ABSTRACT
Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC) or gel ‰ltration chromatography (GFC), is a powerful technique for the puri‰cation, separation, and characterization of natural and synthetic polymers. Over the years, the method has gained wide acceptance as one of the major puri‰cation methods for proteins, polysaccharides, and nucleic
3.1 Introduction .......................................................................................................................... 105 3.2 Separation Mechanism ......................................................................................................... 106 3.3 De‰nitions............................................................................................................................. 107 3.4 Instrumentation and Operation ............................................................................................. 110
3.4.1 Degasser .................................................................................................................... 110 3.4.2 Solvent Delivery System ........................................................................................... 110 3.4.3 Detectors ................................................................................................................... 111
3.4.3.1 UV Detector ............................................................................................... 111 3.4.3.2 Refractive Index Detector .......................................................................... 111 3.4.3.3 Evaporative Light Scattering Detector ....................................................... 112 3.4.3.4 Light Scattering Detector ........................................................................... 112 3.4.3.5 Other Detectors .......................................................................................... 115
3.4.4 Column ..................................................................................................................... 115 3.4.5 Mobile Phase ............................................................................................................ 116 3.4.6 Data Acquisition and Processing .............................................................................. 117 3.4.7 Calibration Curve ..................................................................................................... 118
3.5 Applications of SEC in Oligonucleotide Therapeutics ......................................................... 120 3.5.1 Oligonucleotide Duplexes ......................................................................................... 121
3.5.1.1 Monitoring of Annealing during Manufacturing ...................................... 121 3.5.1.2 Analysis of Liposome Encapsulated siRNA ..............................................124 3.5.1.3 Stability Monitoring of DNA and RNA Duplexes ..................................... 125
3.5.2 Conjugated Oligonucleotides and Oligonucleotide Complexes ................................ 126 3.6 Summary .............................................................................................................................. 132 References ...................................................................................................................................... 133
acids. Although the technique was ‰rst developed more than 50 years ago,1 the original gel matrix, Sephadex (a cross-linked dextran), is still widely used for protein puri‰cation today. Given the popularity of SEC in protein puri‰cation, it is not surprising that the technique has also been widely used in the puri‰cation of DNA,2-8 RNA,9-14 oligonucleotides,15,16 oligonucleotide complexes,17 and oligonucleotide conjugates.18,19
Another area in which SEC ‰nds application is the determination of molecular weight of polymers.20 This is usually accomplished by comparing the unknown sample against a calibration curve (log Mp versus retention time/volume) constructed with reference standards of known molecular weight. The approach is sometimes referred to as conventional SEC and is usually carried out with a concentration detector such as refractive index (RI) or ultraviolet (UV). With the introduction of light scattering (LS) and viscosity detectors, the molecular weight of a polymer can now be determined without a calibration curve. This mode of SEC is sometimes referred to as multidetector SEC because a mass detector is used in conjunction with a concentration detector for molecular weight determination.
