shape for this mixture of polar aromatic compounds. Intensity nm (mau) HALO PFP (μm) P= bar μm PFP P= bar Comparative results presented here may not be representative for all applications. Column:. x mm, HALO Penta-HILIC Part Number: - Mobile Phase A: / Water/acetonitrile with mm Ammonium formate, ph Mobile Phase B: / Water/acetonitrile with mm Ammonium formate, ph Gradient: % B to % B over min.,,,,,,,,,,,,,,, SIM (+) Steviolbioside or rubusoside. (+) Flow Rate:. ml/min. Pressure: bar Temperature: Ambient Injection Volume: Sample Solvent: /: Acetonitrile/water LC System: Shimadzu Nexera MS: Shimadzu LCMS (single quadrupole) Dulcoside A. (+) Stevioside. (+) Column:. x mm, HALO PFP Part Number: - Mobile Phase A: mm Ammonium Acetate, ph. Mobile Phase B: ACN, -% B in min. Flow:.mL/min. Detector: UV at nm Injection:. Oxazepam. Lorazepam. Nitrazepam. Clonazeparm. Flunitrazepam. Diazepam NOTE: Peak widths at half height are labeled for comparable peaks on both columns. Rebaudioside A. (+) Column:. x mm, HALO- RP-Amide Part Number: - Mobile Phase: /: A/B A: mm Potassium Phosphate, ph B: Acetonitrile Flow Rate:. ml/min Pressure: bar Temperature: C Detection: UV nm, VWD Injection Volume: Sample Solvent: /: Water/Acetonitrile Response Time:. sec. Flow Cell: LC System: Optimized Agilent ESI: +. kv Scan Range: - m/z Scan Rate: pps Capillary: Heat Block: Nebulizing Gas Flow:. L/min. Drying Gas Flow: L/min.. Uracil. Benzamide. Aniline. Cinnamyl Alcohol. Dimethyl Phthalate. -Nitroaniline. -Bromoacetanilide., -Biphenol., -Biphenol See page for full list of HALO part numbers.

13 Table F. HALO BioClass Column Specifications HALO PROTEIN, PEPTIDE AND GLYCAN Today, researchers are keenly interested in both fast and high resolution separations of numerous biomolecules to support the development of novel therapeutic proteins and peptides in pharmaceutical drug development, to advance understanding in modern university laboratories, to characterize protein post-translation modifications, and to fully assess subtle differences in biosimilars and other products of bioengineering and manufacture. HALO BioClass columns have been developed to make such tasks easier and more effective, and are aimed at the following areas of bioseparations: Intact proteins, monoclonal antibodies (mabs), biosimilars, and other large biomolecules such as pegylated proteins, antibody drug conjugates (ADCs), etc. Particle Size(s) (μm) Pore Size (Å) Carbon Load (%) Surface Area (m /g) Low ph Limit High ph Limit Max. Temp. Lower ph Limit Max. Temp. Upper ph Limit Endcapped HALO Protein C.. Yes HALO Protein ES-C.. Yes Peptide mapping (analysis of enzyme digests) for characterization and monitoring of synthetic protein drugs Analysis of therapeutic peptides and peptide biomarkers (protein surrogates) HALO Peptide ES-C.... No High resolution separations of complex mixtures of glycans released from N- and O-linked glycoproteins Currently, the HALO BioClass column family is comprised of the following products. Their features and benefits are described below, and their specifications are shown in Table F. HALO Peptide ES-CN... Yes HALO Protein C Stability up to C Can elute very large proteins with good peak shape and recovery Compatible with UHPLC and HPLC Low LC-MS bleed HALO Peptide ES-C Fast, high resolution separations Extremely stable up to C High peak capacity Rugged, reliable performance Use with either UHPLC or HPLC HALO Glycan Improved retention of acidic and zwitterionic analytes Very low sensitivity to buffer concentration Able to separate isobaric oligosaccharides with different linkages HALO Glycan.. No Structure of an antibody showing its heavy chain in blue and its light chain in green. HALO Protein ES-C Extremely stable up to C Can elute very large proteins with good peak shape and recovery Compatible with UHPLC and HPLC HALO Peptide ES-CN Same benefits as Peptide ES-C Alternative selectivity to Peptide ES-C for peptide mapping and proteomic applications Very low LC-MS bleed

14 HIGH RESOLUTION OF LIGHT AND HEAVY CHAIN VARIANTS OF IgG Ångstrom pores to provide unrestricted pore access for polypeptides and proteins as large as kda. μm Fused-Core particles with a very thin. μm outer porous shell - Provides narrower peaks and better recoveries for large biomolecules (vs. smaller pore sizes and non-core particles) - Allows HALO Protein columns to be used with both UHPLC and HPLC instrumentation for fast bioseparations at moderate back pressures C and sterically-protected ES-C phases - Excellent high temperature stability (up to C) for improved peak shape and recovery μm inlet frit HALO Protein Shell with Å pores. μm. μm. μm Figure EE. Very high resolution is obtained between variants of light and heavy chains of a reduced and alkylated monoclonal antibody (IgG) sample using a HALO Protein C column. nm LC:, LC:, LC:,.... Masses deconvoluted using MagTran software*. LC= light chain HC= heavy chain HC:, HC:, HC:, HC:, m/z ULTRAFAST PROTEIN SEPARATION USING HALO PROTEIN ES-C HC:, Column: Part Number: - Mobile Phase A:.% formic acid with mm Ammonium Formate Mobile Phase B: % ACN/% IPA/% A solvent Gradient: % B in min. Detection: nm and MS using pps scan rate from to m/z Injection Volume: alkylated lgg Sample Solvent:.% (v/v) formic acid in water MS Parameters: Positive ion mode, ESI at +. kv, LC-MS System: Shimadzu Nexera and LCMS- (single quadrupole MS) Pressure limit, bar/ psi LARGE PROTEIN SEPARATION USING HALO PROTEIN C FUSED-CORE COLUMN Figure DD. High resolution separation of light and heavy chains of a denatured contractile protein (whole myosin from purified rabbit skeletal muscle) using HALO Protein C at C. Light chain subunits ~- kda Expanded view Heavy chain subunits ~ kda Column:. x mm, HALO. μm Protein C Part Number: - Instrument: Agilent SL Injection Volume: μl Sample: denatured myosin Detection: nm Flow Rate:. ml/min. Mobile Phase A: water/.% TFA Mobile Phase B: acetonitrile/.% TFA Gradient: % B in min. Figure FF. An ultrafast protein separation is achieved on a HALO Protein ES-C Ångstrom column in less than minute. Figure GG. HALO Protein C dramatically outperforms a conventional non-core C column,delivering narrower and taller peaks with improved recovery of holotransferrin, apomyoglobin, catalase and enolase. HALO Protein C,. μm, Å Pressure: bar - HALO Protein ES-C,. μm, Å Pressure: bar Non-core C, μm, Å Pressure: bar Non-core C, μm, Å Pressure: bar Comparative results presented here may not be representative for all applications. Column:. x mm HALO Protein ES-C Part Number: - Instrument: Agilent SL Injection Volume: Detection: nm Temperature: Flow Rate: ml/min. Mobile Phase A: water/.% TFA Mobile Phase B: acetonitrile/.% TFA Gradient: % B in min. Using and μl heat exchangers in column compartment PEAK IDENTITIES. Ribonuclease A. Cytochrome c. Lysozyme. -Lactalbumin. Catalase NOTE: Peak widths at half height are shown above respective peaks. HALO PROTEIN C PROVIDES NARROWER AND TALLER PEAKS THAN TOTALLY POROUS COLUMN Comparative results presented here may not be representative for all applications. Column:. x mm HALO Protein C Part Number: - Instrument: Agilent SL Injection Volume: Detection: nm Flow Rate:. ml/min. Mobile Phase A: water/.% TFA Mobile Phase B: acetonitrile/.% TFA Gradient: % B in min. PEAK IDENTITIES. Ribonuclease A. Cytochrome c. Lysozyme. Holotransterrin. Apomyoglobin. Catalase. Enolase See page for full list of HALO Protein part numbers.

15 COMPARISON OF FUSED-CORE TO NON-CORE COLUMNS FOR PEPTIDE SEPARATIONS HALO Peptide Figure HH. HALO Peptide. column produces significantly taller peaks and higher peak capacity than a non-core μm column. HALO Angstrom columns currently available in three particle sizes (,. and μm) and in two sterically protected phases: ES-C (all particle sizes) and ES-CN (. and μm) (Table F) Extremely stable at high temperatures and low ph Ideal for both ultrafast and ultrahigh resolution separations of peptides and polypeptides up to kda Outperforms non-core μm, Angstrom columns in terms of peak width, peak capacity and peak height (Figure HH) HALO. Peptide μm. μm. μm Shell with Å pores. μm. μm Non-core C μm, Å P max = bar.... HALO Peptide ES-C. μm, Å P max = bar Comparative results presented here may not be representative for all applications..... Columns:. x mm, HALO. Peptide ES-C and. x mm, non-core μm C column Part Number: - Mobile Phase A: % water/% ACN/.% TFA Mobile Phase B: % water/% ACN/.% TFA Gradient: -.% B in min. Flow Rate:. ml/min. Temperature: Injection Volume: LC System: Agilent. Gly-Tyr. Angiotensin / (-) amide. Val-Tyr-Val. Met-Enk. Angiotensin / (-) amide. Angiotensin II. Leu-Enk. Angiotensin (-) human. Angiotensin (-) mouse NOTE: Peak widths at half height are shown above respective peaks. Offers comparable peak capacity to sub- μm non-core columns at % back pressure (. μm) ~ % higher peak capacity than sub- μm non-core columns at comparable back pressure ( μm) Columns (Peptide. and μm) can be used in series to increase peak capacity for UHPLC and HPLC analyses of complex tryptic digest samples (Figure II) HALO Peptide ES-CN (. and μm) offers different selectivity and improved retention for polar peptides (Figure JJ) μm inlet frit (. and μm); μm inlet frit ( μm) provides extra protection from plugging Pressure limit, bar/ psi (. and μm); Pressure limit, bar/ psi ( μm). μm Shell with Å pores HALO Peptide. μm. μm. μm Shell with Å pores COUPLED HALO PEPTIDE COLUMNS FOR MAXIMUM PEAK CAPACITY Figure II. Three HALO Peptide ES-C -mm columns ( mm total length) were connected in series to achieve a peak capacity of for this mixture of tryptic digests of α--glycoprotein and apotransferrin ALTERNATE SELECTIVITY USING HALO PEPTIDE ES-CN Columns: Three -mm. mm HALO Peptide. ES-C columns Part Number: of - Mobile Phase A: water/.% formic acid/ mm ammonium formate Mobile Phase B: A with % acetonitrile Gradient: % B in min. Flow Rate:. ml/min. Detection: nm Injection Volume: --glycoprotein tryptic digest and apotransferrin tryptic digest Figure JJ. HALO Peptide ES-CN offers alternative selectivity to HALO ES-C for this mixture of peptides and polypeptides. Graphical representation of a polypeptide structure having beta-pleated sheet and alpha-helical regions HALO Peptide ES-CN - HALO Peptide ES-C - Column:. x mm Part Numbers: HALO Peptide ES-CN: - HALO Peptide ES-C: - Instrument: Optimized Agilent Injection Volume: Detection: nm Flow Rate:. ml/min Mobile Phase A: water/.% TFA Mobile Phase B: ACN/.% TFA Gradient: % B in min.. Asp-Phe. Angiotensin (-) amide. Tyr-Tyr-Tyr. Bradykinin. Leu-Enk. Angiotensin II. Neurotensin. ß endorphin. Sauvagine. Mellitin See page for full list of HALO Peptide part numbers.

16 SEPARATION OF N-LINKED GLYCANS FROM RIBONUCLEASE B Ångstrom pore size HALO Glycan Figure LL. Gradient HILIC-MS separation of N-linked glycans, which had been released using PNGase from ribonuclease B, using the HALO Glycan column. Incorporates a highly polar ligand that contains hydroxyl groups tethered to. μm Fused-Core silica particles via novel, proprietary linkage chemistry (Table F) Ideal for hydrophilic interaction liquid chromatography (HILIC) separations of oligosaccharides, and particularly, of released and labeled glycans from glycoproteins and proteoglycans Mobile phases typically consist of acetonitrile and aqueous ammonium formate buffer ( mm, ph.) used to form a gradient of increasing water content during elution Shell with Å pores. μm. μm. μm. Man Man Man Man Man Ribonuclease B N-Linked Glycans Column:. x mm,. μm HALO Glycan Part Number: - Mobile Phase A: mm Ammonium Formate, ph. Mobile Phase B: Acetonitrile Gradient:..% B in. min Flow Rate:. ml/min. Temperature: C Detection: UV nm Injection Volume: Each lot of HALO Glycan material is tested for quality assurance (Figure KK) by separation of a procainamidereducing-end-labeled glycan ladder of oligosaccharides having glucose units (GU). - Peaks for oligosaccharides composed of and GU must meet tight specifications for retention and peak width before lot is approved for glycan analysis μm inlet frit Pressure limit, bar/ psi QA ANALYSIS OF HALO GLYCAN nm Figure KK. Example QA Chromatogram for HALO Glycan column. Each HALO Glycan packing lot is tested using this glycan ladder mixture to assess and ensure lot-to-lot reproducibility.. G# = DP of maltooligosaccharide For example, G = maltotriose G G G G G G G G G G Column: Part Number: - Mobile Phase A: mm Ammonium Formate, ph. Mobile Phase B: Acetonitrile Gradient: -% B in min. Flow Rate:. ml/min. Pressure: bar Detection: UV nm Injection Volume: Sample Solvent: / ACN/water Response Time:. sec. Data Rate:. Hz Flow Cell: LC System: Shimadzu Nexera Figure MM. Released- and procainamide-labeled glycans from human IgG were separated using a. x mm HALO Glycan column and detected using UV and selected-ion-monitoring MS detection ,,,,, -.,..... :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) :.(+) GF-N G GF GF GF GF GF..... GFB G... GF GFB SEPARATION OF N-LINKED GLYCANS FROM HUMAN IgG GF+NeuAc UV MS.... G+NeuAc GF+NeuAc GFB+NeuAc GFB+NeuAc Column:. x mm, HALO. μm Glycan Part Number: - Mobile Phase A: mm Ammonium Formate, ph. Mobile Phase B: Acetonitrile Gradient:. % B in min Flow Rate:. ml/min. Temperature: C Detection: UV nm Injection Volume: LC System: Shimadzu Nexera MS: Shimadzu LCMS (single quadrupole) ESI: +. kv Scan range: - m/z Scan rate: pps See page for full list of HALO Glycan part numbers.

17 HALO UHPLC AND HPLC GUARD COLUMNS Collect strongly retained compounds from the sample and minimizes column fouling Finger-tight, direct-connect units that auto-adjust to any column with a inlet port Available for all HALO analytical geometries (.,. and. mm ID) and phases Ultra-low dispersion, easy to use, operate at pressures up to bar Easily replace guard cartridge without removing guard holder from the flow path HALO GUARD COLUMNS: PROTECTION + PERFORMANCE with guard column See figure below for an exploded view of the HALO guard cartridge and guard holder. Please see pages for ordering information. Finger-tight guard cartridge holder, Inlet Provides easy replacement of guard cartridge Finger-tight guard cartridge holder, Outlet Allows for replacement of guard cartridge without removing the guard holder Titanium hybrid ferrule Adds durability for multiple connections Figure NN. HALO guard columns provide optimum protection for your HALO HPLC and UHPLCcolumn without sacrificing column efficiency. nm no guard column Column:. x mm. μm HALO C Mobile Phase: / ACN/water Flow Rate:. ml/min. Detection: nm Injection Volume: μl Pressure: bar with guard column bar without guard column Instrument: Optimized Agilent. ID tubing Hz data rate HALO guard cartridge All HALO phases available Auto-adjusting zero dead volume (ZDV) end fitting Ensures optimum, ZDV connection to the analytical column The Optimize Technologies EXP Titanium Hybrid Ferrule is Patent Pending. The EXP Holder is patented under Patent No.,, B. The OPTI- prefix is a registered trademark of Optimize Technologies, Inc.

18 HALO COLUMNS HALO. COLUMNS The part numbers for HALO columns are presented below. HALO columns are available in the following analytical internal diameters (. and. mm). Guard columns are also available for HALO columns for these IDs for UHPLC to provide additional protection when necessary. HALO. columns are available in nano, capillary and analytical diameters, as well as in a mm semi-preparative diameter. Guard columns are available for analytical diameters of.,., and. mm. μm, Å ID x Length (in mm) C C Phenyl Hexyl RP-Amide PFP ES-CN Penta-HILIC HILIC. x x x x x x x x x x x x ID x Length (in mm) C C Phenyl Hexyl RP-Amide PFP ES-CN Penta-HILIC HILIC. x x Guard Column Holder - Visit our web site ( or contact one of our distributors.. μm, Å (in mm) C C Phenyl Hexyl RP-Amide PFP ES-CN Penta-HILIC HILIC. x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x μm, Å Guard columns, -pack (in mm) C C Phenyl Hexyl RP-Amide PFP ES-CN Penta-HILIC HILIC. x x x Guard Column Holder -

19 HALO COLUMNS HALO PROTEIN COLUMNS HALO GLYCAN COLUMNS HALO columns are available in nano, capillary and analytical diameters, and in a mm semi-preparative diameter. HALO Guard columns are available for analytical diameters of.,. and. mm. μm, Å (in mm) C C Phenyl Hexyl RP-Amide PFP ES-CN Penta-HILIC HILIC. x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x μm, Å Guard columns, -pack (in mm) C C Phenyl Hexyl RP-Amide PFP ES-CN Penta-HILIC HILIC. x x x Part numbers for nano, capillary, analytical and semipreparative HALO Protein columns in both C and ES-C phases are provided below. Guard columns are available for HALO Protein columns in.,. and. mm IDs for UHPLC and HPLC applications to provide additional column protection when desired. (in mm) HALO Protein C HALO Protein ES-C. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - -. x - - Guard Columns, -pack (in mm) HALO Protein C HALO Protein ES-C. x - -. x - -. x - - Guard Column Holder - HALO Glycan columns are available in. and. mm diameters in the following lengths in. μm particle sizes. Guard columns are available for UHPLC and HPLC applications if additional protection is desired. (in mm) HALO Glycan. x -. x -. x -. x -. x -. x - Guard Columns, -pack (in mm) HALO Glycan. x -. x - Guard Column Holder - Visit our web site ( or contact one of our distributors. Guard Column Holder -

20 HALO PEPTIDE COLUMNS The part numbers are provided below for the nano, capillary, analytical and semi-preparative HALO in ES-C, and HALO. and HALO Peptide columns in both ES-C and ES-CN phases. Guard columns are available for.,. and. mm internal diameters for UHPLC and HPLC applications, if additional protection is desired. ID x Length (in mm) HALO Peptide ES-C HALO. Peptide ES-C HALO. Peptide ES-CN HALO- Peptide ES-C HALO- Peptide ES-CN. x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x - - REFERENCES. Adapted from book, Introduction to Modern Liquid Chromatography, rd Edition, L. R. Snyder, J. J. Kirkland and J. W. Dolan,, p., Wiley & Sons.. Orthogonal separations for reversed-phase liquid chromatography; J. Pellett, P. Lukulay, Y. Mao, W. Bowen, R. Reed, M. Ma, R.C. Munger, J.W. Dolan, L. Wrisley, K. Medwid, N.P. Toltl, C.C. Chan, M. Skibic, K. Biswas, K. A. Wells, and L.R. Snyder; Journal of Chromatography A, ().. Column selectivity in reversed-phase liquid chromatography I. A general quantitative relationship; N.S. Wilson, M.D. Nelson, J.W. Dolan, L.R. Snyder, R.G. Wolcott and P.W. Carr; Journal of Chromatography A, ().. Column selectivity in reversed-phase liquid chromatography IV. Type-B alkyl-silica columns; J. J. Gilroy, J. W. Dolan and L. R. Snyder; Journal of Chromatography A, () ( ColumnMatch ).. D.V. McCalley and U.D. Neue, J. Chromatogr. A, ().. A.J. Alpert. Anal. Chem., ().. D.V. McCalley, J. Chromatogr. A, ().. A.J. Alpert et al., Anal. Chem., (). Guard Columns, -pack ID x Length (in mm) HALO Peptide ES-C HALO. Peptide ES-C HALO. Peptide ES-CN HALO- Peptide ES-C HALO- Peptide ES-CN. x x x Guard Column Holder - Visit our web site ( or contact one of our distributors. info@advanced-materials-tech.com

21 Halo and Fused-Core are registered trademarks of Advanced Materials Technology, Inc.