Principles and Practical Aspects of Preparative Liquid Chromatography

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4 STRATEGIES FOR SCALE-UP 9-11 4.1 Analytical chromatography as a starting point for scale-up In this chapter we describe scale-up from a 4.6 by 150 mm analytical column to a 21.2 by 150 mm preparative column, starting with a generic elution gradient. A correct scale-up process between two different column geometries can only be achieved when the same chemistries, pH conditions, particle sizes and column lengths are used. Analytical chromatography is required as a first step to confirm the presence of the target compound and if it could be separated from the other compounds under the selected chromatographic conditions. Typically, a gradient profile from 2 to 98 % of the organic content is best suited to accommodate for a variety of compounds. An additional isocratic step directly after injection makes sure early eluting compounds are retained – especially when the sample has been dissolved in a solvent with high elution strength for better solubility, for example, DMSO. The UV signal or the TIC trace of the mass-selective detector can be used to monitor this step. Generic gradient profiles of 10 %B/min can be used for column lengths of 150 mm. To be as efficient as possible the slope can be increased up to 30 %B/min for a column length of 50 mm. In contrast, the slope must be decreased to 6 %B/min for a column length of 250 mm. Using an appropriate flow rate for the selected column diameters, resolution can be increased by varying the steepness of the gradient slope. Experiments performed on our system showed that a flow rate of 1.5 mL/min delivers highest resolution and number of theoretical plates using a 4.6 by 150 mm, 5 µm column. This flow rate is the starting point for all further scale-up processes. Switching to a preparative system requires changes to flow rates, additional isocratic holding steps, modified gradient slopes, and adjusted injection volumes and run times, whereby the preparative gradient profile should look similar to the analytical gradient profile. Figure 4.1 shows the gradient profile of an analytical scouting run. Figure 4.2 shows the gradient profiles after transferring the method from a system with a small dwell volume to a system with a larger dwell volume and larger column inside diameter. The isocratic holding step has been extended to compensate for the differences in dwell 51
volume of both systems. The linear method transfer from an analytical to the preparative system was accomplished using the formulas in Section 4.2 “Formulas for linear scale-up from analytical to preparative columns”. Equilibration Isocratic steps Gradient Purge 100%B t iso1 0%B Time Figure 4.1 Gradient profile for an analytical scouting run using a 4.6-mm id column, comprising injection, optional isocratic hold, gradient slope and purge phases. Equilibration Isocratic steps Gradient Purge 100%B t h2 t iso2 0%B Time Figure 4.2 Gradient profile after scale-up to a 21.2-mm id column, comprising an extended isocratic step to compensate for the applied flow rate with respect to the difference in dwell volumes between the analytical and preparative system. 52
Page 1:
PRINCIPLES AND PRACTICAL ASPECTS OF
Page 4 and 5:
CONTENTS Foreword IV Introduction
Page 6 and 7:
FOREWORD As a synthetic chemist, bi
Page 8 and 9:
ABOUT THE AUTHORS Helmut Schulenber
Page 10 and 11: 1 INTRODUCING PREPARATIVE LIQUID CH
Page 12 and 13: for each compound. Optimized gradie
Page 14 and 15: Analytical Semi-preparative Prepara
Page 16 and 17: In contrast, we recommend to use st
Page 18 and 19: 3 COMPONENTS OF A PREPARATIVE LC SY
Page 20 and 21: Degassing unit Damper Damper Mixer
Page 22 and 23: Pump Sampling loop Metering device
Page 24 and 25: 3.2.2 Fixed-loop design of autosamp
Page 26 and 27: Figure 3.11 shows a dual-loop autos
Page 28 and 29: Moving the switching valve back to
Page 30 and 31: Plug Plug Sample Figure 3.17 Profil
Page 32 and 33: When deploying a configuration such
Page 34 and 35: [Norm.] 7.584 3000 2000 2.791 5.720
Page 36 and 37: 3.3 Flow splitting 3.3.1 T-piece fl
Page 38 and 39: Split ratio = LC flow rate [μL/min
Page 40 and 41: 3.4.1 Matching concentration range
Page 42 and 43: 3.5.1 Collecting fractions manually
Page 44 and 45: 3.5.3.2 Setting slope parameters Th
Page 46 and 47: Flow splitter UV detector ELS detec
Page 48 and 49: UV t D Injection of calibration sam
Page 50 and 51: Figure 3.41 shows the time differen
Page 52 and 53: When acquiring in scan mode a total
Page 54 and 55: 3.7 System considerations 3.7.1 Sys
Page 56 and 57: • Standard 1/16-inch capillaries
Page 58 and 59: [Norm.] 2000 1500 1000 500 v dwell
Page 62 and 63: 4.2 Formulas for linear scale-up fr
Page 64 and 65: Figure 4.3 shows the results of a m
Page 66 and 67: 4.3.2 Developing focused gradients
Page 68 and 69: 4.4 Describing the entire scale-up
Page 70 and 71: 4.4.2 Step 2 - Applying a focused g
Page 72 and 73: 5 PRACTICAL GUIDELINES AND DETAILED
Page 74 and 75: 1. Prepare slurry 2. Fill column 3.
Page 76 and 77: 5.2 Determining the system dwell vo
Page 78 and 79: 5.3.1 Simplified procedure for colu
Page 80 and 81: 6. Display UV profile at 242 nm in
Page 82 and 83: [mAU] 1600 1400 1200 1000 4.649 4.0
Page 84 and 85: 5.5.1 Volume overload Figure 5.8 sh
Page 86 and 87: Diameter [cm] 0.46 1.09 2.12 3.00 5
Page 88 and 89: REFERENCES 1. Huber, U., Solutions
Page 92: www.agilent.com/chem/purification T
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Principles and Practical Aspects of Preparative Liquid Chromatography

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