AQUASIL C18 Columns. AQUASIL 18 Columns TG Compatible with 100% aqueous mobile phase

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1 AQUASIL C8 Columns AQUASIL 8 Columns TG0-0 Compatible with 00% aqueous mobile phase

2 AQUASIL OH OH AQUASIL C8 columns provide a versatile C8 phase for a wide range of application areas. AQUASIL C8 columns have a hydrophilic endcapping that gives up to twice the retention for polar compounds and alternative selectivity compared to traditional C8 phases. AQUASIL C8 columns are also compatible with 00% aqueous mobile phases without loss of performance. Exhibits different retention & selectivity than conventional C8 Excellent peak shapes for basic, acidic and neutral compounds Retains polar molecules twice as strongly as conventional C8 Compatible with 00% aqueous mobile phase Excellent results with low buffer concentrations Stable for LC/MS applications Applications Highly polar compounds Nucleosides and Nucleotides Organic acids Vitamins Peptides Catecholamines Specifications: Chromatographic Characterization Packings that offer additional modes of interaction give rise to quite different retention behavior and selectivity. In general, analytes with the greatest polar functionality will typically show the greatest changes in selectivity and retention. Figure illustrates the difference in behavior between the AQUASIL C8 column and the BetaBasic 8 column (a highly base deactivated and densely bonded C8 column that has a very similiar percent carbon value). Figure a demonstrates that where analyte interactions are based purely on hydrophobic (or dispersive) interactions, the AQUASIL C8 column is slightly less retentive than the BetaBasic 8 column. The AQUASIL C8 packing was designed for the reversed phase separation of polar molecules. Despite its relatively high concentration of C8 groups, it also has hydrophilic sites that help to provide increased retention of highly polar water soluble compounds (Figure b,c). Figure b shows how the AQUASIL C8 column offers the nearly twice the retention for several polar, basic compounds when compared to a BetaBasic 8 column. The retention of basic compounds and also polar acidic compounds on the AQUASIL C8 column are both significantly increased compared to the BetaBasic 8 column. This illustrates clearly how useful the AQUASIL C8 column can be when increased retention of polar compounds or alternative selectivity is required. Increased Retention of Polar Compounds Phase Particle Size Carbon Load Pore Size Silica Type AQUASIL C8 and µm % 00Å High purity, base deactivated AQUASIL C8 columns combine a C8 phase with polar endcapping to provide a unique material for reversed phase chromatography, offering alternative selectivity, increased retention of polar compounds, and no phase collapse under 00% aqueous conditions. These columns maintain selectivy with reduced concentrations of buffers and additives, making them ideal for use with LC/MS. In this Technical Guide we review the AQUASIL C8 packing, which has been tailored to go beyond the limitations of traditional C8 packing materials. Polar compounds often elute near or at the unretained marker when run on typical C8 HPLC columns. AQUASIL C8 columns provide additional analyte ligand interactions to reversed phase hydrophobic interactions, leading to increased retention of analytes with polar functionality. AQUASIL C8 columns maintain retention of neutral compounds while offering increased retention for both acidic and basic compounds.

3 Figure a) Less retentive for neutral compounds Chromatographic Retention Behavior AQUASIL C8 vs BetaBasic 8 Columns BetaBasic 8 AQUASIL C8 8 8 Columns: µm, 0x.mm Eluent: % H O / % ACN Flow:. ml/min Detector: Uracil. Benzene. Ethylbenzene. Propylbenzene. Butylbenzene. Pentylbenzene. Hexylbenzene 8. Heptylbenzene Dispersive interactions are the primary interactions generally associated with retention on traditional alkyl C8 type packings. Secondary interactions associated with residual silanols have been significantly reduced by endcapping, improvements in silica quality and increased density of the derivatized ligand. Silanol interactions that previously gave rise to broad tailing peaks for basic analytes have therefore, to some extent, been eliminated. These secondary interactions are also responsible in part for the retention of compounds with polar functionality, either by hydrogen bonding interactions or via ion exchange interactions. The progressive elimination of the secondary silanol interactions has resulted in columns that give neucom b) More retentive for basic compounds BetaBasic 8 AQUASIL C8 Columns: µm, 0x.mm Eluent: 90% 0mM KH PO, ph. / 0% ACN Flow:. ml/min Detector: UV@. Uracil. Procainamide. N-Acetylprocainamide. Caffeine. N-Propionylprocainamide. Phenol ABB-00 c) More retentive for polar acidic compounds BetaBasic 8 Columns: µm, 0x.mm Eluent: 80% 0.% Formic Acid / 0% ACN Flow:.0 ml/min Detector: Uracil. Phloroglucinol. Resorcinol. Phenol AQUASIL C8 polcom MIN

4 good peak shape for basic compounds but reduced retention of polar compounds in general. AQUASIL C8 columns provide an excellent combination of traditional reversed phase interactions and polar interactions to retain more polar analytes. Figure demonstrates the capability of the AQUASIL C8 column to separate catecholamines without ion pair reagents in a highly aqueous mobile phase. Highly Aqueous Mobile Phases The inclusion of polar functionality to the stationary phase also increases the wetting characteristics of the packing in highly aqueous mobile phases. The AQUASIL C8 column can be run in 00% aqueous mobile phase conditions and shows no tendency towards phase collapse (Figures and ). Phase collapse is often seen with C8 packings unless a small amount of organic solvent (-%) is added to the mobile phase. As a result of phase collapse, the retention and selectivity of the phase is lost and the column must be regenerated using a pure organic solvent wash. The AQUASIL C8 packing is immune to this folding due its unique polar functionality. Refer to Technical Bulletin TB99-0 for further explanation of phase collapse. Figure Catecholamines Figure AQUASIL C8 Packing Immune to Folding Figure Nucleotides. Epinephrine. L-DOPA. DL-Tyrosine. Vanillylmandelic acid Stable retention even in 00% aqueous buffer Initial Run. Tartaric acid. Malonic acid. Fumaric acid. Succinic acid. '-CTP 0.mg/mL. '-CDP 0.mg/mL. '-CMP 0.mg/mL. '-GDP 0.mg/mL. '-GMP 0.mg/mL. '-ATP 0.mg/mL. '-ADP 0.mg/mL 8. '-AMP 0.mg/mL After Hours of Flushing with Mobile Phase MIN MIN AQUASIL C8, µm, 0x.mm Eluent: 98% 0.M KH PO, ph.0 with H PO / % ACN Flow:.0 ml/min Detector: AQUASIL C8, µm, 0x.mm Eluent: 0.M KH PO, ph.0 with KOH Flow:.0 ml/min Detector: 0 0 MIN AQUASIL C8, µm, 0x.mm Eluent: 0.0M KH PO +0.0M H PO, ph. Flow:. ml/min Detector: 0

5 Reduced Buffer Concentrations and Increased MS Sensitivity At any concentration, additives such as trifluoroacetic acid can cause ion suppression and consequently reduce sensitivity in LC/MS methods. The choice of HPLC column is of key importance for LC-MS applications, since the properties of the bonded stationary phase and underlying silica can strongly influence the concentration of additive required. It is good practice to use volatile mobile phase additives in LC/MS methods to enhance ionization. The addition of acidic modifiers such as formic acid or TFA is commonplace when analyzing proteins and peptides by reversed phase chromatography. The additive solvates the analyte, displacing water molecules and creating a more hydrophobic analyte with stronger retention on traditional C8 packings. The AQUASIL C8 packing can retain peptides in their water soluble state, reducing the need for TFA as an additive. The examples shown in Figure illustrate how the AQUASIL C8 column can be used at very low TFA concentrations while maintaining retention and performance of many of the peptides of interest. Low level additive concentrations (Figure C) also offer the reward of increased sensitivity for MS. This is an important consideration when trying to identify trace quantities of a drug compound or impurity that may normally disappear into the noise of the baseline of the MS response. Figure Tryptic Digest of β-lactoglobulin Aquasdil-digest-after-acn0%TFA- RT: SM: B Aquasil C8, 0xmm, m Eluent: A - HO + TFA B - ACN + TFA Gradient: Time % B DUO Detection:+ESI Thermo Finnigan LCQ Spray Voltage:. kv Source Temp: 0 C Sheath Gas: 80 au Aux Gas: 0 au Cap. Voltage: 8. V Tryptic Digest of β-lactoglobulin % TFA NL:.0E Base Peak F: + c ESI ] MS aquasdil-digest-after-cn0%tfa- Relative Abundance % TFA NL:.0E Base Peak F: + c ESI Full ms [ ] MS aquasdil-digest-after-acn0-0%tfa % TFA NL:.0E Base Peak F: + c ESI Full ms [ ] MS aquasdil-digest-00%tfa- -09

6 Acid Base Mix Water-Soluble Vitamins Fat-Soluble Vitamins. Uracil. Lidocaine. Chlorpheniramine. Benzoic Acid. Doxepin. Vitamin C. Thiamine (Vitamin B). Pyridoxine (Vitamin B). Niacinamide (Vitamin B). Vitamin B. Folic Acid. d-biotin (Vit. H) 8. Vitamin B 9. Vitamin B 9 A). Lutein. Zeaxanthin.&. Lycopene. β-cryptoxanthin. α-carotene. β-carotene B A B). Retinol. Tocol. σ-tocopherol. γ-tocopherol. α-tocopherol A 8 A A A A A 0 8 0MIN AQUASIL C8, µm, 0x.mm Eluent: A: 0.0M KH PO + 0.0M H PO, ph. B: ACN % A / % B Flow:. ml/min Detector: Peptides MIN -0 AQUASIL C8, µm, 0x.mm Gradient: A: 0.M K HPO, ph. B:0% ACN / 0% 0.0M K HPO, ph. % B to 0% B in 9 min Flow:.0 ml/min Detector: 0 Bio-Remediation Acids B B B B -0b 0 0 MIN. AQUASIL C8, µm, 0x.mm Eluent: 8%ACN / %Dioxane/%MeOH Flow:. ml/min Detector: chromatogram A: UV@0 nm chromatogram B: UV@/00 nm Data courtesy of Neal Craft, Craft Technologies, Inc. Clindamycin Alberta Peptide Mix (heptapeptides). Benzoic acid. -Fluoro benzoic acid. -Toluic acid. -Toluic acid.,,-trimethyl benzoic acid.,-dimethyl benzoic acid.,-dimethyl benzoic acid. Lincomycin. Internal Standard. Clindamycin-b. -epiclindamycin. Clindamycin MIN AQUASIL C8, µm, 00x.mm Gradient: A: 0.% TFA in H O B: 0.% TFA in ACN 0%B to 0%B in 0 min. Flow:.0 ml/min Detector: MIN AQUASIL C8, µm, 0x.mm Eluent: 0% MeOH / 0% 0.0M KH PO, ph. Flow:. ml/min Detector: 0 Temp:.0 +/- 0. o C Data courtesy of James D. Stuart and Reena M. Joseph, Department of Chemistry, Univ of Connecticut, Storrs, CT MIN AQUASIL C8, µm, 0x.mm Eluent: A: MeOH B:g/L dl-0-camphorsulfonic acid, g/l NH acetate, 0.% glacial acetic acid. Adjust to ph with HCL or NaOH solution. 0% A / 0% B Flow:.0 ml/min Detector: RI

7 Procainamides Comparison Ground Water Extract. Uracil. Procainamide. N-Acetylprocainamide. Caffeine. N-Propionylprocainamide. Benzoic acid. -Toluic acid. -Toluic acid.,,-trimethyl benzoic acid., &,-Dimethyl benzoic acid MIN BetaBasic 8, µm, 0x.mm Eluent: 0% ACN / 90% 0.0M KH PO, ph. Flow:. ml/min Detector: MIN AQUASIL C8, µm, 0x.mm Eluent: 0% ACN / 90% 0.0M KH PO, ph. Flow:. ml/min Detector: MIN AQUASIL C8, µm, 0x.mm Eluent: 0% MeOH / 0% 0.0M KH PO, ph. Flow:. ml/min Detector: 0 Temp: o C Nucleosides Organic Acids in Highly Aqueous Mobile Phase. Cytidine. Uridine. Adenosine. Guanosine. Thymidine. Oxalic Acid. Tartaric Acid. Pyruvic Acid. Malic Acid. Lactic Acid. Acetic Acid. Citric Acid 8. Maleic Acid 9. Succinic Acid 0. Fumaric Acid. Propionic Acid. Butyric Acid MIN AQUASIL C8, µm, 0x.mm Eluent: A: 0.0M KH PO, & 0.0M H PO, ph. B: H O C: ACN.% A /.% B / % C Flow:.0 ml/min Detector: MIN AQUASIL C8, µm, 0x.mm Eluent: % ACN / 99% 0.0M KH PO, ph.8 Flow:. ml/min Detector: 0 BetaBasic is a registered Trademark of Thermo Hypersil-Keystone. 00 Thermo Hypersil-Keystone. All Rights Reserved. AQUASIL Siliconizing Fluid for treating glass surfaces is sold by PierceChemical Co., Rockford, IL

8 AQUASIL C8 Standard Columns Particle Length Standard bore Standard bore Small bore Small bore Microbore Description Size(µm) (mm) (. mm) (.0 mm) (.0 mm) (. mm) (.0 mm) AQUASIL C AQUASIL C Standard Columns with Integral Guard (COLUMNPLUS Guard, or CPG) Particle Length Standard bore Standard bore Small bore Small bore Microbore Description Size(µm) (mm) (. mm) (.0 mm) (.0 mm) (. mm) (.0 mm) AQUASIL C AQUASIL C Drop-In Guard Cartridges for UNIGUARD Holder and CPG Columns (pk/) Particle Length Standard bore Standard bore Small bore Small bore Microbore Description Size(µm) (mm) (for.mm) (.0 mm) (.0 mm) (. mm) (.0mm) AQUASIL C UNIGUARD Direct-Connect Drop-in Guard Cartridge Holder for use with standard columns NOTE:.0mm drop-ins are used for both.0 and.mm analytical columns. Javelin Guard Columns Available in 0mm and 0mm lengths and NEW.0mm ID! Particle Length Standard bore Small bore Small bore Microbore Description Size(µm) (mm) (.0 mm) (.0 mm) (. mm) (.0 mm) AQUASIL C USA Tel: (800) columninfo@thermo.com UK Tel: + (0) columninfo.uk@thermo.com FRANCE Tel: columninfo.fr@thermo.com GERMANY Tel: 00 /0 0 columninfo.de@thermo.com