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Mobile-Phase Chart

OPTIMUM BED DENSITY PREPARATIVE COLUMNS

Bridging the Performance Gap from Analytical to Prep

Mass Loading

Approximate Mass Loading Capacity (mg) for Prep OBD™ Columns (Gradient Mode) Many factors affect the mass capacity of preparative columns. The listed capacities represent an ‘average’ estimate. Diameter (mm)

Length (mm) 4.6 10 19 30 50 Capacity is: 50 3 15 45 110 310 ■ Higher for strongly retained material ■ Higher for simple mixtures 75 – – – 165 – ■ Lower where higher resolution is required ■ Very strongly dependent on loading conditions 100 5 25 90 225 620 – Limited by loading volume 150 8 40 135 335 930 – Limited by diluent solvent strength

250 13 60 225 560 1550 Reasonable flow rates are based on column diameter. Systems will be limited by increasing backpressure with increasing length and decreasing particle size.

Reasonable Flow 1.4 6.6 24 60 164 Reasonable injection volumes are based on column diameter at a length of Rate (mL/min) 50 mm with relatively strong solvents. Increased length is compatible with larger Reasonable Injection 20 100 350 880 2450 injection, but not proportionately so. Weaker solvents significantly increase Volume (µL) injection volume.

Mass loading capacities for peptide purifications depend strongly on the sequence and may be estimated at 5-20% of listed values. Mobile-Phase Chart

10 mM Concentration pH Adjustment Mobile-Phase Chemical pK * Buffer Range Formula Buffering Equilibrium a Mobile-Phase Preparation** ( or Base)

- pKa 1 4.76 3.8-5.8 CH3COONH4 CH3COOH ↔ CH3COO 0.77 g CH3COOH or NH4OH + pKa 2 9.20 8.2-10.2 CH3COONH4 NH4 ↔ NH3 0.77 g CH3COOH or NH4OH - Ammonium pKa 1 6.35 5.4-7.4 NH4HCO3 H2CO3 ↔ HCO3 0.79 g HCOOH or NH4OH + pKa 2 9.20 7.2-9.2 NH4HCO3 NH4 ↔ NH3 0.79 g HCOOH or NH4OH - -2 Ammonium Bicarbonate pKa 3 10.30 9.3-11.3 NH4HCO3 HCO3 ↔ CO3 0.79 g HCOOH or NH4OH - pKa 1 3.80 2.8-4.8 NH4COOH HCOOH ↔ HCOO 0.64 g HCOOH or NH4OH + Ammonium Formate pKa 2 9.20 8.2-10.2 NH4COOH NH4 ↔ NH3 0.64 g HCOOH or NH4OH 0.70 mL TEA (99% conc.) Triethylammonium Acetate (TEAA) pK 1 4.76 3.8-5.8 (CH CH ) N:CH COONH (1:2) CH COOH ↔ CH COO- TEA or CH COOH a 3 2 3 3 4 3 3 0.57 mL (100% conc.) 3 1.41 mL TEA (99% conc.) Triethylammonium Acetate (TEAA) pK 2 11.01 10.0-12.0 (CH CH ) N:CH COONH (2:1) (CH CH ) NH+ ↔ (CH CH ) N TEA or CH COOH a 3 2 3 3 4 3 2 3 3 2 3 0.29 mL Acetic Acid (100% conc.) 3 0.70 mL TEA (99% conc.) Triethylammonium Formate (TEAF) pK 1 3.75 2.8-4.8 (CH CH ) N:NH COOH (1:2) HCOOH ↔ HCOO- TEA or HCOOH a 3 2 3 4 0.41 mL (88% conc.) 1.41 mL TEA (99% conc.) Triethylammonium Formate (TEAF) pK 2 11.01 10.0-12.0 (CH CH ) N:NH COOH (2:1) (CH CH ) NH+ ↔ (CH CH ) N TEA or HCOOH a 3 2 3 4 3 2 3 3 2 3 0.20 mL Formic Acid (88% conc.) - Acetic Acid (glacial) 4.80 – CH3COOH CH3COOH ↔ CH3COO 0.57 mL (100% conc.) – + Ammonium Hydroxide 9.20 – NH4OH NH4 ↔ NH3 0.65 mL (29% as NH3 conc.) – Formic Acid 3.80 – HCOOH HCOOH ↔ HCOO- 0.41 mL (88% conc.) – + N-Methylpyrrolidine 10.30 – C5H11N C5H11NH ↔ C5H11N 1.04 mL (97% conc.) – + Pyrrolidine 11.30 – C4H9N C4H9NH ↔ C4H9N 0.84 mL (99.5% conc.) – + Triethylamine (TEA) 11.01 – (CH3CH2)3N (CH3CH2)3NH ↔ (CH3CH2)3N 1.41 mL (99% conc.) – - Trifluoroacetic Acid (TFA) 0.30 – CF3COOH CF3COOH ↔ CF3COO 0.74 mL (100% conc.) –

* pKa and Buffer Range determined in aqueous . Addition of organic modifiers may change the apparent pKa value. ** Addition of volume or mass per 1 liter. Silica-based columns (SunFire™ Prep and Atlantis® Prep) should not be operated at high pH.

UPLC to Preparative HPLC

■ Achieve high throughput with focused gradient HPLC purification ■ Gain high resolution and speed by screening with UPLC® Technology ■ Reduce steps – alleviate need for method re-development

Rapid Screening by UPLC % organic 1.06 UPLC Methods 1.5 Rapid Screening by UPLC ® m/z 216 Column: ACQUITY UPLC CSH™ C18, 2.1 x 30 mm, 1.7 µm UPLC linear gradient method for fast 0.51 ACQUITY UPLC CSH C18 0.1% Ammonium Hydroxide Part Number: 186005295 screening of purification samples. 1.0 0.67

Determine elution time of target AU Mobile Phase A: 0.1% ammonium hydroxide in compound which is used to determine 5.0e-1 1.27 Mobile Phase B: 0.1% ammonium hydroxide in acetonitrile focused gradient method for 0.78 0.0 Gradient: 2 to 98% B in 2.0 minutes, then 0.5 minute hold purification run. min time 0.00 0.50 1.00 1.50 2.00 2.50 Flow Rate: 0.9 mL/min Temp.: 30 °C Detection: UV at 210 and 254 nm 4.75 Determine HPLC Determine Prep HPLC Method ToF-MS (in ES+ mode) 1.52 Purification Method Determine 2.0e+2 and Collect Fraction system volumes 1.67 XSelect CSH C

18 m/z=216.0 HPLC Prep Method Calculate Prep HPLC focused gradient 0.1% Ammonium Hydroxide AU 1.0e+2 method from the UPLC screen using Enter elution 1.02 Column: XSelect CSH C18, 19 x 100 mm, 5 µm the Focused Gradient time from 7.10 10.72 11.45 Part Number: 186005421 screening run 0.0 UPLC to Prep Calculator. min Gradient: 2 to 30% B in 0.81 minutes, 40% B at 10 minutes, 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 % organic 98% B at 11 minutes, hold for 1.0 minutes, Collect Fractions and reset conditions for 3.0 minutes 1.07 Flow Rate: 25.0 mL/min Purification run with Prep HPLC 1.0 focused gradient method. Confirm Purity Temp.: Ambient UPLC Screening Method Total Mass Load: 50 mg 5.0e-1 time AU 0.1% Ammonium Hydroxide Purification System: AutoPurification™ Prep HPLC with FractionLynx™ Detection: UV at 210 and 254 nm and ESI+ MS 0.0 min Confirm Purity by UPLC 0.00 0.50 1.00 1.50 2.00 2.50 Compounds: 5-hydroxyindole acetic acid acetylation products

Scaling Rules Key M1 = Mass of Column 1 L1 = Length of Column 1 d1 = Diameter of Column 1 t1 = Gradient Duration for Column 1 F1 = Flow Rate for Column 1 dp1 = Particle Size in Column 1 M2 = Mass of Column 2 L2 = Length of Column 2 d2 = Diameter of Column 2 t2 = Gradient Duration for Column 2 F2 = Flow Rate for Column 2 dp2 = Particle Size in Column 2

Mass Load Flow Rate Gradient Duration Injection Volume ■ Proportional to column volume ■ Proportional to cross-sectional area ■ Slope is % change/column volume, not time ■ Approximately proportional L2 d22 L2 d22 F1 for constant linear velocity to column volume ■ Limited by in mobile phaseM2 = M1 x x t2 = t1 x x x ■ Resolution is constant with the same number of column L1 d12 ■ Increased linear velocityL 1gives shorterd12 runs F2 volumes from initial to final conditions ■ Approximately proportional to both 2 2 L2 d2 L2 ■ dUltimately2 F 1restricted by flow and pressure ■ Must include initial hold to mimic the system delay, expressed as length and cross-sectional area M2 = M1 x x t2 = t1 x x limits xof system number of column volumes, observed on small-scale separation ■ Most strongly dependent 2 2 L1 d1 L1 d1 F2 on sample solvent 2 2 2 2 d2 d2 L2 d p1 d2 L2 d2 F1 F2 = F1 x F2M =2 F=1 Mx 1 x x x t2 = t1 x x x 2 2 2 2 d1 d1 L1 d p2 d1 L1 d1 F2 2 2 d2 d2 dp1 F2 = F1 x F2 = F1 x x 2 2 d1 d1 dp2 System Volume 2 2 Recommended Method for Measurement Programmed Time = d5.002 minutes d2 dp1 System volume is important in scaling separations because it F2 = F1 x FFlow2 = Rate F =1 1.5x mL/minx 1. Remove column. 0.70 2 2 creates an isocratic hold at the start of every run. This hold is 1 1 p2 2. Use acetonitrile as mobile-phase A, and acetonitrile with 0.65 d d d often several column volumes on a small scale, but a fraction 5.69 minutes 0.05 mg/mL uracil as mobile-phase B (eliminates non-additive 0.60 - 5.00 minutes of the volume of a prep column. Compensation for this volume 0.55 mixing and viscosity problems). 0.69 minutes must be included in planning a scaling experiment to avoid 0.50 3. Set UV detector at 254 nm. 0.45 distorting the chromatography. 4. Use the flow rate in the original method and the intended 0.40 AU 0.35 50% Time = 5.69 minutes flow rate on the target instrument. 0.30 5. Collect 100% A baseline for 5 minutes. 0.25 6. Program a step change at 5 minutes to 100% B, 0.20 System Volume: and collect data for an additional 5 minutes. 0.15 0.69 min x 1.5 mL/min = 1.04 mL 0.10 7. Measure absorbance difference between 100% A and 100% B. 0.05 8. Measure time at 50% of that absorbance difference. 0.00 9. Calculate time difference between start of step and 50% point. 0 2 4 6 8 10 12 14 16 18 20 min 10. Multiply time difference by flow rate.

Waters Preparative OBD Columns Calculator

■ Convenient scale-up tool provides: – Mass load scaling – Gradient scaling with appropriate flow rate scale-up and predicting volume consumption – Calculations for split flow ratios for those using mass spectrometer driven chromatography – Focused gradient UPLC to preparative method transfer

■ Scale-up calculations are carried out using the equations shown above

To try this tool, visit www.waters.com/prepcalculator

©2012 Waters Corporation. Waters, Atlantis, UPLC and ACQUITY UPLC are registered trademarks of Waters Corporation. XBridge, XSelect, SunFire, OBD, CSH, AutoPurification, FractionLynx, and The Science of What’s Possible are trademarks of Waters Corporation. 720002117EN October 2012 LB-AP

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