ACQUITY UPLC BEH Amide Columns - Waters Corporation

1 [HydropHilic interaction cH romatograpHy]Since 2003, Waters has developed innovative stationary phases for Hydrophilic Interaction chromatography [HILIC] to overcome the challenge of retaining and separating extremely polar compounds. Based on Waters novel ethylene bridged hybrid [BEH] particle technology, New ACQUIT Y UPLC BEH Amide Columns utilize a chemically stable, trifunctionally-bonded Amide phase, enabling a new dimension in stability and versatility for HILIC separations. Designed to retain polar analytes and metabolites that are too polar to retain by reversed-phase [RP] chromatography , ACQUIT Y UPLC BEH Amide Columns facilitate the use of a wide range of mobile phase pH [2 11] to facilitate the exceptional retention of polar analytes spanning a wide range in polarity, structural moiety and pKa.

2 In addition to enhanced retention of polar compounds, ACQUIT Y UPLC BEH Amide Columns provide increased mass spectrometry response, direct compatibility with sample preparation eluates [PPT, LLE and SPE] as well as orthogonal selectivity compared to reversed-phase materials, making HILIC an attractive alternative separation UPLC BEH Amide Columns [ product solution ]Separation of Water Soluble Vitamins0123 min12345678 Column: ACQUITY UPLC BEH Amide , m x 50 mmPart Number: 186004800 Mobile Phase A: 50/50 ACN/H2O with 10 mM CH3 COONH4 and NH4OH, pH Phase B: 90/10 ACN/H2O with 10 mM CH3 COONH4 and NH4OH, pH Rate: mL/min Gradient: Time Profile (min) %A %B Volume: L (PLNO) Sample Diluent: 75/25 ACN/MeOH with HCOOH Column Temp.

3 : 30 C Weak Ndl. Wash: ACN/H2O 95/5 Detection: UV @ 265 nm Sampling Rate: 20 points/secFilter Time Constant: NormalInstrument: Waters ACQUITY UPLC with ACQUITY UPLC PDA DetectorCompounds:1. Nicotinamide 25 g/mL2. Pyridoxal 50 g/mL 3. Riboflavin 50 g/mL 4. Nicotinic acid 50 g/mL5. Thiamine 50 g/mL6. Ascorbic Acid 25 g/mL7. B12 50 g/mL8. Folic Acid 25 g/mLParticle Type: Ethylene Bridged Hybrid [BEH]Ligand Type: Trifunctional AmideParticle Size: mLigand Density: mol/m2 Carbon Load: 12 %Endcap Style: NonepH Range: 2 -11 Pressure Tolerance: 1000 barO Si O OO NH2 LinkerWaters Corporation 34 Maple Street Milford, MA 01757 T: 1 508 478 2000 F: 1 508 872 1990 2009 Waters Corporation .

4 Waters , T he Science of W hat s Possible, ACQUIT Y Ultra Performance LC and ACQUIT Y UPLC are trademarks of Waters Corporation . All other trademarks are the property of their respective owners. October 2009 720003122EN IH-PDF[carboHydrate analysis]One of the most abundant and diverse classes of compounds analyzed by HILIC are carbohydrates [ , monosaccharides, disaccharides, oligosaccharides and polysaccharides].The ACQUIT Y UPLC BEH Amide column is an exceptional tool for the analysis of carbohydrates, providing the following benefits: The m particle size enables high resolution, high speed analysis of carbohydrates in complex sample matrices while maintaining or improving chromatographic resolution.

5 Increased chemical stability enables the use of high pH and high temperature to collapse reducing sugar anomers, shorten analysis times and improve MS detection. BEH particle technology, in combination with a trifunctional-ly-bonded Amide phase, provides exceptional column lifetime, thus improving assay robustness. Unlike amine-based Columns used for carbohydrate analysis, the ACQUIT Y UPLC BEH Amide column is not susceptible to Schiff base formation, thus improving quantitation accuracy. High pH mobile phase compatibility facilitates MS detection without the need for derivatization, post column addition or complexing with a metal cation, thus dramatically simplifying sample pre-treatment before LC/MS analysis and improving assay Analysis of Saccharides901234567 min12345678 Column: ACQUITY UPLC BEH Amide , m x 50 mmPart Number: 186004800 Mobile Phase A: 30/70 MeCN/H2O with NH4OH Mobile Phase B: 80/20 MeCN/H2O with NH4OH Flow Rate: mL/min Gradient.

6 Time Profile (min) %A %B Initial 0 100 60 40 0 100 Injection Volume: L (PLNO) Sample Diluent: 50/50 MeCN/H2 OColumn Temp.: 35 C Needle Wash: ACN/H2O 75/25 Instrument: Waters ACQUITY UPLC with ACQUITY UPLC TQDI onization Mode: ES-Capillary: kVCone Voltage: 25 V Source Temp.: 120 CDesolv. Temp.: 350 CDesolv. Gas Flow: 500 L/HrSIR m/z: (fructose, glucose); (sucrose, maltose); , , , , 115 (maltooligosaccharides [n=1 to 5])Dwell Time: sCompounds:1. Fructose 10 g/mL2. Glucose 10 g/mL3. Sucrose 10 g/mL4.

7 Maltose 10 g/mL5. Maltotriose 10 g/mL6. Maltotetraose 10 g/mL7. Maltopentaose 10 g/mL8. Maltohexahose 10 g/mL9. Maltoheptaose 10 g/mLACQUITY UPLC BEH Amide ColumnsChemistryParticle SizeDimensionPart Number 1 packPart Number 3 packPart Number Method Validation KitBEH x 50 mm186004848176001914 BEH x 100 mm186004849176001915 BEH x 150 mm186004850176001916 BEH x 30 mm186004839176001906 BEH x 50 mm186004800176001907186004807 BEH x 100 mm186004801176001908186004808 BEH x 150 mm186004802176001909 BEH x 30 mm186004803176001910 BEH x 50 mm186004804176001911186004809 BEH x 100

8 Mm186004805176001912186004810 BEH x 150 mm186004806176001913 BEH x 5 mm* 186004799 * VanGuard Pre-column 3-pack