HPLC Columns from Supelco RP-Amide C18 C8 Elevated HPLC Performance sigma-aldrich.com/supelco 2 Supelco – A Pioneer in Chromatography upelco’s 30-year history in pioneering Fluorinated phases are another area of pioneering Schromatography products has led to research for Supelco. Discovery® HS F5 is a market worldwide recognition of its products. leader providing reversed phase as well as aqueous normal phase (ANP) or hydrophilic interaction (HILIC) Supelco introduced SUPELCOSIL™ LC-18-DB, the fi rst separations. base deactivated HPLC column, in 1982. It provided Supelco, along with Sigma-Aldrich, Fluka, and improved peak shape for basic compounds that Riedel-de Haën, provides thousands of chromatogra- traditionally tailed with early generation columns. phy related products through a worldwide R&D, Supelco also innovated with the amide-type embed- manufacturing, distribution and sales operation. This ded polar group (EPG) column, Suplex™ pKb-100, and includes sales and distribution in over 35 countries and has been a leader in EPG bonding chemistry. R&D and production on four continents. Sigma-Aldrich... The World’s Leading Supplier of Analytical Products sigma-aldrich.com/supelco ASCENTIS™ HPLC COLUMNS 3 The Ascentis Family of HPLC Columns The Ascentis family of columns is the fourth genera- tion provides quality oversight into all aspects of the tion of HPLC column technology from Supelco scien- manufacturing process leading to a product that tists. Ascentis columns are bonded on high purity, consistently meets exacting specifi cations. 100 angstrom silica including 3, 5, and 10 micron Supelco’s bonding chemistry advances have particle size. Columns are designed for small molecule achieved: applications and are scalable from micro columns ● Improved stability for extremely long column life (1.0 mm I.D.) to preparative dimensions (50 mm I.D.). ● Better reproducibility for easy method validation The family includes C18, C8 and an embedded polar ● group phase, RP-Amide. Low bleed for sensitive LC-MS applications ● Columns are manufactured at our ISO 9001 regis- Excellent selectivity and increased retention tered facility in Bellefonte, PA. This ISO 9001 registra- for polar compounds Ascentis Separates the Entire Polarity Spectrum / 814-359-3041 Ascentis HPLC Columns (US and Canada only) Column Specifi cations C18 C8 RP-Amide USP Code L1 L7 L60 Bonded Phase Octadecyl Octyl Amido embedded Endcapped Yes Yes Yes Particle Shape Spherical Spherical Spherical Particle Purity <5 ppm metals <5 ppm metals <5 ppm metals Particle Size 3,5,10 µm 3,5 µm 3,5,10 µm Pore Size 100 Å 100 Å 100 Å Surface Area 450 m2/g 450 m2/g 450 m2/g Carbon Load 25% 15% 19.5% pH Range 2 - 8 2 - 8 2 - 8 / 814-359-3441 technical service: 800-359-3041 (US only) ordering: 800-247-6628 ordering: Elevated HPLC Performance 4 Extended Column Life Save time and money One of the most important parameters surface area of the silica help to create a more consistent in choosing a column is lifetime. and well-packed column that minimizes shifting of the Columns that last longer save time and money. Column column bed during use. This reduces the chance of voiding. failure often occurs because of hydrolytic instability of the Supelco scientists have performed extensive column stationary phase and silica as well as mechanical instability lifetime studies under conditions employed in most HPLC of the column packing (voiding). Ascentis columns are laboratories. Studies included the use of pH modifi ers such designed to last beyond competitive columns. as TFA, phosphate, and ammonia coupled with both The high phase coverage of all Ascentis products plus acetonitrile and methanol conditions. A range of tempera- proprietary endcapping create a phase that is more stable tures was explored across a variety of probe analytes toward hydrolytic attack. Supelco’s narrow specifi cations on including neutrals, acids, bases, and pharmaceuticals. Extended Column Life Even Under Harsh pH 1.5 Conditions 12 Extended Column Life 10 Toluene 8 k’ 6 4 2,4-dichlorophenoxyacetic acid 2 0 0 2000 4000 6000 8000 Number of Injections* *Equivalent to 80,000 column volumes 3 Quality Control Application First Run Symmetry and selectivity remain unchanged on 1 this sensitive application even after 80 days▲ of pH 5 4 1.5 conditions. (▲100 injections a day) 2 column: Ascentis C18, 5 cm x 3.0 mm I.D., 5 µm particles (581329-U) mobile phase: 60:40, methanol:100mM ammonium acetate, pH 6.8 fl ow rate: 0.43 mL/min. G003379 temp.: 25 °C 3 det.: 230 nm inj.: 5 µL After 80 Days 1. Uracil 2. Hydroquinidine 3. Quinidine 1 4. Fluoxetine 5 4 5. Diphenhydramine 2 G003380 sigma-aldrich.com/supelco ASCENTIS™ HPLC COLUMNS 5 Highly Reproducible HPLC Columns Minimize troubleshooting Reproducibility is imperative to any Bonding procedures are transferred to production through application in any laboratory. a manufacturing transfer validation procedure. Each bonded lot is analyzed by QC and must pass bond lot specifi cations A novel column may be interesting, but if the retentivity before a single column is packed. Once the bond lot meets and selectivity are not consistent, it has little value in a specifi cations, each individual column must meet QC validated method. Supelco has designed reproducibility into specifi cations prior to being tagged with the Ascentis label. the Ascentis family starting with tight specifi cations on the Detailed production and QC documents are maintained for surface area, pore size, and particle size of the silica. every column that receives the Ascentis label. Carbon Load on Ascentis C18 Since Inception Columns 30.0 NOTE: 1.0% RSD for carbon load. 28.0 Highly Reproducible Highly Reproducible / 814-359-3041 26.0 % Carbon 24.0 (US and Canada only) 22.0 20.0 Carbon load data obtained from 3, 5, and 10 micron bond lots. Ascentis RP-Amide Reproducibility - Aromatic Compounds column: Ascentis RP-Amide, 15 cm x 4.6 mm I.D., 5 µm particles (565324-U) mobile phase: 30:70, water:acetonitrile NOTE: Less than 1.0% fl ow rate: 1.0 mL/min RSD for retention times. temp.: 35 °C det.: UV, 254 nm inj.: 2 µL 1. Butylbenzene 2. o-Terphenyl 3. Triphenylene 4. Amylbenzene Butylbenzene Triphenylene / 814-359-3441 technical service: 800-359-3041 BL Rt USP Tail Factor N Rt USP Tail Factor N 5403A 8.66 0.950 13657 13.03 0.959 13746 5422A 8.69 0.923 12527 12.88 0.919 12594 (US only) 5433 8.76 1.000 14101 13.06 1.010 14350 Avg. 8.700 0.957 13428 12.980 0.959 13563 St. Dev. 0.051 0.039 811 0.102 0.040 892 % RSD 0.580 4.070 6.04 0.780 4.220 6.57 Bond Lot 5433 Bond Lot 5422A Bond Lot 5403A G003390 ordering: 800-247-6628 ordering: Elevated HPLC Performance 6 Ideal for LC-MS Short, Fast Columns Multiplying Productivity with lower solvent consumption. Ascentis columns work well for Elevated Performance fast LC because they are rugged and more retentive than Imagine facing more and more samples each month other reversed phase columns. Greater reversed phase without an increase in chemists or instruments. The solution retention allows more organic solvent that lowers viscosity to this dilemma is here today with Ascentis fast HPLC and pressure. Another potential benefi t of operating with columns. Fast LC performs rapid analysis using short (2-10 higher organic mobile phases is increased performance from cm) columns operating in the 1-4 mL/min range. Fast LC has evaporative detectors such as MS. Ascentis fast LC columns many benefi ts including faster analysis, reduced method are available with all three phases (C18, C8, and RP-Amide) LC-MS development time, increased mass spec sensitivity, and with column lengths down to 2 cm. Fast Gradient Analysis on Ascentis Columns A comparison of all three Ascentis phases under fast 8 Ascentis RP-Amide 3 gradient conditions is shown. A set of aromatics with a 5 variety of functional groups was utilized. The interactions 4 7 NOTE: Elution between the anilines, phenols, and carboxylic acids and the order is reversed Ascentis RP-Amide give different selectivity as compared to for peaks 4 and 5. the Ascentis C18 and C8. When comparing the Ascentis C18 6 and C8, the Ascentis C8 is more retentive at lower organic 1 2 while the Ascentis C18 is more retentive at higher organic. column: Ascentis RP-Amide, 3 cm x 3.0 mm I.D., 5 µm particles Ascentis C18, 3 cm x 3.0 mm I.D., 5 µm particles Ascentis C8, 3 cm x 3.0 mm I.D., 5 µm particles G003375 mobile phase A: 95:5, 10 mM ammonium phosphate:acetonitrile, pH 2.5 mobile phase B: 40:60, 10 mM ammonium phosphate:acetonitrile, pH 2.5 4 fl ow rate: 3.5 mL/min Ascentis C18 5 8 temp.: 35 °C 3 det.: 220 nm inj.: 10 µL 7 gradient: 0% B to 100% B in 4 min. 1. p-Toluidine 2. p-Nitroaniline 3. p-Nitrophenol 6 4. p-Cresol 5. p-Nitrobenzoic acid 1 2 6. p-Methylbenzoic acid 7. p-Nitrotoluene 8. p-Xylene G003376 4 Ascentis C8 5 8 3 7 6 1 2 G003377 sigma-aldrich.com/supelco ASCENTIS™ HPLC COLUMNS 7 Fast Gradient Analysis on an Ascentis RP-Amide Column with MS Detection A column lifetime study was performed using a fast 2 5 gradient to separate caffeine (major component) and catechins from green tea extract. Even after 500 injections, 1st Injection all seven peaks are still resolved in under two minutes. 3 7 6 column: Ascentis RP-Amide, 2 cm x 4.6 mm I.D., 3 µm particles (565316-U) mobile phase A: 10 mM ammonium formate, pH 3.0 with concentrated formic acid mobile phase B: methanol 4 fl ow rate: 1 mL/min., split to the mass spectrometer temp.: 35 °C det.: MS, ESI (-) and (+) in selected ion recording (SIR) 1 mode (combined) inj.: 5 µL sample: water extract of green tea gradient: Min.