Intra-tube effect

As we will see in the next section, when field-inversion electrophoresis is used to separate nucleic acids in certain size ranges, intra-tube relaxation effects take place. Of course, as the angle between the two fields increases from 90 to 180 , we expect that the crossed field effects discussed earlier will be modified by more and more of the intra-tube effects that sometimes dominate in field-inversion electrophoresis. It is not the goal of this article to discuss the angles between 90 to 180 . The next section presents a discussion of field-inversion electrophoresis as well as the ways to optimize the reorientation-induced separation obtained with this experimental arrangement. 

In continuous fields, these intra-tube effects change the effective values of the parameters Brown needs to fit experimental... 

One exception to that is apparently the steady-state orientation factor measured by LD. Our BRM predicts that this orientation factor should be slightly molecular-size dependent in the vicinity of the band-inversion phenomenon, but otherwise size-independent. Experiments showed a fairly strong size dependence up to large molecular sizes, even if these large molecules had a size-independent mobility. This is possibly due to an intra-tube effect which can induce local fluctuations in the average tube orientation but, since the tube stays oriented, retain the molecular-size independence of the mobility. 

Although intra-tube effects may be expected to play a larger role in pulsed field techniques and transient effects, the form of the model which averages over these effects offers a good framework to discuss the experimental results even in these cases. The orientation overshoot observed with linear dichroism is a transient effect that occurs at a time which is smaller than the tube-renewal time we thus conclude... 

Crossed-field electrophoresis provides an interesting alternative to intermittent-field electrophoresis. The idea is simply to change by 90 the direction of the field each time the tube is fully oriented in the field direction because we know that this orientation destroys the molecular-size dependence of the electrophoretic velocity. Although some intra-tube effects can be expected to play a role here, especially because crossed fields may induce the creation of a large number of transverse tube leakages, the BRM reproduces most experimentally observed effects up... 

The biased reptation model provides a good framework to discuss the experimental results of the various gel electrophoresis techniques used to separate nucleic acids. Although more experiments are needed to fully characterize these techniques, available results indicate that the simplified version of the model discussed in this paper is satisfactory when low-frequency pulsed fields are used, or when transient intra-tube effects are not dominant. This is the case in continuous fields, for small molecules in intermittent fields, and possibly also for crossed fields. However, intra-tube effects are observed to play a role in field-inversion electrophoresis, for long molecules in intermittent fields, and during the first stages of an experiment (where an orientation overshoot is observed). 

The large number of cases where the biased reptation model is reliable indicates that the intra-tube effects do not rule out reptation as the basic migration mechanism. Further theoretical advances will include the effects of both intra-tube molecular orientation and tube orientation on the electrophoretic properties of large nucleic acids. 

However, three remarks concerning the effects of short wavelength modes should be made here. First, the over- and under-shoot stretching phenomena clearly point out that some intra-tube DMA modes, neglected in the BRM, are excited if the field is applied on a truly random coil DNA. This transient effect does not seem to affect the overall reptative behaviour of DNA, but may be very important in pulsed field techniques where separation is increased by coupling the external pulsed field(s) to transient effects that may affect the molecular electrophoretic mobility. 

In the meantime the quasiplanarity, predicted in Ref. [2], has theoretically been confirmed by several authors . On the other hand, Perkins el af. experimentally confirmed that, by breaking intra-icosahedral bonds on the (100) surface of icosahedral solid YBee crystals, hexagonal and pentagonal pyramidal forms are observed as open umbrellas . Due to the fact that pure boron spheres like B32 are less stable in comparison to boron sheets or tubes, in the present work we look for the stabilizing effect of the hydrogen atoms by eliminating the dangling bonds in the boron spheres. 

To minimize the effect of sample volume on dispersion, and ensure that there was minimum dispersion from the valve and valve connections, a 0.2 jxl Valeo internal loop valve was employed to place the sample on the column. In addition, the sample valve was used with an intra-column injection system [2] that ensured the sample was placed in the center of the column about 5 mm below the packing surface. TMs device also determined that, even if the packing settled, there would be no void above the point of injection and the effective length of the column was not changed. All connecting tubes were 0.007 in. I.D. and their lengths were kept to a minimum (Le., <5 cm). The detector employed was the Perkin Elmer LC-85B fitted with a cell 1.4 pi volume and was used in conjunction with an electronic amplifier that had an effective time constant of 24 millisec. The columns employed had internal diameters of 8-9 mm to ensure the peak volumes were very large compared to any dispersion introduced by extra-column dispersion. As a result, the extra-column dispersion was maintained at a level of less than 2% of the peak volume for all measurements and, in most instances, was maintained at a level of less than 1%. 

---