Trefoil Columns a. Safety Considerations b. Column Connectors and Installation c. eCord Installation d. Column Equilibration e. Initial Column Efficiency Determination a. Sample Preparation b. Solvents c. Additives d. Pressure e. Temperature f. Band Spreading Minimization IV. T ROUBL E SHOOT ING V. COLUMN C L EANING, A DDIT IV E S, AND STO RAG E a. Cleaning b. Effects of Additives c. Storage a. Introduction b. Installation c. Column Use Information I. INT RODUC T ION Thank you for choosing a Trefoil® Column. Trefoil Columns are specifically designed to use the complete range of capabilities of the ACQUITY UPC 2® System to achieve fast, robust chiral separations to enable both selectivity and speed and to reduce method development time. Trefoil Columns consist of modified polysaccharide coated stationary phases for broad-spectrum chiral selectivity. The higher order structure of the polysaccharides have specific spatial orientations that impart chiral selectivity through well-chosen modifications or selectors. Trefoil AMY1, CEL1, and CEL2 Column chemistries are complementary to each other and independently offer different retention characteristics for separating chiral compounds. Selectivity can be further enhanced by using co-solvents and additive blends that most favorably modulate chiral recognition. These columns are designed to separate enantiomers, stereoisomers, metabolites, degradants, and impurities with greater resolution and speed.

Trefoil Columns for Chiral Separations O Trefoil AMY1 2.5 µm Columns Amylose tris-(3,5-dimethylphenylcarbamate) Trefoil CEL1 2.5 µm Columns Cellulose tris-(3,5-dimethylphenylcarbamate) 0.0 Trefoil CEL2 2.5 µm Columns Cellulose tris-(3-chloro-4-methylphenylcarbamate) Chiral separations were all run using the 2-minute screening method. Trefoil Column: 2.1 x 50 mm Co-solvent: 1:1 MeOH:IPA w/20 mM NH4OH Gradient: 3–60% in 1.5 min, 60% for 0.5 min Flow rate: 1.2 mL/min Temperature: 40 °C ABPR: 3,200 psi UV Detection: 220 nm The Trefoil packing materials are designed for use with the ACQUITY UPC 2...

Any small leak could produce a situation where the LEL (lower exposure limit) is reached. Laboratories should be equipped with CO2 and/or O2 sensors when carbon dioxide (CO2) is in use. b. Column Connectors and Installation ACQUITY UPC 2 Systems utilize tubing and connectors which have been designed to meet stringent tolerance levels and to minimize extra-column volume. For information on system tubing and connectors, please refer to the ACQUITY UPC 2 System Operator’s Guide (Literature Number 720004226EN). Note: Scale the flow rate up or down accordingly based upon the column I.D., length, particle...

III. COLUMN US E To ensure the continued high performance of Trefoil Columns, follow these guidelines: a. Sample Preparation 1. Sample impurities often contribute to column contamination. Waters offers both solid-phase extraction (SPE) and supercritical fluid extraction (SFE) options. For SPE, use Oasis ® Solid-Phase Extraction Cartridges/Columns or Sep-Pak ® Cartridges of the appropriate chemistry to cleanup the sample before analysis. For more information, visit www.waters.com/sampleprep. Alternatively, Waters offers the MV-10 ASFE Supercritical Fluid Extraction System for high throughput extractions...

Trefoil Column hardware and packing materials are packed and tested to pressures of up to 6,000 psi (414 bar or 41 Mpa). 1. One of the most common problems with regards to columns is incorrect or insufficient priming of the co-solvent/modifier pump. If no peaks are observed after an injection or unusually long retention times are observed, check the priming of the co-solvent/modifier pump first. e. Temperature The recommended maximum operating temperature for Trefoil Columns is 40 °C. f. Band Spreading Minimization The ACQUITY UPC 2 System has been designed to minimize band spreading. Deviation...

Retest the column after using the cleaning procedure to determine if the specific problem has been fixed. If so, continue using the column, avoiding samples and solvents that may clog the column inlet. b. Effects of Additives Use of co-solvent additives (eg, ammonia, organic amines, formic acid, trifluoroacetic acid) can be used successfully with all Trefoil Columns. Consider the volatility and detector compatibility when choosing an appropriate additive. Additives tend to improve peak shape and control the retention characteristics of analytes, but can also impart different selectivities. c....

V II. ACQUIT Y U P C 2 COLUMN FAMILY The eCord Chip provides the user with column use data, column dimensions, and serial number. The overall column usage information includes the total number of samples, total number of injections, total sample sets, date of first injection, date of last injection, maximum pressure, and temperature. The information also details the column history by sample set including date started, sample set name, user name, system name, number of injections in the sample set, number of samples in the sample set, maximum pressure, and temperature in the sample set and if...