Monolithic matrix for separating bio-organic molecules

Details Monolithic matrix for separating bio-organic molecules Monolithic matrix for separating bio-organic molecules

1998-09-16

2001-05-29

Therkorn, Ernest G. (Department: 1723)

Liquid purification or separation

Processes

Liquid/liquid solvent or colloidal extraction or diffusing...

C210S656000, C210S659000, C210S198200, C435S006120, C536S023100, C536S025400

Reexamination Certificate

active

06238565

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the separation of bio-organic molecules. In particular, the invention provides a monolithic polymer matrix for the separation of polynucleotides by reversed-phase ion-pairing chromatography.
BACKGROUND OF THE INVENTION
Polynucleotide separations have become increasingly important in recent years. Separating polynucleotide species contained in a sample is useful, for example, in the detection andlor quantification of DNA that is the product of amplification reactions, and in the detection of variant DNA (e.g. polymorphisms or mutations). Due in large part to the complex structures and large sizes of such molecules, however, none of the separation techniques devised to date have proven wholly satisfactory.
Traditionally, polynucleotide separations have been performed using electrophoretic methods, such as slab gel electrophoresis or, more recently, capillary electrophoresis. Generally, these methods involve passing an electric current through a medium into which a mixture containing the species of interest has been injected. Each kind of molecule travels through the medium at a different rate, depending upon its electrical charge and size. Unfortunately, the electrophoretic methods are associated with certain disadvantages. For example, slab gel electrophoresis suffers the drawbacks of relatively low speed, difficulty in detection of samples, poor quantitation, and is labor intensive. Although faster and less labor intensive, capillary electrophoresis has suffered from irreproducibility of separations due to changes in the capillary performance and relatively poor quantitation.
Liquid chromatography has provided an alternative to the electrophoretic separation methods. Generally, the liquid chromatographic methods rely on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the components in a mixture. A column tube, or other support, holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster. As the components elute from the column they can be quantified by a detector and/or collected for further analysis.
Typical stationary phases, and their interactions with the solutes, used in liquid chromatography are:
STATIONARY
NAME
PHASE
INTERACTION
Size-
Porous inert
Samples are separated by virtue of their size
Exclusion
particles
in solution; different sized molecules will
have different total transit times through
the column.
Ion-
Ionic groups
Sample ions will exchange with ions already
Exchange
on a resin
on the ionogenic group of the packing;
retention is based on the affinity of different
ions for the site and on a number of other
solution parameters (pH, ionic strength,
counterion type, etc.).
Reverse-
Non-polar
Samples are separated based on hydrophobic
Phase
groups on
interactions with the stationary phase.
a resin
Although the liquid chromatographic methods have some advantages over electrophoretic separation techniques, they are not without their shortcomings. Size-exclusion chromatography suffers from low resolution, as, typically, DNA molecules must differ in size by 50-100% in order to obtain acceptable resolution. Although ion-exchange chromatography offers higher resolution, it can be affected by anomalous elution orders based on DNA sequence composition. Also with ion-exchange chromatography, the eluted DNA is often heavily contaminated with nonvolatile buffers that can further complicate sample recovery. Reverse-phase chromatography is capable of relatively high resolution but, unless special small-diameter particulates are used as the stationary phase, it cannot be performed at a very high speed. Silica-based particulates used in reverse-phase chromatography have suffered from low speed and instability at high pH conditions. Polymeric particulates have not been able to provide a high recovery of sample components or high separation speeds.
With particular regard to DNA separations, a technique known as reverse-phase ion-pair high performance liquid chromatography (RP-IP HPLC) has provided a limited amount of relief from some of the problems discussed above. For example, RP-IP HPLC avoids the problem of anomalous elution orders often encountered with packed beds bearing strong-anion exchangers. In RP-IP HPLC, the stationary phase typically consists of discrete particles bearing hydrophobic surface groups that are packed into a column. The eluent contains a cationic species, such as triethylammonium ion (0.1M), capable of interacting with the negatively charged phosphate groups on DNA and also with the hydrophobic surface of the particles in the column. Thus, the cationic species can be thought of as a bridging molecule between DNA and the column. As the mobile phase is made progressively more organic, e.g., with increasing concentration of acetonitrile, the DNA fragments are eluted in order of size.
Despite the advantages of RP-IP HPLC for DNA separations, the technique nevertheless suffers from problems common to all liquid chromatographic techniques wherein small particles (e.g., beads) are packed to form a bed in a column tube. For example, the production of particulate separation media can be complex and time-consuming. Once prepared, it can be difficult to pack the particles in columns in a reproducible and efficient manner. In particular, it has been difficult to pack efficient columns of small dimensions, such as columns less than 1 mm in diameter. Columns having norcclrcular cross-sections, such as polygonal cross-sections (e.g., thin-layer or rectangular), would be extremely difficult to prepare from particulate packing materials. Also, columns based on packed beads can fail due to shifting of packing material and the development of channels or voids. As an additional disadvantage, the use of small particles often leads to high column operating pressures, which necessitate column tubes, pumps, injectors and other components capable of containing fluid pressures of 3,000 psi or greater.
Current chromatographic theory predicts that, for beds of packed particles, separation efficiency will be determined by the diffusional distance for mass transfer of sample molecule to the stationary phase. This effect is easily modeled based on the known geometry of the particles used in such beds. According to such theory, maximum resolution will occur when the stationary phase has pore diameters of at least 3 times the Stokes' diameter of the molecule to be separated. This theory is based on the assumption that the molecule will be transported to the stationary phase by a diffusive process and that smaller pores would cause hindered diffusion. For DNA separations, this means that for a sample of 1,000 base pairs in size, a pore diameter of at least 1 micrometer will be needed. The need for such large pores practically eliminates the use of porous particulate materials packed in a support for the separation of DNA, as such matrices lack the required physical strength to be used at the operating pressures encountered in high performance liquid chromatography.
Nonporous particulate materials are preferred for DNA separations due to the reduction in diffusion distance compared to porous packings. For nonporous packings, the pore diameter is determined by the interstitial dimensions of the packed bed. The interstitial dimension is approximately ⅓ of the diameter of a spherical packing particle. A lower limit to the pore diameter may be imposed by the need to avoid trapping or shearing of larger DNA molecules. A higher limit will be imposed by the loss of efficiency that occurs when using larger particles. Separation using larger particles would be advantageous because of the lower operating pressures involved, but the loss in separation efficiency is too great.
As a further disadvantage, spherical packings can be packed i

Affiliated with

Also associated with

Rating

Monolithic matrix for separating bio-organic molecules does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Monolithic matrix for separating bio-organic molecules, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Monolithic matrix for separating bio-organic molecules will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2473187