Principles and Practical Aspects of Preparative Liquid Chromatography

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[Norm.] 7.584 3000 2000 2.791 5.720 1000 0 1 2 3 4 5 6 7 8 Time [min] Figure 3.20 High-volume injection with strong eluents using the alternative injection mode. A broad solvent peak is obtained unless the DMSO has been washed out of the column. The two compounds at 5.72 and 7.58 minutes have been retained on-column and eluted as baseline separated peaks. No sample loss has been observed. Sample: 50 mg acetamidophen and 50 mg caffeine in 5000 µL DMSO Column: Agilent ZORBAX SB C18, 21.1 x 150 mm, 5 µm Applied gradient profile for sandwich injection mode – a longer isocratic holding step is needed to transfer the sample to the column prior to ramping up the gradient: Time Flow %A %B 0 37 93 7 4.0 37 93 7 10.0 37 78 22 10.1 37 2 98 13.0 37 2 98 13.1 37 93 7 16.0 37 93 7 3.2.3.3 Injecting large sample volumes using an injection pump For separation of highly diluted samples in aqueous solvents, large injection volumes that exceed the typical loop sizes of autosamplers are often required. In these situations an injection pump can be used to load the sample onto the column. Injection pumps can also be used for applications such as enantiomer separation. Here, the same sample solution must be loaded periodically and an injection pump can achieve this much faster than an autosampler. After sample transfer to the column, thoroughly purge the injection pump, the sampling and injection valves, and all connection capillaries to avoid sample carryover. Figure 3.21 shows a typical system configuration for sample introduction using an injection pump. 25
Gradient pump Preparative column Channel A Detector Channel B 2 3 1 4 6 5 Injection (valve in pos. 1) Fraction collector 2 3 4 5 1 6 12 7 11 10 9 8 Sampling valve Waste Injection pump Sample container Gradient pump Preparative column Channel A Detector Channel B 2 3 1 4 6 5 Elution (valve in pos. 2) Fraction collector 2 3 4 5 1 6 12 7 11 10 9 8 Sampling valve Waste Injection pump Sample container Figure 3.21 Schematics of a system configuration for sample introduction using an injection pump. With the valve in position 1, the injection pump draws the sample from the container and delivers to the column. For elution, the valve is switched to position 2 so that channels A and B combine, generating the required eluent composition for compound separation. 26
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 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 60 and 61: 4 STRATEGIES FOR SCALE-UP 9-11 4.1
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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